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In our CAS experience encouraging long term results seem to derive from both neurological event free rate and restenosis incidence. Because of residual stenosis after CAS is a strongest independent predictor of restenosis, adequate recanalization of the treated vessel seems an important goal to limit the development of restenosis. Multiple stents deployment and with less evidence, diabetes or neoplasm have to be considered to facilitate in-stent restenosis after CAS. Figure 1 Kaplan Meyer analysis curve of the cumulative freedom from stroke and death. Figure 2 Kaplan Meyer analysis curve of the cumulative freedom from restenosis. Figure 3 Kaplan Meyer analysis curves of the cumulative freedom from stroke and death in patients with (dotted line) and without (continuous line) restenosis.

1. Introduction We report two cases where an anti-gravity suit (also named MAST: Medical Antishock Trousers [1, 2]) was applied with a low gradient of pressure during the acute phase of symptomatic carotid occlusion to amplify the blood volume shift towards the craniothoracic territory [3, 4], improving cerebral haemodynamic conditions and neurological symptoms. The impact of anti-gravity suit application on cerebral vascularisation was measured by transcranial Doppler.

1. Introduction Despite recent advances in minimally invasive imaging techniques for carotid vessels like Doppler ultrasonography (US), MR angiography (MRA), and CT angiography (CTA), digital subtraction angiography (DSA) provides the highest spatial resolution and still remains the “gold standard” for diagnosis of carotid artery stenosis, but it is associated with risk for procedure-related neurologic complications. In fact, therapeutic decisions in large clinical trials [1–4] have been based on maximal internal carotid artery (ICA) stenosis depicted with conventional DSA. It is well known that vulnerable carotid plaque is an atheromatous plaque that is particularly prone to disruption, fracture, or fissuring with a higher risk for embolization, occlusion, and consequent ischemic neurological events [5]. Although disruption of such unstable plaques has been commonly implicated as a risk for procedure-related neurological complications in patients undergoing DSA, most resultant stroke events are clinically silent or transient [6, 7], and there are few descriptions of the affected vessel walls. We report a case of direct visualization and histolopathologic examination of carotid plaque disruption associated with the diagnostic DSA for therapeutic consideration of asymptomatic moderate-grade carotid stenosis, which was incidentally detected later during carotid endarterectomy (CEA).

It is well known that vulnerable carotid plaque is an atheromatous plaque that is particularly prone to disruption, fracture, or fissuring with a higher risk for embolization, occlusion, and consequent ischemic neurological events [5]. Although disruption of such unstable plaques has been commonly implicated as a risk for procedure-related neurological complications in patients undergoing DSA, most resultant stroke events are clinically silent or transient [6, 7], and there are few descriptions of the affected vessel walls. We report a case of direct visualization and histolopathologic examination of carotid plaque disruption associated with the diagnostic DSA for therapeutic consideration of asymptomatic moderate-grade carotid stenosis, which was incidentally detected later during carotid endarterectomy (CEA). 2. Case Report A 64-year-old man with past medical history of hypertension, type 2 diabetes mellitus, hypercholesterolemia, and symptomatic ischemic stroke in the territory of the thalamoperforate artery diagnosed to have an asymptomatic moderate (approximately 50%) right ICA stenosis that was observed on MRA was referred to our center for consideration of surgical intervention. Carotid Doppler ultrasonography demonstrated hypoechoic plaques with an irregular surface at the carotid bifurcation extending to the proximal ICA with stenosis of 83% (by area method) and peak systolic flow velocity at 1.89 m/s (Figures 1(a) and 1(b)). Resting single photon emission CT (SPECT) showed severe hypoperfusion in the right ICA territory (Figure 1(c)), presumably due to less prevalence of collateral flow via the anterior or posterior communicating artery (Figures 1(d) and 1(e)) [8].

ethod) and peak systolic flow velocity at 1.89 m/s (Figures 1(a) and 1(b)). Resting single photon emission CT (SPECT) showed severe hypoperfusion in the right ICA territory (Figure 1(c)), presumably due to less prevalence of collateral flow via the anterior or posterior communicating artery (Figures 1(d) and 1(e)) [8]. For therapeutic decision-making, diagnostic carotid angiography was then performed via a femoral approach. It was difficult to cannulate a 5-Fr. JB-2 catheter (Cook, Bloomington, IN) over an angled 0.035 inch Radifocus guidewire (Terumo, Tokyo, Japan) selectively advanced into the right common carotid artery (CCA). The procedure was repeated with a 5-Fr. Simmons II catheter (Cook, Bloomington, IN) but failed to engage in the CCA due to severe vascular elongation. Therefore, the guidewire was advanced carefully, with special attention not to cross the stenotic lesion at the proximal ICA, into the lingual branch of the external carotid artery (ECA) for support, and the catheter was successfully advanced to the CCA. The DSA revealed a 60% stenosis of the proximal right ICA with wall irregularities (Figure 2(a)), calculated according to the North American Symptomatic Carotid Endarterectomy Trial (NASCET) method [2]. The contralateral carotid angiogram demonstrated a mild stenosis in the posterior wall of the ICA. Vertebral angiography was discontinued as it was difficult to probe the bilateral vessels due to elongation.

2(a)), calculated according to the North American Symptomatic Carotid Endarterectomy Trial (NASCET) method [2]. The contralateral carotid angiogram demonstrated a mild stenosis in the posterior wall of the ICA. Vertebral angiography was discontinued as it was difficult to probe the bilateral vessels due to elongation. The patient developed numbness and mild weakness of the left hand immediately after the procedure. Diffusion-weighted MR imaging showed multiple, small hyperintense lesions in the distal ICA territory of the right front-parietal lobe indicative of an embolic origin from the carotid plaques (Figure 2(c)). The symptoms were transient and resolved within 24 hours of the procedure with supplemental intravenous fluids followed by oral clopidogrel (Plavix, Sanofi Pharmaceuticals, New York, NY) 75 mg once daily. Based on the results of the Asymptomatic Carotid Atherosclerosis Study (ACAS) [1] and the Medical Council Asymptomatic Carotid Surgery Trial (ACST) [4], the patient is considered to be a good candidate for elective surgery and given informed consent about CEA.

ix, Sanofi Pharmaceuticals, New York, NY) 75 mg once daily. Based on the results of the Asymptomatic Carotid Atherosclerosis Study (ACAS) [1] and the Medical Council Asymptomatic Carotid Surgery Trial (ACST) [4], the patient is considered to be a good candidate for elective surgery and given informed consent about CEA. Two weeks later, the patient underwent successful right CEA. Fragile atherosclerotic plaque with sharp surface laceration, somewhat different from atheromatous plaque rupture, accompanied by intraplaque hemorrhage was observed at the proximal ICA close to the bifurcation (Figures 3(a) and 3(b)). There was no evidence of perforation outside the wall. Microscopic examination of the endarterectomy specimen revealed a large atheromatous plaque with fibrous hypertrophy and intraplaque hemorrhage filled with recent hemorrhagic debris that stained red to brown with Elastica-Masson stain, cholesterol crystal formation, and speckled calcification (Figure 3(c)). The postoperative course was uneventful and the stenosis had improved significantly on follow-up MRA (Figure 2(b)) with no apparent distal embolization (Figure  2(d)). The patient was asymptomatic at his neurological baseline without any postoperative complications and was discharged on postoperative day 10.

ure 3(c)). The postoperative course was uneventful and the stenosis had improved significantly on follow-up MRA (Figure 2(b)) with no apparent distal embolization (Figure  2(d)). The patient was asymptomatic at his neurological baseline without any postoperative complications and was discharged on postoperative day 10. 3. Discussion Although minimally invasive MRA and CTA have partially replaced conventional DSA in clinical routine, it is still not a standardized method for detection and grading of carotid artery stenosis, especially for asymptomatic patients with moderate carotid stenosis (50%–69%) where the quantification of stenosis can seriously impact on clinical decision-making. According to the current United States guidelines from the American Academy of Neurology, surgical treatment of asymptomatic patients with carotid stenosis 60%–99% in patients with a 5-year life expectancy are recommended if the operator has a perioperative complication rate of <3% (level A) [9]. By contrast, there is no treatment recommendation for asymptomatic patients with stenosis 50%–59%. This patient has already diagnosed as 50% moderate stenosis on MRA, and thus confirmatory imaging was required for therapeutic decision-making. Although multidetector CT had initially been taken into consideration for assessment of carotid stenosis; however, conventional DSA was chosen after all because we were worried about misclassification of patients within the surgical range associated with greater underestimation with CTA in moderate grades of stenosis [10].

ough multidetector CT had initially been taken into consideration for assessment of carotid stenosis; however, conventional DSA was chosen after all because we were worried about misclassification of patients within the surgical range associated with greater underestimation with CTA in moderate grades of stenosis [10]. It should be noted that the high diagnostic accuracy of DSA before deciding on carotid intervention must be balanced against the risk of neurological complications. The neurologic complications are more common when indication for DSA is carotid stenosis or ischemic stroke (1.8%) [11]. Furthermore, it has also been pointed out that such clinically overt neurological symptoms are only the “tip of the iceberg” since the rates of DSA-related silent microemboli detected by diffusion-weighted MR abnormalities are considerably higher [7, 12, 13]. Although it remains a matter of debate, the level of operator experience (procedural and fluoroscopy time, multiple catheters use, and aortography) and the nature of the underlying disease are thought to be predictors of the occurrence of cerebral ischemic events following diagnostic DSA [7, 11]. At the authors' institution, diagnostic cerebral angiographies are generally performed by neurosurgical fellows who had already performed at least 250 cerebral angiographies and allowed to perform the procedures on their own, with an acceptable neurologic complication rate (0.8%) compatible to those of recent data [7, 11, 14].

uthors' institution, diagnostic cerebral angiographies are generally performed by neurosurgical fellows who had already performed at least 250 cerebral angiographies and allowed to perform the procedures on their own, with an acceptable neurologic complication rate (0.8%) compatible to those of recent data [7, 11, 14]. We postulate that the periprocedural manipulation could have resulted in the symptomatic neurological event. This might be related to the difficulties in probing the vessels, the presence of vulnerable atherosclerotic plaque located at the carotid bifurcation that might be impinged and scraped off the vessel wall presumably during the guidewire maneuver in the ECA, and the instability of fresh thrombus in exulcerating plaques that might embolize during the procedure. Unfortunately, the surface morphology of the excised carotid plaque corresponding to the affected intimal lesion could not be well characterized in this case because of artifacts introduced during the removal of the lesion, and nor could we distinguish the hemorrhagic lesion caused directly by the angiographical procedure and/or by its natural course of bleeding. During CEA, the full thickness of the plaque was incised, thereby disrupting the luminal surface of the lesion. However, the disrupted plaque showed sharp laceration (Figure 2(b)) that cannot be explained simply by atheromatous plaque rupture.

ly by the angiographical procedure and/or by its natural course of bleeding. During CEA, the full thickness of the plaque was incised, thereby disrupting the luminal surface of the lesion. However, the disrupted plaque showed sharp laceration (Figure 2(b)) that cannot be explained simply by atheromatous plaque rupture. 4. Conclusion This is, to the authors' knowledge, the first report of direct visualization and histolopathologic examination of DSA-related carotid plaque disruption. The impact of the invasive diagnostic carotid angiography on vessel wall injuries warns the danger of serious procedure-related complications, and thus the highest level of practitioner training and technical expertise that ensures precise preprocedural assessment of plaque characteristics by multimodal methods (e.g., ulcerations, surface irregularities, number of plaques, echolucent plaques, plaque distribution along the carotid bifurcation, intraplaque hemorrhage, and lipid-rich necrotic core) [5, 15, 16] should be encouraged in the patient subgroup where the DSA remains necessary. Figure 1 (a) B-mode ultrasound with color flow Doppler image on the longitudinal display of the carotid plaque with irregular surface. (b) The transverse display of the plaque at the carotid bifurcation. (c) 123I-IMP SPECT transaxial slices of a patient with right ICA stenosis. MR angiography ((d) submental vertical projection; (e) lateral projection) with a hypoplastic or absent anterior and posterior communicating arteries.

e with irregular surface. (b) The transverse display of the plaque at the carotid bifurcation. (c) 123I-IMP SPECT transaxial slices of a patient with right ICA stenosis. MR angiography ((d) submental vertical projection; (e) lateral projection) with a hypoplastic or absent anterior and posterior communicating arteries. Figure 2 (a) Peroperative common carotid angiogram (lateral projection) of a 64-year-old asymptomatic patient with a 60% stenosis of the right ICA by NASCET criteria (arrow). (b) Postoperative MR angiography confirmed disappearance of the stenosis. (c) Axial diffusion-weighted MR imaging of the brain immediately after the occurrence of neurologic events following the DSA. Ipsilateral hyperintense lesions are appreciable at the cortical-subcortical junction of right front-parietal lobes. (d) Postoperative diffusion-weighted imaging indicative of no ischemic lesions associated with CEA. Postoperative MR imagings ((b) and (d)) were performed on the next day after CEA. Figure 3 (a) An intraoperative view of an atheromatous plaque originating from the proximal ICA during CEA with a shunt. (b) Magnified image revealing the sharp laceration of the plaque surface (black arrow) and intraplaque hemorrhage (white arrow). (c) Elastica-Masson stain matching histology cross section of the carotid plaque showing a large lateralized atheroma and intraplaque hemorrhage. A ditch shown in blue arrow suggests a part of the lacerating injury. Asterisks indicate the lumen.

1. Introduction Based upon data of multicenter trials, carotid endarterectomy (CEA) is a proven treatment in carotid artery stenosis and it is considered the most effective method to prevent stroke occurrence in patients with symptomatic and asymptomatic high-grade carotid artery disease [1, 2]. For high risk surgical patients, percutaneous carotid transluminalangioplasty and stenting (CAS) are emerging with encouraging results as alternative method to carotid endarterectomy [3]. The endovascular treatment of carotid artery stenosis with CAS has an acceptable periprocedural complication rate and stroke/death rate at one year especially with the use of cerebral protection devices [4–6]. On the basis of randomized [3] and non randomized [7] multicenter trials results, in high surgical risk patients, protected CAS is actually considered equal to surgery in the hands of experienced operators [8]. The efficacy of CAS over time is under clinical evaluation. Data regarding efficacy of CAS over a longer period of time (until five years) are recently emerging [9]. In a recent meta-analysis [10] the early restenosis rates after CAS compare well with those reported for CEA, nevertheless due to the short followup period of many published works the long term durability of CAS needs of further studies. Moreover few information are available about independent clinical, radiological or procedural predictors of restenosis after CAS in patients with long followup.

ll with those reported for CEA, nevertheless due to the short followup period of many published works the long term durability of CAS needs of further studies. Moreover few information are available about independent clinical, radiological or procedural predictors of restenosis after CAS in patients with long followup. The aim of our study was to assess the incidence of long term in-stent restenosis after CAS and to identify some clinical, radiological or procedural predictors of restenosis. 2. Materials and Methods 2.1. Patients Selection and Data Collection We conduct a retrospective study including patients that underwent CAS in our centre from 1997 to 2004. Out of 400 procedures we select from our data base 189 patients that underwent to a complete clinical and CD-US followup exclusively in our centre avoiding to include patients that were followed from other hospitals and operators. Previously, we have obtained for this study the approval from our Istitutional Review Board. Indications to CAS were ICA stenosis higher than 70% in symptomatic patients and asymptomatic patients with one of the following co-morbidities: two or more coronary vessels with >70% stenosis, an ejection fraction <30%, bronchopulmonary obstructive disease, recurrent stenosis after CEA, previous radical neck surgery or radiation therapy, surgically inaccessible lesions and controlateral carotid artery occlusion. Diagnosis of ICA stenosis was made by CD-US and by MRA or CTA as a confirmatory test and definitively stated with DSA.

<30%, bronchopulmonary obstructive disease, recurrent stenosis after CEA, previous radical neck surgery or radiation therapy, surgically inaccessible lesions and controlateral carotid artery occlusion. Diagnosis of ICA stenosis was made by CD-US and by MRA or CTA as a confirmatory test and definitively stated with DSA. One hundred forty eight patients were males and 41 females with a mean age of 72.31 ± 8.17 (range 37–87 y). 200 vessels were treated with CAS (in 11 patients (5.8%) a bilateral treatment was performed). In 169 vessels the carotid stenosis was atherosclerotic (84.5%), in 23 it was postsurgical (restenosis after endarterectomy) (11.5%) and in 8 post radiotherapy (4%). Ninety eight vessels (49%) did not cause neurological ipsilateral symptoms while 102 (51%) did it. Clinical, radiological and procedural data supposed to interfere with in-stent restenosis were collected. Clinical variables included age, sex, symptomatic status of patient, and presence of supposed risk factors and comorbidity (smoke, chronic pulmonary diseases, hypertension, diabetes, hypercholesterolemia, coronary arterial disease, neoplasms). Radiological variables were obtained with the evaluation of pre and post-procedural diagnostic DSA. They included the grade of stenosis, presence of ulcerated plaques, nature of stenosis (atherosclerotic versus not atherosclerotic), percentage of residual stenosis after CAS. Procedural variables included postdilatation, type of stent, number of deployed stents.

he evaluation of pre and post-procedural diagnostic DSA. They included the grade of stenosis, presence of ulcerated plaques, nature of stenosis (atherosclerotic versus not atherosclerotic), percentage of residual stenosis after CAS. Procedural variables included postdilatation, type of stent, number of deployed stents. 2.2. CAS Procedure All procedures were performed with local anesthesia and percutaneous transfemoral access F8. Patients were premedicated with aspirin (100 mg/die) and ticlopidine (500 mg die) at least three days preintervention.

he evaluation of pre and post-procedural diagnostic DSA. They included the grade of stenosis, presence of ulcerated plaques, nature of stenosis (atherosclerotic versus not atherosclerotic), percentage of residual stenosis after CAS. Procedural variables included postdilatation, type of stent, number of deployed stents. 2.2. CAS Procedure All procedures were performed with local anesthesia and percutaneous transfemoral access F8. Patients were premedicated with aspirin (100 mg/die) and ticlopidine (500 mg die) at least three days preintervention. The patients received intra-arterial administration of 70 IU/Kg of heparin to achieve an activated clotting time (ACT) longer than 200–250 sec. By using a 100-cm long guiding catheter, the filter guidewire was introduced crossing the stenosis and the cerebral protection device, a self-expanding basket type filter, was deployed in the cervical portion of internal carotid artery. Out of 200 procedures, 69 (34.5%) were performed with a cerebral protection device (Filter Wire EZ Boston Scientific). A self-expandable stent was mounted on the protection device guidewire and placed and deployed across the stenosis. In our study group the type of stent in the 200 procedure was: 132 (66%) Carotid WallStent Monorail (Boston Scientific), 39 (19.5%) Precise Stent (Cordis Corp., Johnson & Johnson Company), 17 (8.5%) Easy WallStent (Boston Scientific), 7 (3.5%) Smart Stent (Cordis Corp., Johnson & Johnson Company), 3 (1.5%) Wallstent (Boston Scientific Corp.), 1 (0.5%) Acculink (Guidant Corp), 1 (0.5%) Omnilink (Guidant Corp). The stent was dilated to reach an adequate vessel recanalization by using an appropriate size angioplasty balloon. One mg of atropine sulphate was intravenously administered during angioplasty balloon insufflations to prevent carotid sinus stimulation and bradycardia. Then the cerebral protection device, when used, was removed. Predilation with a 3 mm PTA balloon catheter (generally Ultrasoft 5 V Boston Scientific Natick MA USA) was performed in 34 patients before stent placement in tight stenoses with a residual lumen smaller than the diameter of the stent delivery catheter.

ardia. Then the cerebral protection device, when used, was removed. Predilation with a 3 mm PTA balloon catheter (generally Ultrasoft 5 V Boston Scientific Natick MA USA) was performed in 34 patients before stent placement in tight stenoses with a residual lumen smaller than the diameter of the stent delivery catheter. In 19 patients the misplacement of the first stent necessitates of the deployment of a further stent. A permanent daily medication with acetylsalicylic acid (100 mg) and a prevention therapy with 500 mg/die of ticlopidine was started for one month, after endovascular recanalization. Before the interventional procedure, all patients were submitted to diagnostic DSA by selective injection of both common carotid arteries and vertebral arteries of at least one side. Intracranial vessels and carotid bifurcation evaluation of the treated side was repeated after endovascular procedure. In case of unprotected CAS, the procedure differed exclusively for use of guidewire instead of filter guidewire to encompass the stenosis. Patients remained in a monitored setting overnight and discharged in the following day.

Before the interventional procedure, all patients were submitted to diagnostic DSA by selective injection of both common carotid arteries and vertebral arteries of at least one side. Intracranial vessels and carotid bifurcation evaluation of the treated side was repeated after endovascular procedure. In case of unprotected CAS, the procedure differed exclusively for use of guidewire instead of filter guidewire to encompass the stenosis. Patients remained in a monitored setting overnight and discharged in the following day. 2.3. CD-US Follow-Up All patients were followed with CD-US examination at 24 hours, 1, 3, 6 months after the procedure and every 6 months thereafter. In each diagnostic session a clinical interview was made. The restenosis detection was based on CD-US by using modified velocity criteria of Washington University [11] developed in our centre and validated with digital subtraction angiography. Moreover B-mode imaging of the arterial lumen and spectral waveform analysis were used to assess possible restenosis occurrence. Elevations in peak systolic velocity or ICA/CCA ratio with respect to the first post procedural CD-US examination were considered as suggestive of progressive in-stent restenosis. A peak systolic velocity higher than 220 cm/sec was interpreted as a stenosis higher than 50%. A in stent stenosis higher than 50% was considered a restenosis. 2.4. Statistical Analysis Continuous data are presented as mean value ± SD, and categorical data are presented as frequencies.

2.3. CD-US Follow-Up All patients were followed with CD-US examination at 24 hours, 1, 3, 6 months after the procedure and every 6 months thereafter. In each diagnostic session a clinical interview was made. The restenosis detection was based on CD-US by using modified velocity criteria of Washington University [11] developed in our centre and validated with digital subtraction angiography. Moreover B-mode imaging of the arterial lumen and spectral waveform analysis were used to assess possible restenosis occurrence. Elevations in peak systolic velocity or ICA/CCA ratio with respect to the first post procedural CD-US examination were considered as suggestive of progressive in-stent restenosis. A peak systolic velocity higher than 220 cm/sec was interpreted as a stenosis higher than 50%. A in stent stenosis higher than 50% was considered a restenosis. 2.4. Statistical Analysis Continuous data are presented as mean value ± SD, and categorical data are presented as frequencies. Overall survival curves (Kaplan-Meier) were obtained using as end point, respectively, death, occurrence of major related event (death and stroke) and occurrence of restenosis. Patients dead for nonrelated causes were considered as lost to the observation. Clinical, radiological and procedural variables were used as group variables in an univariate survival analysis (Kaplan-Meier, log-rank Mantel Cox test for significance of difference). This analysis was performed by patient (n = 189) relative to the clinical variables and by vessel (n = 200) relative to the radiological and procedural variables.

procedural variables were used as group variables in an univariate survival analysis (Kaplan-Meier, log-rank Mantel Cox test for significance of difference). This analysis was performed by patient (n = 189) relative to the clinical variables and by vessel (n = 200) relative to the radiological and procedural variables. Finally a multivariate survival analysis was performed by using the proportional hazard stepwise Cox model (P to enter = .15). This analysis was performed by patient and excluding the single patient bilaterally treated with a single vessel restenosis (n = 188). Statistical analysis was performed with Stat View 5.02 software package (Abacus Concepts). 3. Results All patients were observed for a mean of 29.9 months (range 0–99) median 26 months. The overall periprocedural complications were 5 (2.6%): 2 fatal strokes (1.05%), 1 major stroke (0.5%) and 2 minor strokes (1.05%). The overall survival rate was 98% at 1 year, 94% at 3, and 92% at 5 years. The freedom from stroke and death defined as the freedom from all ipsilateral strokes and related deaths, was 95.1% at one year, 91.4% at 3 years and 89.1% at five years from treatment (Figure 1). During the followup we observed 5 periprocedural complications and 9 further strokes (4.8%), all of them were homolateral to the treated vessel. Out of 9 patients with stroke, 7 deceased (3.7%).

ateral strokes and related deaths, was 95.1% at one year, 91.4% at 3 years and 89.1% at five years from treatment (Figure 1). During the followup we observed 5 periprocedural complications and 9 further strokes (4.8%), all of them were homolateral to the treated vessel. Out of 9 patients with stroke, 7 deceased (3.7%). Twenty-three patients for a total of 23 treated vessels (23/200, 11.5%) developed a in-stent restenosis. Six restenosis occurred after stenting of 31 not-atherosclerotic plaques while 17 restenosis followed the stenting of 169 atherosclerotic stenosis. Restenosis occurred proximally to the stent implantation (common carotid artery) in four vessels, distally to the stent implantation (internal carotid artery) in one, and in the middle segment of the stent in 18 vessels. The grading of the restenosis was moderate (50%–79%) in 18 vessels (18/200, 9%), and severe (>80%) in 5 vessels (5/200, 2.5%). Only three patients (3/23, 12%) had a neurological event homolateral to the treated and restenosed vessel. The remaining patients with restenosis were asymptomatic. The cumulative rate of freedom from restenosis was, respectively, of 87%, 82.5% and 82.5%, respectively, at 1, 3 and 5 years (Figure 2).

els (5/200, 2.5%). Only three patients (3/23, 12%) had a neurological event homolateral to the treated and restenosed vessel. The remaining patients with restenosis were asymptomatic. The cumulative rate of freedom from restenosis was, respectively, of 87%, 82.5% and 82.5%, respectively, at 1, 3 and 5 years (Figure 2). Out of 23 restenosis, 10 were retreated with angioplasty alone (7 restenosis) or combined angioplasty and stenting (3 restenosis). In one patient with restenosis the intention to treat failed for the highly deformed stent that did not permit the crossing of the stenosis with the angioplasty balloon catheter. In three cases the treatment of restenosis was performed with a cerebral protection device placement. No periprocedural complications occurred in all retreated patients. The cumulative rate of survival did not differ from patients with restenosis with respect to patients without it (P = .48). The comparison of the Kaplan-Meyer curves in patients with and without restenosis does not reveal a significant difference (P = .37) in the free neurological event rate (Figure 3) that was 95%, 80% and 80% in the first group with respect to 95%, 93% and 90% in the latter, respectively at 1, 3 and 5 years. Out of all the clinical radiological and procedural variables, the univariate analysis revealed that residual stenosis after stenting and the number of deployed stents for lesion are the only variables that correlate with the restenosis occurrence (P = .0007 and P = .04, resp.).

The cumulative rate of survival did not differ from patients with restenosis with respect to patients without it (P = .48). The comparison of the Kaplan-Meyer curves in patients with and without restenosis does not reveal a significant difference (P = .37) in the free neurological event rate (Figure 3) that was 95%, 80% and 80% in the first group with respect to 95%, 93% and 90% in the latter, respectively at 1, 3 and 5 years. Out of all the clinical radiological and procedural variables, the univariate analysis revealed that residual stenosis after stenting and the number of deployed stents for lesion are the only variables that correlate with the restenosis occurrence (P = .0007 and P = .04, resp.). Multivariate analysis showed that the residual stenosis after stenting is a positive predictor of restenosis with relative hazard 1.091 per percent unit of residual stenosis, (CI 95% 1.050–1.130) P < .0001. Another significant predictors was double stent deployment with relative hazard 5.2, (CI 95% 1.49–18.5) P = .0084. Suggestive but not significant variables, included in the stepwise model, were diabetes with relative hazard 2.30, P value = .070 and neoplasms with relative hazard 2.53, P value = .085. 4. Discussion The overall periprocedural complications in our series are comparable to the literature results [4] and within the range of the acceptable perioperative risks after CEA [12].

Another significant predictors was double stent deployment with relative hazard 5.2, (CI 95% 1.49–18.5) P = .0084. Suggestive but not significant variables, included in the stepwise model, were diabetes with relative hazard 2.30, P value = .070 and neoplasms with relative hazard 2.53, P value = .085. 4. Discussion The overall periprocedural complications in our series are comparable to the literature results [4] and within the range of the acceptable perioperative risks after CEA [12]. The stroke or death rate (4.9% at one year, 8.6% at 3 years and 10.9% at five years from treatment) is in line with former publications that report 4%–7% at one year [9, 13, 14], 10.1%–11% at 3 years [9, 15] and 15.1% at 5 years [9] indicating encouraging prospective for CAS also over a long period of time.

4. Discussion The overall periprocedural complications in our series are comparable to the literature results [4] and within the range of the acceptable perioperative risks after CEA [12]. The stroke or death rate (4.9% at one year, 8.6% at 3 years and 10.9% at five years from treatment) is in line with former publications that report 4%–7% at one year [9, 13, 14], 10.1%–11% at 3 years [9, 15] and 15.1% at 5 years [9] indicating encouraging prospective for CAS also over a long period of time. The overall incidence of restenosis >50% is 11.5% and it is comparable to the value reported for CEA (10%) at one year [16] and lower to 20% at one year of angioplasty alone [17] confirming the competitiveness of CAS and CEA and the improvement of angioplasty durability after a stent deployment. Our cumulative restenosis rate at one and two years is 13 and 13.8%, respectively, slightly higher to the values derived from a recent meta-analysis (6 and 7.5% at one and two years) [10] but within the range reported in the literature varying from 0.6% [18] to 20.8% [19]. If we consider a significant restenosis as higher to 80%, we obtain an overall incidence of restenosis of 2.5% that is lower to the 4% reported in the aforementioned meta-analysis study [10]. Lower restenosis rate are reported in the single long-term multi-centre study of ELOCA registry [9] (1%, 2% and 3.4%, resp. to 1, 3 and 5 years). The variable incidence of restenosis in the different centres means that restenosis is not however negligible and it induce us to monitor the patients submitted to CAS over time.

r restenosis rate are reported in the single long-term multi-centre study of ELOCA registry [9] (1%, 2% and 3.4%, resp. to 1, 3 and 5 years). The variable incidence of restenosis in the different centres means that restenosis is not however negligible and it induce us to monitor the patients submitted to CAS over time. We outline that the Kaplan-Meyer analysis for survived and free from neurological events does not differ between patients with and without restenosis confirming the benign course of in-stent restenosis [20] as occur for restenosis after CEA [21]. Although there are few and often asymptomatic significant (>80%) in-stent restenosis, probably they will increase in the next time and may became, in future, a clinical problem due to the widespread application of CAS [22]. Concerning to the treatment of in-stent restenosis, angioplasty has been recently recommended as the primary approach to hyperplastic lesions with repeat stenting in cases of suboptimal results [23]. Although further experiences in carotid artery restenosis after stent are necessary to formulate standardized approaches, in our limited experience, the endovascular treatment of restenosis seems to be safety and when it is not feasible, surgical treatment can be considered [24].

ng in cases of suboptimal results [23]. Although further experiences in carotid artery restenosis after stent are necessary to formulate standardized approaches, in our limited experience, the endovascular treatment of restenosis seems to be safety and when it is not feasible, surgical treatment can be considered [24]. According to previous studies, restenosis occurs preferentially in the first year from the endovascular procedure (20 out of 23 in the first 12 months) and reduces its prevalence in the subsequent years. It is generally accepted that the major cause of restenosis and of its benign course is neointimal hyperplasia with smooth muscle cells proliferation [25] that prevailed in the first 12 months after CEA [26].

ndovascular procedure (20 out of 23 in the first 12 months) and reduces its prevalence in the subsequent years. It is generally accepted that the major cause of restenosis and of its benign course is neointimal hyperplasia with smooth muscle cells proliferation [25] that prevailed in the first 12 months after CEA [26]. Specific risk factors in the development of restenosis after CAS remains to be elucidated: some studies have identified advanced age [27, 28], hyperglycemia [27, 29], smoke [27] and previous CEA [30]. In our study group, out of all the evaluated clinical radiological and procedural variables, multivariate analysis reveal that the residual stenosis after CAS is a stronger predictor of restenosis with a consistent increased risk of restenosis (1.091 per % unit of residual stenosis). Suboptimal technical results after CAS was previously described as clinical predictor of restenosis in the first six months of followup [31] and a small post procedural stent dimension evaluated with intravascular ultrasound imaging has been demonstrated to be associated with a higher risk of restenosis also in carotid artery stenting [32]. Although larger studies with long term followup do not reveal [33] this radiological variable as predictor, recently suboptimal result with residual stenosis has been reported as a restenosis predictor [34] Carotid neointimal proliferation and stent auto-expansion have counteracting effects: the first one predominates during the first year after stenting, whereas the latter in the subsequent year [35]. We may suppose that in some cases, unexpanded carotid stent could imbalance these complex counteracting effects in favour of neointimal proliferation inducing post procedural stenosis and a following long term restenosis.

predominates during the first year after stenting, whereas the latter in the subsequent year [35]. We may suppose that in some cases, unexpanded carotid stent could imbalance these complex counteracting effects in favour of neointimal proliferation inducing post procedural stenosis and a following long term restenosis. Our results suggest that post procedural stenosis has a higher risk of restenosis and it conflicts with the opinion that to pursue a perfect angiographic result after CAS is not necessary [36]. Nevertheless, small post procedural lumen dimension as a restenosis predictor have to be considered with caution and have not to induce an opposite aggressive behaviour because it is known that high post dilation pressures increase the risk of embolization and neointimal proliferation [37]. To our knowledge the implantation of multiple stents as a restenosis predictor after CAS has been mentioned in a brief report [28]. In our experience, similarly to the results of the coronary stenting, by both univariate [38] and multiple logistic regression analysis [39] the number of stents per lesion is a significant variable and independent predictor of restenosis. Probably the higher risk of in-stent restenosis may be due to a larger surface area covered with stent material or to the overlapped edges of the stent inducing a more enunciated intimal hyperplasia.

ression analysis [39] the number of stents per lesion is a significant variable and independent predictor of restenosis. Probably the higher risk of in-stent restenosis may be due to a larger surface area covered with stent material or to the overlapped edges of the stent inducing a more enunciated intimal hyperplasia. Our study identify other clinical variables with a less relevant influence on restenosis such as diabetes and neoplasms. The role of diabetes has been described as a predictor of carotid [27, 28] and coronary in-stent restenosis [40] and may be due to smooth muscle cell proliferation common in diabetic patients [41]. The role of neoplasms in the restenosis occurrence is prone to speculative interpretations, in fact a series of cell proliferation regulatory pathways have been associated with plaque progression, stenosis and restenosis after angioplasty as well as in cancer progression [42]. The main limitations of the study are the small number of patients and events sourcing by a single centre study. However the detection of independent predictors after CAS that evoke the predictors after coronary stenting [43], may be useful to be largely studied in prospective randomized trials.

Our study identify other clinical variables with a less relevant influence on restenosis such as diabetes and neoplasms. The role of diabetes has been described as a predictor of carotid [27, 28] and coronary in-stent restenosis [40] and may be due to smooth muscle cell proliferation common in diabetic patients [41]. The role of neoplasms in the restenosis occurrence is prone to speculative interpretations, in fact a series of cell proliferation regulatory pathways have been associated with plaque progression, stenosis and restenosis after angioplasty as well as in cancer progression [42]. The main limitations of the study are the small number of patients and events sourcing by a single centre study. However the detection of independent predictors after CAS that evoke the predictors after coronary stenting [43], may be useful to be largely studied in prospective randomized trials. In our CAS experience encouraging long term results seem to derive from both neurological event free rate and restenosis incidence. Because of residual stenosis after CAS is a strongest independent predictor of restenosis, adequate recanalization of the treated vessel seems an important goal to limit the development of restenosis. Multiple stents deployment and with less evidence, diabetes or neoplasm have to be considered to facilitate in-stent restenosis after CAS. Figure 1 Kaplan Meyer analysis curve of the cumulative freedom from stroke and death. Figure 2 Kaplan Meyer analysis curve of the cumulative freedom from restenosis.

ssure during the acute phase of symptomatic carotid occlusion to amplify the blood volume shift towards the craniothoracic territory [3, 4], improving cerebral haemodynamic conditions and neurological symptoms. The impact of anti-gravity suit application on cerebral vascularisation was measured by transcranial Doppler. 2. Case  1 A 28-year-old woman was seen 2 hours after the sudden onset of total right brachial monoplegia, right facial palsy, and mutism (NIH Stroke Scale: 17) due to a left middle cerebral artery (MCA) infarct. She received intravenous recombinant tissue plasminogen activator (rt-PA) 2.5 hoursafter stroke onset, without subsequent improvement. Cerebral MRI diffusion-weighted images and T2 flair-weighted images performed 24 hours later showed increased signal in the whole superficial left MCA territory (Figures 2(a) and 3(a)). The MR Angiography (MRA) showed a proximal left internal carotid artery (ICA) occlusion, a tight stenosis on the distal right ICA, no signal in the right siphon and right MCA, while there was a weak signal in the left MCA (Figure 4). T1-FAT-SAT-weighted images showed bilateral ICA dissection (Figure 5). Doppler study showed a high resistance to flow in both common carotid arteries and low bilateral MCA flow with low systolic and diastolic velocities. Intravenous heparin treatment was started 24 hours after rt-PA administration and the patient was lying in a strict −20 degrees head down tilt [5].

dissection (Figure 5). Doppler study showed a high resistance to flow in both common carotid arteries and low bilateral MCA flow with low systolic and diastolic velocities. Intravenous heparin treatment was started 24 hours after rt-PA administration and the patient was lying in a strict −20 degrees head down tilt [5]. Two days after stroke onset, there was no clinical improvement and the patient developed bradycardia (40 pulses/min) and hypotension (100/70 mmHg). Lower body positive pressure (LBPP) was applied using an anti-gravity suit (Trauma Air Pants; Life Support Products Inc., St Louis, Missouri, USA) for 150 minutes. The anti-gravity suit included three independent bladders connected to three pneumatic extremities with manometers providing instantaneous pressure values. The garment was applied from ankle to costal margin and the bladder pressures were set at 20 mmHg on the lower limbs and 10 mmHg on the abdominal area, to keep a positive pressure gradient [3, 4]. During the procedure the patient was lying in a supine position. The following parameters were recorded at baseline, after intravenous infusion of 500 mL of fluid load (colloid), 15 minutes and 150 minutes during LBPP application and 10 minutes after gravity suit deflation: right arm mobility, blood pressure, heart rate, systolic and diastolic velocities recorded on both MCAs and resistance index (RI) recorded on both common carotids arteries (CCAs).

infusion of 500 mL of fluid load (colloid), 15 minutes and 150 minutes during LBPP application and 10 minutes after gravity suit deflation: right arm mobility, blood pressure, heart rate, systolic and diastolic velocities recorded on both MCAs and resistance index (RI) recorded on both common carotids arteries (CCAs). During LBPP application and up to 48 hours later, there was a 50% increase in systolic and diastolic MCA velocities (Figure 1). The RI decreased on both CCAs from 0.93 to 0.86 and continued to decrease on the right side after the procedure. Heart rate and blood pressure remained stable during LBPP application and during the proceeding days. Three days after LBPP application the patient started to recover her right hand mobility and her facial palsy started to improve. One week after LBPP application she had completely recovered her right arm and facial mobility. She finally went back home 3 weeks after hospitalisation. On MRI performed 5 days after stroke onset, diffusion-weighted images showed reduced diffusion abnormalities and reduced infarct size on T2 flair-weighted images (Figures 2(b) and 3(b)). The MRA showed recanalisation of the right ICA with good visualisation of siphon and MCA but a persistent left ICA occlusion (Figure 6).

On MRI performed 5 days after stroke onset, diffusion-weighted images showed reduced diffusion abnormalities and reduced infarct size on T2 flair-weighted images (Figures 2(b) and 3(b)). The MRA showed recanalisation of the right ICA with good visualisation of siphon and MCA but a persistent left ICA occlusion (Figure 6). 3. Case  2 A 61-year-old. man was admitted in our stroke unit after 2 reversible episodes of left monocular blindness associated with a total right distal upper limb paralysis. His medical past history was hypertension, hypercholesterolemia, and tobacco use. At the admission the clinical examination showed a total right wrist extension paresis, the so-called “pseudoradial paresis” related to a small left frontal infarction visible on MRI (DWI). The MR angiography (MRA) and the duplex scanning showed an occlusion of the left internal carotid artery due to atherosclerosis and a negative flow in the ophthalmic artery downstream. The intracranial MRA showed poor collateral supply by anterior communicating artery and an apparent nonfunctional posterior communicating artery. We decided to apply LBPP process for 2 reasons: (1) the worsening of the right hand deficit (see movie), (2) this worsening was not related to a fall in systemic blood pressure (167/84 mmHg). The LBPP was applied once during 90 minutes following the same procedure than in case one. The repeated measurements of blood pressure and heart rate did not show any significant changes along the procedure. The transcranial Doppler showed a decreased of the left MCA resistance to flow index ([systolic velocity-diastolic velocity]/systolic velocity, 0.5 at baseline to 0.4 at the end of the procedure) while there was slight changes on the MCA velocities (64/32 to 75/45 cm/sec). The left ophthalmic artery flow remained negative during and after LBPP application. Heart rate and blood pressure remained stable during LBPP application. The clinical improvement was sustained after anti-G suit deflation (as presented on the movie) consisting in the recovery of the pseudoradial palsy with persisting ataxia lasting few days. The total recovery was obtained before discharge.

plication. Heart rate and blood pressure remained stable during LBPP application. The clinical improvement was sustained after anti-G suit deflation (as presented on the movie) consisting in the recovery of the pseudoradial palsy with persisting ataxia lasting few days. The total recovery was obtained before discharge. 4. Discussion We report two patients with symptomatic carotid artery occlusion in whom LBPP was applied with an anti-gravity suit during the acute phase of stroke. The patients made a remarkable recovery in parallel with significant cerebral hemodynamic improvement not related to global systemic hemodynamic changes. LBPP is known to shift blood from the lower body compartment towards the upper part of the body, which improves cardiac preload and output [4, 6, 7] and potentially cerebral perfusion [8]. The addition of an abdominal compression to lower limbs was supposed to be more efficient to push more blood to the brain than compression of lower limb only [6]. Several mechanisms might be discussed for neurological improvement: (1) recanalization of carotid occlusion, (2) development of collaterals, and (3) recruitment of smaller vessels in watershed fields. For the first patient, it is unlikely that rt-PA administration played a major role in this dramatic improvement since there was no improvement 24 hours after treatment, which decreases the likelihood of a favourable outcome to 20% [9, 10], and the efficacy of rt-PA in carotid occlusions is debatable [11, 12].

fields. For the first patient, it is unlikely that rt-PA administration played a major role in this dramatic improvement since there was no improvement 24 hours after treatment, which decreases the likelihood of a favourable outcome to 20% [9, 10], and the efficacy of rt-PA in carotid occlusions is debatable [11, 12]. The reopening of the carotid artery, which was unusually rapid for the spontaneous evolution of a dissection [13], may have played an important role in case 1 evolution. Whether LBPP application contributed to this reopening, though likely with RI decrease on right CCA, is open to debate and we cannot rule out coincidental recanalization of the ICA occlusion. In case 1, the parallel improvement in the clinical status and flow parameters on transcranial Doppler (TCD) during and after LBPP application suggests a beneficial effect of LBPP application on brain perfusion, particularly through restitution of blood flow in the ischemic left MCA territory. It could be due to the recruitment of collateral pathway through Willis circle explaining the increase of systolic and diastolic velocities recorded on left MCA during LBPP application (case 1, Figure 1), as well as the improvement of anterior cerebral circulation shown by the MRA performed 5 days after stroke onset (right siphon and right MCA were visible and the posterior communicating arteries were functional on Figure 6). The second case reported which showed a clinical improvement (film) during the LBPP application showed a weak improvement in cerebral velocities. The temporal relationship between clinical recovery and LBPP application suggests a benefit.

re visible and the posterior communicating arteries were functional on Figure 6). The second case reported which showed a clinical improvement (film) during the LBPP application showed a weak improvement in cerebral velocities. The temporal relationship between clinical recovery and LBPP application suggests a benefit. Another possible beneficial effect could be the recruitment of smaller vessels in watershed fields especially in case 2. The clinical improvement during the LBPP application could be related to the new partition in the frontal region of blood which could play a role in the recruitment of small vessels by an efficient loading or even a reopening. Two recent reports showed the impact of a mechanical external counter-pulsation on cerebral velocities in healthy volunteers [8] and on functional outcome in patients with stroke [14]. In this study the LBPP application was limited to the lower limbs by the mean of a cyclic external counter-pulsation at diastolic time of heart cycle which is more complex than our classical anti-gravity suit. Furthermore, we can expect that the anti-gravity suit could mobilised a higher blood volume in respect of the abdominal compartment implication.

was limited to the lower limbs by the mean of a cyclic external counter-pulsation at diastolic time of heart cycle which is more complex than our classical anti-gravity suit. Furthermore, we can expect that the anti-gravity suit could mobilised a higher blood volume in respect of the abdominal compartment implication. Even if this paper is limited to two patients, without control group, the remarkable recovery observed in these two patients together with the improvement in hemodynamic parameters on TCD might be related to LBPP application through improvement of cerebral perfusion. We need definitively, further investigation during the acute phase of ischemic stroke to conclude on the beneficial effect of this noninvasive tool. Acknowledgment This work was supported in part by the Plan quadriennal EA 322 2002-2006. Figure 1 (a) Patterns of intracranial MCA blood flow velocities with time in both the right and left sides, before, during, and after (10 minutes and 48 hours) LBPP application. Note the left side improvement in systolic and diastolic blood flow velocity induced by LBPP, which was sustained for at least 2 days. (b) Vascular resistance index (RI) evolution (method for computation: systolic V/diastolic V). A clear difference between right and left RI occurred only after 48 hours of LBPP application. Corresponding mean arterial pressure (mean AP) and heart rate (HR) at each time. Figure 2 MRI diffusion-weighted images showing left superficial MCA infarct. (a) 24 hours after stroke onset, (b) 5 days after stroke onset.

Figure 1 (a) Patterns of intracranial MCA blood flow velocities with time in both the right and left sides, before, during, and after (10 minutes and 48 hours) LBPP application. Note the left side improvement in systolic and diastolic blood flow velocity induced by LBPP, which was sustained for at least 2 days. (b) Vascular resistance index (RI) evolution (method for computation: systolic V/diastolic V). A clear difference between right and left RI occurred only after 48 hours of LBPP application. Corresponding mean arterial pressure (mean AP) and heart rate (HR) at each time. Figure 2 MRI diffusion-weighted images showing left superficial MCA infarct. (a) 24 hours after stroke onset, (b) 5 days after stroke onset. Figure 3 MRI T2 flair-weighted images showing left superficial MCA infarct. (a) 24 hours after stroke onset, (b) 5 days after stroke onset. Figure 4 (a) Intracranial MRA of Willis circle: right siphon, right and left MCAs were not visible. Posterior communicating arteries were also not visible, with visible posterior cerebral arteries. (b) Cervical MRA showing proximal left internal carotid artery occlusion and distal right internal carotid artery tight stenosis (arrows). Figure 5 MRI T1-FAT-SAT-weighted images showing the dissecting process visible as a hypersignal in the wall of both ICAs as well as an enhancement of ICA diameter (arrows). Figure 6 Intracranial MRA showing recanalisation of the right ICA; the right and left MCAs are visible as well as both posterior communicating arteries (arrows).

1. Introduction Obstructive sleep apnea (OSA) is the most common form of sleep disordered breathing in patients with acute ischemic stroke and associated with early neurological deterioration, increased mortality rates, and poor functional outcomes [1, 2]. Given that the rate of neurological deterioration is the highest during the acute stroke setting [3, 4], urgent treatment with noninvasive ventilatory correction may provide a novel therapeutic target to improve outcomes [5]. 2. Case Report A 55-year-old female (weight 233 pounds, height 5 feet 7.75 inches, and body mass index 35.7) with a history of untreated OSA and arterial hypertension woke up with fluctuating right-hemispheric stroke symptoms. On admission, she had mild left-sided hemiparesis and facial palsy with dysarthria (National Institutes of Health Stroke Scale (NIHSS) Score 6). Cerebral computed tomography (CT) revealed early ischemic changes involving less than 1/3 of the right anterior middle cerebral artery (MCA) distribution and a hyperdense MCA sign (Figure 1). Transcranial Doppler (TCD) demonstrated a blunted flow of the right proximal/middle MCA (Thrombolysis in Brain Ischemia (TIBI) score 2, Figure 1) indicating an MCA occlusion. We initiated intravenous thrombolysis with tissue plasminogen activator (tPA) 200 minutes after symptom onset. After initiation of intravenous tPA infusion, the patient was enrolled in a thrombolytic research study (involving a direct thrombin inhibitor, permission of the sponsor was obtained to release this case report).

initiated intravenous thrombolysis with tissue plasminogen activator (tPA) 200 minutes after symptom onset. After initiation of intravenous tPA infusion, the patient was enrolled in a thrombolytic research study (involving a direct thrombin inhibitor, permission of the sponsor was obtained to release this case report). At the end of tPA infusion, the patient developed excessive sleepiness with repetitive episodes (each lasting approximately 30 seconds) of irregular breathing and desaturation (with oxygen saturation levels below 90% under 2 to 4 liters supplemental oxygen delivered by a nasal cannula), and her neurological symptoms worsened rapidly (in less than 1 minute) to complete left-sided hemiplegia (NIHSS score 24). Her blood pressure (BP) and heart rate ranged from 126/61 to 164/93 and 72 to 98 bpm, respectively, during the entire monitoring period, and did not drop significantly during the neurological worsening. There were no clinical signs or lab results suggestive of a systemic inflammatory response syndrome (e.g., elevated white blood cell count or CRP, abnormal body temperature). An urgent CT (within 15 minutes after neurological worsening) ruled out intracerebral hemorrhage and edema progression. TCD monitoring showed persisting right MCA occlusion (TIBI score 2) and paradoxical as well as transient velocity decreases (>10 cm/s) during hypoventilation episodes consistent with intracranial blood flow steal. Because of continuation of excessive sleepiness with apnea periods, the patient was placed on biphasic positive airway pressure (BiPAP). Within the next hour, the patient improved to an NIHSS score of 13, and TCD showed continuing improvement in flow velocities suggestive of slow and partial recanalization (Figure 1). Next day, CT demonstrated an infarction involving 1/3 of the MCA territory (Figure 1). On TCD, right MCA appeared completely recanalized (TIBI score 5, Figure 1). Further clinical workup revealed a moderate stenosis (50 to 69%) in the right internal carotid artery strongly suggesting large-artery thrombosis and artery-to-artery embolism as the likely mechanism of her stroke.

the MCA territory (Figure 1). On TCD, right MCA appeared completely recanalized (TIBI score 5, Figure 1). Further clinical workup revealed a moderate stenosis (50 to 69%) in the right internal carotid artery strongly suggesting large-artery thrombosis and artery-to-artery embolism as the likely mechanism of her stroke. BiPAP was transitioned to night-time ventilation only, and her neurological status improved to an NIHSS of 6 at discharge. One month later, she had a residual minor left-sided hemiparesis and was functionally independent (NIHSS 3, modified Rankin Scale 1). An overnight sleep study in our sleep-wake disorder center four months later demonstrated a significant OSA (apnea-hypopnea index > 10/h), which improved on continuous positive airway pressure.

th later, she had a residual minor left-sided hemiparesis and was functionally independent (NIHSS 3, modified Rankin Scale 1). An overnight sleep study in our sleep-wake disorder center four months later demonstrated a significant OSA (apnea-hypopnea index > 10/h), which improved on continuous positive airway pressure. 3. Discussion In this paper, an early neurological deterioration was noticed that was not related to proximal vessel patency changes but coincidentally with the development of drowsiness and sleep apnea. Noninvasive ventilatory correction was applied in the hyperacute phase of ischemic stroke, and this could have improved cerebral hemodynamics distal to the occlusion. The latter is likely to have occurred as evident from her fast neurological improvement on BiPAP without complete vessel recanalization. Although tPA infusion was bridged with an experimental protocol that involves a direct thrombin inhibitor, the specific agent here was not at question. Our case highlights possible significance of ventilatory management in acute stroke, and the magnitude at which ventilatory compromise can affect both routine treatment and any hyperacute clinical trial.

ith an experimental protocol that involves a direct thrombin inhibitor, the specific agent here was not at question. Our case highlights possible significance of ventilatory management in acute stroke, and the magnitude at which ventilatory compromise can affect both routine treatment and any hyperacute clinical trial. OSA is frequently present in patients with acute ischemic stroke and associated with a higher likelihood of early clinical deterioration [1, 2]. However, the underlying pathophysiologic mechanism for irregular breathing and early neurological deterioration in acute ischemic stroke patients is poorly understood. Under ischemic conditions, cerebral vasomotor reserve may become exhausted with occurrence of an intracranial blood flow steal, and affected vessels are less responsive to vasodilator stimuli like carbon dioxide leading to an intracranial blood flow steal from the affected to the nonaffected side [6–8]. This intracranial hemodynamic steal phenomenon has been documented on continuous TCD monitoring in up to 14% of stroke patients, leading to clinical deterioration in 7% of cases (termed reversed Robin Hood syndrome) [7]. Moreover, patients with proximal arterial occlusion and excessive daytime sleepiness were more vulnerable to intracranial blood flow steal than patients without these conditions.

s TCD monitoring in up to 14% of stroke patients, leading to clinical deterioration in 7% of cases (termed reversed Robin Hood syndrome) [7]. Moreover, patients with proximal arterial occlusion and excessive daytime sleepiness were more vulnerable to intracranial blood flow steal than patients without these conditions. We cannot rule out that early neurological deterioration in our case simply reflected a natural course of proximal MCA occlusion and failure of collateral flow [9]. However, at the time of neurological worsening, our patient developed excessive sleepiness with recurrent and witnessed episodes of hypoventilation/apnea. Unfortunately, we did not measure arterial blood gases as arterial stick after intravenous tPA infusion was deemed risky. Nevertheless, early clinical recovery after application of BiPAP may represent a clinical surrogate of arterial carbon dioxide correction and increased blood flow in vessels supplying ischemic tissue. We considered that early clinical recovery might also have reflected a partial recanalization after intravenous thrombolysis. However, at the time of significant clinical improvement, MCA was still occluded. In conclusion, noninvasive ventilatory correction should be considered more aggressively as a complementary treatment option in selected acute stroke patients. Further research is needed to determine whether early initiation of this treatment may improve functional outcomes and unmask the true potential of other therapies.

onclusion, noninvasive ventilatory correction should be considered more aggressively as a complementary treatment option in selected acute stroke patients. Further research is needed to determine whether early initiation of this treatment may improve functional outcomes and unmask the true potential of other therapies. Acknowledgment Dr. A. V. Alexandrov and University of Alabama Hospital, Comprehensive Stroke Center are funded by the National Institute of Neurological Disorders and Stroke (NINDS) University of Texas-Houston Medical School to conduct studies on experimental combinatory therapies (SPOTRIAS Program). The other coauthors have no disclosures. Figure 1 (a) Baseline noncontrast CT: a long thromboembolus within the right MCA trunk (arrow). (b) Baseline TCD: flattened systolic flow acceleration (TIBI 2) at a depth of 48 mm, indicating right proximal MCA occlusion. (c) One-hour TCD: improved flow velocities/waveforms (TIBI 2) at a depth of 58 mm suggestive of slow and partial recanalization. (d) Followup CT reveals a small cortical infarction (arrows). (e) 24-hour TCD: normal flow velocities and waveforms (TIBI 5) at a depth of 46 mm.

1. Introduction Cerebral venous thrombosis (CVT) is a well-recognized condition presenting in multiple different forms. It was first described in 1825 [1] in a man who suffered from headaches, seizures, and delirium for six months. His autopsy revealed superior sagittal sinus thrombosis. Since that initial description nearly two centuries ago, several case reports and case series have come out, but the condition continues to be a diagnostic and therapeutic challenge. The estimated incidence is about 2 to 4/million/year [2] and about 75% of these are reportedly women [3]. A recent study in children <18 years found a higher incidence of 6.7/million/year [4]. Also CVT seems to be a bigger problem in the Asian world compared to the west. A report from India by Panagariya et al. suggests that CVT accounts for up to 40% of strokes in women and 50% of all young strokes [5, 6]. Risk factors for CVT have been highlighted in several studies and case series. The largest trial to date was the International study on cerebral vein and dural sinus thrombosis (ISCVT) [7]. This was a prospective, observational study from 21 different countries in which 624 patients with CVT were followed up for 16 months. In this study, 43.6% patients had more than one risk factors. The commonest cause was genetic or acquired thrombophilia (34.1%), followed by use of oral contraceptives or hormonal replacement therapy (58.6% of female patients) and local or systemic infection in 12.3%.

4 patients with CVT were followed up for 16 months. In this study, 43.6% patients had more than one risk factors. The commonest cause was genetic or acquired thrombophilia (34.1%), followed by use of oral contraceptives or hormonal replacement therapy (58.6% of female patients) and local or systemic infection in 12.3%. Another study carried out by Khealani et al. reported systemic and central nervous system infection as the commonest predisposing cause in patients from Pakistan and Middle East. These were followed by postpartum state, homocysteinemia, and genetic thrombophilia. The contribution of oral contraceptive pill use was much lower at 3% [8]. The etiologic factors are therefore diverse and may remain obscure in up to 40%, most of whom are believed to have a genetic predisposition [9]. Broadly speaking, the common settings for CVT are puerpurium, infectious and inflammatory disease, malignancy, and oral contraceptives use [10]. CVT presents most commonly with headaches, focal or generalized seizures, neurologic deficits, or coma [11–14]. The course of the illness in CVT varies with death and dependency rates ranging from 8 to 40% [7, 15–17]. The ISCVT has identified several predictors of poor outcome, most notable being older age ( >37 years), male gender, seizures at admission, rapid evolution of thrombosis, the presence of focal deficits, and CNS infection and cancer [7].

ess in CVT varies with death and dependency rates ranging from 8 to 40% [7, 15–17]. The ISCVT has identified several predictors of poor outcome, most notable being older age ( >37 years), male gender, seizures at admission, rapid evolution of thrombosis, the presence of focal deficits, and CNS infection and cancer [7]. Therapeutic options for CVT have been explored in several studies. At present, anticoagulation is the mainstay of treatment. However, ~40% of patients with CVT have concomitant propensity for intracranial hemorrhage and hence the apprehension on the part of the clinicians. In this paper, we intend to review the evidence for and against the available therapeutic options for CVT. 2. General Measures The most dreaded complications of CVT are intracranial hypertension and cerebral herniation. The raised intracranial pressure is secondary to obstructed venous drainage leading to cerebral edema and intracranial hemorrhage which is often accompanying this condition. A review paper on mechanisms of damage in CVT [18] highlights multiple factors such as increased pressure in the dural sinus as well as increased venous flow velocities, rate of occlusion of the sinus, and superimposed cytotoxic as well as vasogenic edema. The paper also suggests that the extent of damage depends on the rate at which occlusion occurs and collaterals are formed. Hydration is of paramount importance when patients present with signs and symptoms suggestive of CVT. Dehydration will make the condition worse by making the hypercoagulability worse.

2. General Measures The most dreaded complications of CVT are intracranial hypertension and cerebral herniation. The raised intracranial pressure is secondary to obstructed venous drainage leading to cerebral edema and intracranial hemorrhage which is often accompanying this condition. A review paper on mechanisms of damage in CVT [18] highlights multiple factors such as increased pressure in the dural sinus as well as increased venous flow velocities, rate of occlusion of the sinus, and superimposed cytotoxic as well as vasogenic edema. The paper also suggests that the extent of damage depends on the rate at which occlusion occurs and collaterals are formed. Hydration is of paramount importance when patients present with signs and symptoms suggestive of CVT. Dehydration will make the condition worse by making the hypercoagulability worse. Another important measure to prevent sudden elevations of intracranial pressure would be to give antiepileptic cover to these patients. Focal or generalized seizures are more frequent in CVT than in any other type of stroke and this includes status epilepticus. The ISCVT reported seizures in 39% of their patients. Again no consensus exists on use of antiepileptic agents in CVT, but the risk is higher for those who present initially with seizures and in those who have supratentorial brain lesions including hemorrhage and edema [19]. The risk of developing seizures after CVT diagnosis is very low in patients who do not have these risk factors. Therefore, for the high-risk group, we would recommend antiepileptic drug use. The guidelines for treatment of seizures and for status epilepticus are the same as the standard epilepsy guidelines.

edema [19]. The risk of developing seizures after CVT diagnosis is very low in patients who do not have these risk factors. Therefore, for the high-risk group, we would recommend antiepileptic drug use. The guidelines for treatment of seizures and for status epilepticus are the same as the standard epilepsy guidelines. In this acute phase, medical management to decrease intracranial pressure (ICP) is recommended. This includes head elevation, hyperventilation to a target PaCO2 of 30–35 mmHg, and use of mannitol [20]. Glucocorticoids like dexamethasone are used by many for controlling ICP. There is, however, no randomized trial to prove their efficacy. A study [21] analyzed data from the ISCVT on the use of glucocorticoids and failed to find any benefit. Therefore, routine use of these agents cannot be recommended. It is not known whether use of mannitol or hyperventilation improves outcomes, but it definitely buys time before a more definitive procedure for controlling ICP (like hematoma evacuation and decompressive craniectomy) can be undertaken. There are no randomized controlled trials to support their use, however. Isolated intracranial hypertension is also a well-recognized form of presentation in patients with CVT [22]. Given the difference in prognosis, its management also differs from simple management of intracranial hypertension, which includes measures to decrease ICP. For those with underlying venous thrombosis, in addition to these measures, anticoagulation is recommended although more aggressive measures like mechanical thrombectomy are seldom required.

gnosis, its management also differs from simple management of intracranial hypertension, which includes measures to decrease ICP. For those with underlying venous thrombosis, in addition to these measures, anticoagulation is recommended although more aggressive measures like mechanical thrombectomy are seldom required. 3. Specific Measures These would be tailored around the underlying cause of cortical venous thrombosis. The major causes include prothrombotic conditions, either genetic or acquired which are the commonest cause, oral contraceptives and other drugs that cause a hypercoagulable state, pregnancy and the puerperium, malignancy, infections including CNS infections, ear, sinus, mouth, face, and neck infections and even systemic infectious disease, head injury, and mechanical precipitants like lumbar puncture and neurosurgical procedures [7]. For genetic prothrombotic conditions, there is no specific therapy available. For acquired conditions, it is tailored accordingly. For drug-induced CVT, the drug has to be discontinued along with other measures that follow in this paper. Malignancy requires its own specific therapy besides treatment for CVT.

dures [7]. For genetic prothrombotic conditions, there is no specific therapy available. For acquired conditions, it is tailored accordingly. For drug-induced CVT, the drug has to be discontinued along with other measures that follow in this paper. Malignancy requires its own specific therapy besides treatment for CVT. Spread of infection from a contiguous site is a well-known cause of dural sinus thrombosis. Particularly important sources are mastoidits and other middle-ear infections as well as ethmoid and frontal sinusitis. For these infections, leading to septic dural sinus thrombosis, high-dose antibiotics are the mainstay of treatment. Additionally, local collections of pus at these sites may have to be drained. Anticoagulation is not well studied but has been used particularly in cavernous sinus thrombosis with good results [23, 24]. For lateral and superior sagittal sinus thrombosis, the data is even more sparse with mixed results [25, 26]. Therefore, till more evidence is available, the physician will have to balance the risk of benefit of anticoagulation with the risk of hemorrhage in septic dural sinus thrombosis.

is with good results [23, 24]. For lateral and superior sagittal sinus thrombosis, the data is even more sparse with mixed results [25, 26]. Therefore, till more evidence is available, the physician will have to balance the risk of benefit of anticoagulation with the risk of hemorrhage in septic dural sinus thrombosis. Patients with nephrotic syndrome have a much higher incidence of arterial as well as venous thrombosis compared to the general population [27]. There are several underlying mechanisms that lead to this increased risk. The general therapeutic measures for treatment of CVT in patients with nephrotic syndrome do not differ from management of the condition due to other causes. However, the underlying disease condition leading to nephrotic syndrome has to be separately addressed. Those patients who achieve remission of nephrotic syndrome may discontinue anticoagulation after six months following remission if there is no other indication for anticoagulation. 4. Antiplatelets There are only anecdotal reports of use of aspirin for cortical venous thrombosis. No randomized trial or even a case series exist on the use of antiplatelet agents. 5. Anticoagulation In 1941, Lyons [28] for the first time described the successful use of heparin in cerebral venous thrombosis. He reported two cases of infective cavernous sinus thrombosis that greatly benefited from a combination of antibiotics and heparin.

4. Antiplatelets There are only anecdotal reports of use of aspirin for cortical venous thrombosis. No randomized trial or even a case series exist on the use of antiplatelet agents. 5. Anticoagulation In 1941, Lyons [28] for the first time described the successful use of heparin in cerebral venous thrombosis. He reported two cases of infective cavernous sinus thrombosis that greatly benefited from a combination of antibiotics and heparin. In 1985, Bousser et al. [29] retrospectively reviewed 38 patients with angiographically proven cerebral venous thrombosis. Twenty three of these patients received heparin, and clinical improvement was reported in all with 19 making a complete recovery. No deaths occurred in the heparin arm, and based on these findings the authors concluded that heparin was both safe and efficacious in patients with CVT.

y proven cerebral venous thrombosis. Twenty three of these patients received heparin, and clinical improvement was reported in all with 19 making a complete recovery. No deaths occurred in the heparin arm, and based on these findings the authors concluded that heparin was both safe and efficacious in patients with CVT. In 1991, the first randomized controlled trial [15] on anticoagulation in CVT was published. Twenty patients with aseptic CVT were randomized, 10 to a placebo arm and 10 to heparin arm. A CVT severity scale was used to monitor the clinical course. Patients in the heparin arm showed a clear improvement at day 3 (P < .05), and the difference remained significant after 8 days of treatment (P < .01). After 3 months, 8 of the heparin-treated patients had a complete clinical recovery, and 2 had slight residual neurological deficits. In the placebo group, only 1 patient had a complete recovery, 6 patients had neurological deficits, and 3 patients died (P less than  .01). Three patients in the treatment group and 2 in the control group had ICH when therapy was started. However, no new cases of ICH occurred after initiation of heparin. Three patients in the control group experienced a new or worsened hemorrhage.

patients had neurological deficits, and 3 patients died (P less than  .01). Three patients in the treatment group and 2 in the control group had ICH when therapy was started. However, no new cases of ICH occurred after initiation of heparin. Three patients in the control group experienced a new or worsened hemorrhage. In this same report [15], Einhaupl et al. described an additional retrospective study on the relation between heparin treatment and ICH in CVT patients. 43 patients with CVT and ICH were studied. 27 of these patients were treated with dose-adjusted intravenous heparin after the ICH. Of these 27 patients, 4 died (mortality 15%), and 14 patients completely recovered. Of the 13 patients that did not receive heparin after ICH, 9 died (mortality 69%) and only 3 patients completely recovered. The authors concluded that anticoagulation with dose-adjusted intravenous heparin is an effective treatment in patients with CVT and that ICH is not a contraindication to heparin treatment in these patients. The study was criticized for its small sample size, use of an outcome measure that was not previously validated, and a significant delay from symptom onset to the initiation of therapy.

heparin is an effective treatment in patients with CVT and that ICH is not a contraindication to heparin treatment in these patients. The study was criticized for its small sample size, use of an outcome measure that was not previously validated, and a significant delay from symptom onset to the initiation of therapy. The other randomized trial to improve on the results of the above trial came in 1999 [30]. This was a double-blind placebo-controlled multicenter trial. Thirty patients were randomized to subcutaneous nadroparin (180 antifactor Xa units/kg per 24 hours) and 29 to matching placebo for 3 weeks (double-blind part of trial), followed by 3 months of oral anticoagulants for patients allocated nadroparin (open part). Patients with cerebral hemorrhage caused by sinus thrombosis were also included. After 3 weeks, 6 of 30 patients (20%) in the nadroparin group and 7 of 29 patients (24%) in the placebo group had a poor outcome, defined as death or Barthel Index score of <15 (risk difference, −4%; 95% CI, −25 to 17%; NS). After 12 weeks, 4 of 30 patients (13%) in the nadroparin group and 6 of 29 (21%) in the placebo group had a poor outcome, defined as death or Oxford Handicap Score of ≥3 (risk difference, − 7%; 95% CI, − 26% to 12%; NS). There were no new symptomatic cerebral hemorrhages. One patient in the nadroparin group had a major gastrointestinal hemorrhage, and 1 patient in the placebo group died from clinically suspected pulmonary embolism. Authors concluded that patients with cerebral sinus thrombosis treated with anticoagulants (low-molecular-weight heparin followed by oral anticoagulation) had a favorable outcome more often than controls, but the difference was not statistically significant. Anticoagulation proved to be safe, even in patients with cerebral hemorrhage.

d that patients with cerebral sinus thrombosis treated with anticoagulants (low-molecular-weight heparin followed by oral anticoagulation) had a favorable outcome more often than controls, but the difference was not statistically significant. Anticoagulation proved to be safe, even in patients with cerebral hemorrhage. A Cochrane review [2] was published in 2005 using these two trials for meta-analysis, and this concluded that based upon the limited evidence available, anticoagulant treatment for cerebral sinus thrombosis appeared to be safe and was associated with a potentially important reduction in the risk of death or dependency which did not reach statistical significance. The results from the ISCVT [7] also support the use of anticoagulation, as in a retrospective review done recently showed a nonsignificant but definite trend towards improvement with anticoagulation. No trials have compared unfractionated heparin with low-molecular-weight heparin. The recently published EFNS guidelines [20] based on Cochrane review; MEDLINE search; Cochrane Central Register of Controlled Trials (CENTRAL) recommend that anticoagulation should be given to all patients with CVT who do not have contraindications for anticoagulation. The recommended duration of oral anticoagulation varies depending on the underlying etiology. For transient risk factors, the recommendation is for 3 months, for idiopathic variety and for mild thrombophilia, 6–12 months, and for those with recurrent episodes of CVT or severe thrombophilia, the therapy should continue indefinitely.

ed duration of oral anticoagulation varies depending on the underlying etiology. For transient risk factors, the recommendation is for 3 months, for idiopathic variety and for mild thrombophilia, 6–12 months, and for those with recurrent episodes of CVT or severe thrombophilia, the therapy should continue indefinitely. Most of the reviews suggest that anticoagulation is safe even in patients who have evidence of intracranial hemorrhage, be it intracranial or subarachnoid. In one review published by Wasay and Kamal in 2008 [31] on anticoagulation, however, the authors were strongly of the opinion that in the absence of randomized trials and hence statistical significance, anticoagulation cannot be recommended across the board for all CVT patients. They have stressed on studies where patients have recanalized even in the absence of any treatment. In the Paediatric population, uncertainty regarding the therapeutic options continues. In the International Paediatric Stroke study [32], 84 neonates were diagnosed with CVT from 10 countries. In these patients, there were significant differences regarding use of antithrombotics and their indications. A recent single center prospective study [33] evaluated the safety and efficacy of anticoagulation in neonates and children and concluded that in this age group also, AC is safe and nontreatment results in thrombus propagation. Based on these trials and reviews, anticoagulation continues to be the mainstay of treatment for CVT.

In the Paediatric population, uncertainty regarding the therapeutic options continues. In the International Paediatric Stroke study [32], 84 neonates were diagnosed with CVT from 10 countries. In these patients, there were significant differences regarding use of antithrombotics and their indications. A recent single center prospective study [33] evaluated the safety and efficacy of anticoagulation in neonates and children and concluded that in this age group also, AC is safe and nontreatment results in thrombus propagation. Based on these trials and reviews, anticoagulation continues to be the mainstay of treatment for CVT. 6. Fibrinolytic Agents Despite the use of anticoagulant therapy, some patients continue to worsen. For these patients, use of fibrinolytic agents, particularly locally, has been tried. For many years, agents like urokinase and streptokinase were of interest for clot lysis. Initially, people tried systemic urokinase and reported mixed results [34, 35]. A series of 5 patients affected by aseptic dural sinus thrombosis were treated with a combination of heparin sodium and urokinase, and complete clinical recovery was reported [36]. Then in the late 80s, some investigators tried local instead of systemic urokinase. In 1988 to improve the safety profile, a patient with dural sinus thrombosis was successfully treated with local urokinase infusion continued for 8 hours [37]. The patient recovered with very minimal deficits despite a small temporal hemorrhage.

he late 80s, some investigators tried local instead of systemic urokinase. In 1988 to improve the safety profile, a patient with dural sinus thrombosis was successfully treated with local urokinase infusion continued for 8 hours [37]. The patient recovered with very minimal deficits despite a small temporal hemorrhage. Following this report, several case reports and series with this modality were published. Barnwell and colleagues [38] reported three patients who were treated with a transjugular direct infusion of urokinase. The period of infusion ranged from 4 to 10 days. Two patients had both angiographic and clinical improvement of signs and symptoms, whereas the third only showed angiographic improvement. Another series reported seven patients treated with direct infusion of urokinase into the thrombosed sinus. The duration of infusion was a mean of 163 hours. All had angiographic improvement, and six out of seven had clinical benefit also [39]. In 1995, Horowitz et al. [40] reported a case series of 12 patients with CVT treated with selective catheterization and urokinase infusion. 4 of these had hemorrhages on preinfusion scans. Despite this, there was no major therapeutic morbidity. Eleven out of the 12 patients had sinus patency restored, and 10 of these had excellent clinical outcome.

reported a case series of 12 patients with CVT treated with selective catheterization and urokinase infusion. 4 of these had hemorrhages on preinfusion scans. Despite this, there was no major therapeutic morbidity. Eleven out of the 12 patients had sinus patency restored, and 10 of these had excellent clinical outcome. For superior sagittal sinus thrombosis, Wasay et al. [41] conducted a nonrandomized comparison of local urokinase infusion with systemic heparin anticoagulation. Forty patients were enrolled, 20 received local urokinase followed by systemic heparin, and 20 received only systemic heparin. Discharge neurological function was better in the thrombolysis group than in the heparin group (P = .019), but hemorrhagic complications were also more with the thrombolysis group (P = .49). The authors concluded that local urokinase may be superior to systemic heparin alone in the treatment of superior sagittal sinus thrombosis. Other agents have also been tried for thrombolysis. In one patient, local thrombectomy followed by streptokinase infusion proved very beneficial [42]. In yet another series [43], 12 patients were treated with local recombinant tissue plasminogen activator along with systemic heparin. The authors concluded that this combination shows promise but should be reserved for those without obvious hemorrhage. The time taken to restore flow was faster than with urokinase.

42]. In yet another series [43], 12 patients were treated with local recombinant tissue plasminogen activator along with systemic heparin. The authors concluded that this combination shows promise but should be reserved for those without obvious hemorrhage. The time taken to restore flow was faster than with urokinase. Subsequently, a systematic review was published in 2003 [44] that included cases of cerebral venous and dural sinus thrombosis treated with fibrinolytics. 72 studies were included and no randomized clinical trial was found. Urokinase was the thrombolytic most frequently administered (76%) and majority had it locally infused (88%). ICH was reported in 17% of the patients, and in 5% it caused clinical deterioration. The conclusion, however, was that although thrombolytics appeared to be safe, their efficacy cannot be assessed from the published literature. In 2004, Cochrane review [45] also failed to identify any randomized controlled trial on thrombolytic use in CVT and concluded that there is no concrete evidence of the safety and efficacy of thrombolytic therapy in dural sinus thrombosis.

Subsequently, a systematic review was published in 2003 [44] that included cases of cerebral venous and dural sinus thrombosis treated with fibrinolytics. 72 studies were included and no randomized clinical trial was found. Urokinase was the thrombolytic most frequently administered (76%) and majority had it locally infused (88%). ICH was reported in 17% of the patients, and in 5% it caused clinical deterioration. The conclusion, however, was that although thrombolytics appeared to be safe, their efficacy cannot be assessed from the published literature. In 2004, Cochrane review [45] also failed to identify any randomized controlled trial on thrombolytic use in CVT and concluded that there is no concrete evidence of the safety and efficacy of thrombolytic therapy in dural sinus thrombosis. More recently, some more reviews have come out on the use of local thrombolytics. A series of 168 patients of CVT [46] treated with individualized endovascular treatment was published in January 2009. These included direct thrombolysis via internal jugular vein, injection of urokinase via common carotid artery, and stent angioplasty in venous sinus. They reported favorable outcomes with these individualized procedures. Another Chinese study [47] reported five patients with deteriorating neurological condition who underwent endovascular thrombolysis. The recovery was excellent in four of the five patients; one, however, could not be saved despite a decompressive craniectomy.

ted favorable outcomes with these individualized procedures. Another Chinese study [47] reported five patients with deteriorating neurological condition who underwent endovascular thrombolysis. The recovery was excellent in four of the five patients; one, however, could not be saved despite a decompressive craniectomy. In November 2009, another review of studies done on thrombolysis was published [48]. The authors concluded that although there was some evidence of beneficial effects of chemical thrombolysis from the reported literature, there is a need for prospective trials. Till then most institutions continue to combine direct thrombolysis with systemic anticoagulation. An even more recent experience with 19 patients from India [49] suggests that intrasinus thrombolysis is safe and effective in patients with CVT who fail to respond to the conventional anticoagulation. Based on the available evidence and in the absence of any randomized controlled trial on fibrinolytic use in CVT, its safety and efficacy are still questionable. 7. Mechanical Thrombectomy Mechanical thrombectomy has been used to augment the effects of chemical thrombolysis and sometimes to avoid fibrinolytics due to high risk of hemorrhage. Several methods have been tried for mechanical thrombectomy. These include balloon angioplasty, stenting, clot maceration, and rheolytic thrombectomy [50, 51].

ctomy Mechanical thrombectomy has been used to augment the effects of chemical thrombolysis and sometimes to avoid fibrinolytics due to high risk of hemorrhage. Several methods have been tried for mechanical thrombectomy. These include balloon angioplasty, stenting, clot maceration, and rheolytic thrombectomy [50, 51]. In 2003, Soleau et al. [52] published a review of 31 patients retrospectively evaluated from one institution. Four treatment strategies were identified, and complications and clinical outcomes were assessed for each group. Chemical thrombolysis was very effective in restoring sinus patency; however, 30% experienced hemorrhagic complications. 60% in this group made good clinical recovery. Patients who underwent mechanical thrombectomies demonstrated low hemorrhagic complications, and 88% had good clinical recovery. Kirsch et al. in 2007 [51] published a retrospective review of four patients with CVT treated with transfemoral intravenous rheolytic thrombectomy. All four had good restoration of blood flow, and in three it was completely normalized. In these three, there was a rapid clinical improvement also. The authors concluded that this was a safe and effective method for selected patients with dural venous sinus thrombosis.

transfemoral intravenous rheolytic thrombectomy. All four had good restoration of blood flow, and in three it was completely normalized. In these three, there was a rapid clinical improvement also. The authors concluded that this was a safe and effective method for selected patients with dural venous sinus thrombosis. There is only a single prospective review of endovascular treatment in CVT which was published in 2008 [53]. 20 patients were enrolled, 12 of those were comatose, and 14 had hemorrhagic infarcts before thrombolysis. Thrombolysis was done by introducing a catheter through the internal jugular vein into the superior sagittal sinus, most underwent thrombosuction with a rheolytic catheter, combined with thrombectomy with a Fogarty catheter, followed by urokinase infusion. 6 patients died, 5 of whom had large infarcts and impending herniation even prior to thrombolysis. The authors concluded that thrombolysis can be effective for severe sinus thrombosis, but patients may deteriorate because of increased cerebral hemorrhage. Therefore, endovascular therapy is a promising modality but lacks randomized controlled trials for its evaluation. However, the available evidence supports its use along with chemical thrombolysis as well as alone for treatment of selected cases of CVT.

There is only a single prospective review of endovascular treatment in CVT which was published in 2008 [53]. 20 patients were enrolled, 12 of those were comatose, and 14 had hemorrhagic infarcts before thrombolysis. Thrombolysis was done by introducing a catheter through the internal jugular vein into the superior sagittal sinus, most underwent thrombosuction with a rheolytic catheter, combined with thrombectomy with a Fogarty catheter, followed by urokinase infusion. 6 patients died, 5 of whom had large infarcts and impending herniation even prior to thrombolysis. The authors concluded that thrombolysis can be effective for severe sinus thrombosis, but patients may deteriorate because of increased cerebral hemorrhage. Therefore, endovascular therapy is a promising modality but lacks randomized controlled trials for its evaluation. However, the available evidence supports its use along with chemical thrombolysis as well as alone for treatment of selected cases of CVT. 8. Decompressive Surgery This modality has limited utilization so far in the treatment of CVT. Decompression has been used in cases of malignant elevations in intracranial pressure to abort impending herniation. Despite treatment with endovascular thrombolytics, it was observed that there was poor outcome in patients with impending herniation. For these patients, decompressive craniectomy was introduced. Coutinho et al. [54] reports 3 such patients who underwent decompression, two had excellent outcome, the third died.

iation. Despite treatment with endovascular thrombolytics, it was observed that there was poor outcome in patients with impending herniation. For these patients, decompressive craniectomy was introduced. Coutinho et al. [54] reports 3 such patients who underwent decompression, two had excellent outcome, the third died. Another prospective series [55] of three patients with malignant sinus thrombosis reported good recovery in two of these patients, and one was left with moderate disability. The authors reviewed prior literature on decompressive surgeries in these patients between 1970 and 2008. Eight such patients had been reported in three reviews [56–58], and the outcome in four was good. Three patients were left with moderate and one with severe disability. The authors concluded that decompressive surgery in these patients with severe CVT and evidence of malignant intracranial hypertension can be life saving, and the outcome can also be reasonably good. A retrospective review [59] of 12 patients with malignant cerebral venous thrombosis has recently been published. Of these, 8 underwent decompressive surgery. The four patients who did not undergo surgery died. Of these 8 who did, one died of pulmonary embolism, but the other 7 not just survived, but 6 of them made excellent neurological recovery. Based on the current available literature, decompressive surgery can be a very promising option for patients with malignant cerebral venous thrombosis.

A retrospective review [59] of 12 patients with malignant cerebral venous thrombosis has recently been published. Of these, 8 underwent decompressive surgery. The four patients who did not undergo surgery died. Of these 8 who did, one died of pulmonary embolism, but the other 7 not just survived, but 6 of them made excellent neurological recovery. Based on the current available literature, decompressive surgery can be a very promising option for patients with malignant cerebral venous thrombosis. 9. Conclusion After reviewing all the available therapeutic modalities for CVT, the question remains which modality to use in which situation. Generally speaking, the choice of therapy, which is at the discretion of the treating physician in the absence of clear cut guidelines, depends largely on the clinical presentation. This may vary from mild headache to focal deficits and even coma. The presentation broadly speaking can be acute, subacute, or chronic. For subacute or chronic cases, headache is often the most prominent presenting feature, and the need is for urgent but not emergent therapy. Anticoagulation with heparin is the best studied and the only recommended therapy in these cases. Acute presentation can be more striking with encephalopathy and focal deficits dominating the picture. In these patients, again the best evidence is in favor of anticoagulation, but more aggressive measures to recannulate the sinus may be undertaken in case the patient continues to deteriorate. These include endovascular thrombolysis and mechanical thrombectomy.

encephalopathy and focal deficits dominating the picture. In these patients, again the best evidence is in favor of anticoagulation, but more aggressive measures to recannulate the sinus may be undertaken in case the patient continues to deteriorate. These include endovascular thrombolysis and mechanical thrombectomy. Escalation of therapy is recommended in case of clinical worsening. A patient presenting with mild symptoms may just be managed on anticoagulation. However, if there is evidence of deterioration in terms of new deficits and drowsiness, therapy may be escalated to use of systemic or local thrombolytics, and in the presence of intracranial hemorrhage and malignant intracranial hypertension, surgical options may also be considered. To summarize, overall therapeutics for CVT need larger randomized controlled trials. Anticoagulation with heparin is the only modality with reasonable evidence to support its use in CVT, even in patients with cerebral hemorrhage. Endovascular thrombolysis is a promising option for patients with a severe form of CVT or following a failure of anticoagulation therapy. Mechanical thrombectomy is reserved for selected cases and decompression surgery for malignant CVT with impending herniation.

1. Evidence for Genetic Factors in Stroke Risk Stroke is the third commonest cause of death and the major cause of adult neurological disability, affecting both the developed world and increasingly having an impact in the developing world as well. It is also a major cause of dementia and the commonest cause of late onset epilepsy. Therefore, increasing our understanding of the risks, causes, and treatment of ischaemic stroke is of great importance. Stroke is itself a syndrome cause by a number of different disease processes. About 80% of strokes are ischemic and 20% are due to primary hemorrhage. In this paper we will only address the genetics of ischaemic stroke. While much is known about conventional risk factors such as hypertension, diabetes, and incidence of smoking, studies suggest these only account for a proportion of ischaemic stroke risk. Considerable evidence suggests genetic predisposition may explain some of the remaining risk, including evidence from both twin and family studies [1]. Family studies have shown differential association with different subtypes of stroke, suggesting these may have different underlying genetic risk factors [2, 3].

risk. Considerable evidence suggests genetic predisposition may explain some of the remaining risk, including evidence from both twin and family studies [1]. Family studies have shown differential association with different subtypes of stroke, suggesting these may have different underlying genetic risk factors [2, 3]. Further evidence for a genetic contribution to ischaemic stroke risk comes from animal models [4] and from the study of intermediate phenotypes such as carotid artery intima-media thickness (IMT) as a marker for large artery disease and MRI white matter hyperintensities as a marker for small vessel stroke. Twin and family history studies have shown these both have significant heritability (the proportion of stroke risk attributable to genetic risk factors) with estimates ranging from 55–71% for IMT [5–7] and 30–68% for WMH [8–10]. The identification of genetic variants predisposing to known stroke risk factors such as atrial fibrillation (AF) [11] and myocardial infarction (MI) and coronary artery disease (CAD) [12] further highlights the role of genetic predisposition in stroke risk.

ranging from 55–71% for IMT [5–7] and 30–68% for WMH [8–10]. The identification of genetic variants predisposing to known stroke risk factors such as atrial fibrillation (AF) [11] and myocardial infarction (MI) and coronary artery disease (CAD) [12] further highlights the role of genetic predisposition in stroke risk. The clearest evidence that genetics can cause ischaemic stroke comes from monogenic forms of the disease, although these account for only a relatively small percentage of overall ischaemic stroke incidence [13] and appear to have limited relevance to common polygenic stroke. As such they will not be considered as part of this paper in detail, but are covered in reviews elsewhere [14]. Therefore, considerable evidence suggests genetic factors do play an important role in ischaemic stroke, so why have so few genes been identified that contribute to this risk and why have other fields, including related cardiovascular disease phenotypes, been more successful?

but are covered in reviews elsewhere [14]. Therefore, considerable evidence suggests genetic factors do play an important role in ischaemic stroke, so why have so few genes been identified that contribute to this risk and why have other fields, including related cardiovascular disease phenotypes, been more successful? 2. Identification of Genetic Risk: Candidate Gene and Familial Linkage Studies Until recently, identification of genetic variants contributing to disease has been attempted by 2 main techniques—candidate gene studies and familial linkage studies (See Box 1 for details of the different types of genetic investigation and their use). Of these, the candidate gene study has been the mainstay of genetic investigation into the vast majority of polygenic diseases thought to have a genetic component. Typically, a gene identified as a “candidate” is hypothesised to be involved in stroke risk, and then, genetic variants, usually single nucleotide polymorphisms (SNPs), are identified within that gene. The frequency of the SNPs is then determined in a series of cases and controls and the two compared.

enetic component. Typically, a gene identified as a “candidate” is hypothesised to be involved in stroke risk, and then, genetic variants, usually single nucleotide polymorphisms (SNPs), are identified within that gene. The frequency of the SNPs is then determined in a series of cases and controls and the two compared. The vast majority of candidate gene studies in ischaemic stroke have turned out to be disappointing. Reasons for this include insufficient sample size, a failure to replicate results initially reported as significant, poor stroke subtyping or phenotyping, and a failure to look for associations with specific subtypes of stroke [15]. Meta-analysis of published candidate gene studies has revealed some consistently positive findings however, such as Factor V Leiden Arg506Gln, MTHFR C677T and the ACE insertion/deletion polymorphism [16], although caution is required in interpretation due to the possible effect of publication bias meaning positive studies are more likely to be published. Although still useful when explaining specific hypotheses, candidate gene studies have now been largely superseded by the genome-wide association study (GWAS) technique.

lthough caution is required in interpretation due to the possible effect of publication bias meaning positive studies are more likely to be published. Although still useful when explaining specific hypotheses, candidate gene studies have now been largely superseded by the genome-wide association study (GWAS) technique. Familial linkage studies examine genetic variants through multiple generations of families and correlate these with disease incidence. Associations with a specific gene are not sought using this approach, but rather one looks for variants anywhere in the entire genome, and they are therefore referred to as “nonhypothesis driven” experiments. The technique has had, and continues to have, great success in identifying genes underlying Mendelian disorders in monogenic conditions where a single gene contributes the entirety of genetic risk. Familial linkage studies rely on collection of families with the disease however, and this is a challenge in stroke where the late age of onset means parents are often not alive; this has hampered collection of cases in studies such as the siblings with ischaemic stroke study (SWISS) [17, 18].

entirety of genetic risk. Familial linkage studies rely on collection of families with the disease however, and this is a challenge in stroke where the late age of onset means parents are often not alive; this has hampered collection of cases in studies such as the siblings with ischaemic stroke study (SWISS) [17, 18]. One notable exception to this has been in Iceland, where the DeCode group reported identification of the first genetic risk for common polygenic ischaemic stroke via such a familial linkage study, which they named STRK1 [19]. This study used the unique national collection of genealogical samples and family structures tracked in the Icelandic population to retrospectively determine cause of death and provide material for genotyping. The STRK1 locus was identified as overlying the gene phosphodiesterase4D (PDE4D), a cyclic AMP regulator which is a plausible biological candidate [19]. Subsequent replication in European cohorts failed to confirm these findings [20]. This study was undertaken as large-scale genome-wide experiments were being developed as a mainstream technique. By current standards the DeCode finding would today be considered underpowered as it failed to exceed the currently agreed statistical threshold for such studies.

horts failed to confirm these findings [20]. This study was undertaken as large-scale genome-wide experiments were being developed as a mainstream technique. By current standards the DeCode finding would today be considered underpowered as it failed to exceed the currently agreed statistical threshold for such studies. 3. Identification of Genetic Risk: The Genome Wide Association Study The field of complex genetics has been revolutionized by the advent of the genome-wide association study (GWAS) [21]. This can be thought of as a large series of candidate gene studies performed in a single experiment on an array based format. As many as 1.2 million polymorphisms at a time can now be studied in this manner. Crucially, these are spread throughout the entire genome and such experiments are thus nonhypothesis driven, overcoming a major limitation of the candidate gene study. Such a large number of experiments in a single study requires a large sample sizes to allow sufficient power, even after statistical correction for multiple comparisons. Also crucial to progress has been the realisation that careful phenotyping is important, and that associations should be replicated in a second population before publication.

xperiments in a single study requires a large sample sizes to allow sufficient power, even after statistical correction for multiple comparisons. Also crucial to progress has been the realisation that careful phenotyping is important, and that associations should be replicated in a second population before publication. An early demonstration of the power of this technique was in age-related macular degeneration, a late onset eye disorder leading to blindness in which conventional cardiovascular risk factors play a part. Applying a GWAS approach to this condition revealed associations with the complement factor H gene, and identification of a single amino acid substitution which proved to be the causal variant in this condition [22]. Interestingly, the same locus had been identified by a familial linkage approach in previous studies, but refinement of the region and identification of the causal variant via familial linkage had been impossible.

fication of a single amino acid substitution which proved to be the causal variant in this condition [22]. Interestingly, the same locus had been identified by a familial linkage approach in previous studies, but refinement of the region and identification of the causal variant via familial linkage had been impossible. As a consequence of this and other studies, the enormous potential of GWAS to identify common variants associated with common diseases became recognised, with perhaps the seminal GWAS publication by the Wellcome Trust Case Control Consortium 1 study making GWAS a mainstream technique in disease gene identification [23]. This study examined 14,000 cases of seven common diseases and 3,000 shared controls in an effort to identify genetic variants in human disease. Investigating bipolar disorder, coronary artery disease, Crohn's disease, hypertension, rheumatoid arthritis, and type I and type II diabetes, this single study identified over 58 novel loci as potentially contributing genetic risk in these conditions. To date, the GWAS technique has identified over 1212 new genetic loci predisposing to common polygenic disease (http://www.genome.gov/gwastudies). Novel genetic associations with a range of cardiovascular phenotypes including myocardial infarction, coronary artery disease, diabetes and hyperlipidaemia have been reported, but few variants have been confirmed for ischaemic stroke.

tic loci predisposing to common polygenic disease (http://www.genome.gov/gwastudies). Novel genetic associations with a range of cardiovascular phenotypes including myocardial infarction, coronary artery disease, diabetes and hyperlipidaemia have been reported, but few variants have been confirmed for ischaemic stroke. It should be noted that while GWAS is a powerful technique, it requires very large, well phenotyped case series—typically in the thousands of samples, and even with these sample sizes is powered only to detect modest risks, typically with odds ratios in the region of 1.2–1.5. Thus the contribution of each risk locus to overall disease incidence is likely to be minor, although these risks are additive and as such identification of multiple loci may allow individual risk profiles to be determined. Identification of high risk individuals could be useful in early intervention to reduce conventional risk factors, more rigorous screening for early signs of disease and in investigating severity of disease at onset as well as associations with disease recurrence.

ci may allow individual risk profiles to be determined. Identification of high risk individuals could be useful in early intervention to reduce conventional risk factors, more rigorous screening for early signs of disease and in investigating severity of disease at onset as well as associations with disease recurrence. 4. GWAS and Ischaemic Stroke While GWAS has contributed greatly to identification of genetics of many complex diseases over the last 5 years, application of the technique to ischaemic stroke has been slower, with large-scale collaborative efforts only now beginning to emerge. An early study applied the GWAS technique to 249 ischaemic stroke cases and 268 controls, but we now realize this was underpowered [24]. A more recent study in prospective population-based cohorts identified a region on Chromosome 12 overlying the NINJ2 gene in ischaemic stroke cases [25], although a subsequent large replication failed to confirm this finding [26].

9 ischaemic stroke cases and 268 controls, but we now realize this was underpowered [24]. A more recent study in prospective population-based cohorts identified a region on Chromosome 12 overlying the NINJ2 gene in ischaemic stroke cases [25], although a subsequent large replication failed to confirm this finding [26]. The collection of large, well phenotyped sample cohorts for genetic analysis in stroke presents major challenges. In particular phenotyping, which we now realize is essential, requires detailed and expensive investigations. As in other complex diseases, collection of sufficiently large sample sizes depends on larges scale international collaborations, and to address this the International Stroke Genetics Consortium (ISGC—http://www.strokegentics.org/) was established. Currently an ischaemic stroke GWAS in 4000 cases is near completion as part of the Wellcome Trust Case Control Consortium 2 study (WTCCC2) in collaboration with the ISGC. GWAS studies in countries including the US and Australia are also ongoing with results expected in 2011. A lesson from other disease areas is that, even with sample sizes of several thousands, power is limited and meta-analysis of multiple GWAS studies has become standard practice. The Meta-stroke collaboration has been formed in ischaemic stroke to address this.

d Australia are also ongoing with results expected in 2011. A lesson from other disease areas is that, even with sample sizes of several thousands, power is limited and meta-analysis of multiple GWAS studies has become standard practice. The Meta-stroke collaboration has been formed in ischaemic stroke to address this. These collaborative efforts have achieved early success in ischaemic stroke via examination of genetic associations already identified in related cardiovascular diseases. The identification of a region on Chromosome 9p21 in myocardial infarction and coronary artery disease, which surrounds the CDKN2A and CDKN2B genes, has generated a large amount of interest [27]. An examination of this locus in a candidate gene study in ischaemic stroke cases revealed an association with large artery stroke, but not the other ischaemic stroke subtypes [28]. This association persisted across multiple populations and importantly emphasises the likely differing contributions of genetic risks to different ischaemic stroke subtypes. Two genetic variants identified as contributing to the risk of atrial fibrillation (AF), in the genes PITX2 and ZFHX3, have also been shown to associate with cardioembolic stroke risk for which AF is an important risk factor [29, 30]. As new loci are identified for other cardiovascular diseases which themselves are associated with stroke, rapid testing these in stroke populations via large International collaborations is possible.

HX3, have also been shown to associate with cardioembolic stroke risk for which AF is an important risk factor [29, 30]. As new loci are identified for other cardiovascular diseases which themselves are associated with stroke, rapid testing these in stroke populations via large International collaborations is possible. 5. Recommendations for Future Genetic Studies in Stroke Previous studies in stroke genetics have been disappointing. There are a number of reasons for this, most significant of which are poor phenotyping, small sample sizes, and failure to replicate initial findings in a second population. Any future genetic study, whether hypothesis driven or nonhypothesis driven, should address each of these issues prior to publication. Power calculations demonstrating the number of cases required for confirmation or refutation of a finding should be included to allow an estimate of the significance and robustness of the findings presented. Genetic risks in stroke are usually estimated to be between 1.1 and 1.5 for a single loci, and studies should be adequately powered (i.e., be comprised of sufficient cases) to detect risks of this size. Replication of positive associations prior to publication is important. This should be in a separate case series using a different control set.

e usually estimated to be between 1.1 and 1.5 for a single loci, and studies should be adequately powered (i.e., be comprised of sufficient cases) to detect risks of this size. Replication of positive associations prior to publication is important. This should be in a separate case series using a different control set. Increasing evidence suggests genetic risks differ depending on ischaemic stroke subtype. Future genetic studies should therefore include reference to subtypes and subtype specific risks. Evidence of genetic risk in a homogenous population of ischaemic stroke without subtype investigation is likely to lead to spurious associations. While these measures lead to increased cost and complexity of studies, it is only through such robust experimental procedures that we will truly begin to understand the genetic risks of stroke and how these are manifest. 6. The Post GWAS Era in Stroke Genetics Genome-wide association studies have been specifically conceived to address the common variant, common disease (CVCD) hypothesis. This concept underlies the majority of genetic studies to date not just in stroke but in other common diseases. According to the CVCD hypothesis, multiple genetic risk factors contribute to disease, each with a small additional increase in risk. These risks are additive in nature and together provide an individual risk profile that allows for a significant genetic contribution. In order for this hypothesis to hold true however, variants have to be common in the population.

netic risk factors contribute to disease, each with a small additional increase in risk. These risks are additive in nature and together provide an individual risk profile that allows for a significant genetic contribution. In order for this hypothesis to hold true however, variants have to be common in the population. Despite the success of GWAS in identifying susceptibility loci, for the vast majority of diseases these account for only a fraction of the heritability initially attributed to genetic risk factors. Each risk identified carries a much smaller risk than originally thought under the CVCD hypothesis. For this reason the CVCD theory of genetic risk is now being questioned, and various mechanisms have been suggested to account for this “missing heritability” [31]. An alternative hypothesis is that rare variants are important in common disease risk (RVCD). This states that as well as common variants that each contribute very small risks, susceptible individuals may carry variants of higher risk which are rare and perhaps even private to themselves or closely related family members. Such risks would not be detectable via classical familial linkage since there would be multiple risk variants contributing to an individuals susceptibility to disease, but neither would they be detectable via GWAS since they would be specific to individuals or closely related family members and therefore not carried by the rest of the population. Under this hypothesis any one individual would be expected to carry many variants detectable by GWAS, and a handful of higher risk alleles in a private manner. Together these combine to produce an individuals overall risk profile of disease susceptibility, and may account for the so called “hidden heritability” conundrum which persists after GWAS. Identification of these rare variants requires a sequencing approach which provides information on every base pair across the region of interest, and this approach is provided by next generation sequencing (NGS).

usceptibility, and may account for the so called “hidden heritability” conundrum which persists after GWAS. Identification of these rare variants requires a sequencing approach which provides information on every base pair across the region of interest, and this approach is provided by next generation sequencing (NGS). NGS has arisen from both advances in technology, and from our ability to sequence the human genome as a consequence of the human genome project (http://www.hapmap.org/) and the 1000 genome (http://www.1000genomes.org/) project among others. It is now possible to obtain the entire coding sequence of the human genome in less than a week, and to make comparisons between genomes due to advances in computational methods and processing power. Sequencing of targeted regions at a previously unparalleled depth and fold coverage without the need for generation of vector libraries or bacterial culture is now routine and provided by a number of service providers using a variety of techniques [32]. While currently expensive, such techniques give access to perhaps the majority of the information that conventional genetics can be expected to provide, namely the entire coding sequence of the genome. Interpretation of that sequence is still in its relative infancy.

ervice providers using a variety of techniques [32]. While currently expensive, such techniques give access to perhaps the majority of the information that conventional genetics can be expected to provide, namely the entire coding sequence of the genome. Interpretation of that sequence is still in its relative infancy. 7. Beyond Genomics in Identifying Ischaemic Stroke Risk Factors Understanding the genetic basis of disease risk requires an understanding of the way in which these genes have their effects in the body. Genes code for RNA, which is then translated into protein. These proteins can act alone, in multiples of themselves as homodimers or in conjunction with other proteins to form heterodimers. Similarly genes may interact with each other, so called epigenetics, or may interact with environmental factors in gene-environment interactions which only affect disease risk when both the environmental and genetic components of the interaction are present. Detecting such gene-gene or gene-environment interactions requires much larger sample sizes. Their importance has been shown in other cardiovascular diseases [33] and in association studies with the quantitative trait continuous carotid IMT [34].

the environmental and genetic components of the interaction are present. Detecting such gene-gene or gene-environment interactions requires much larger sample sizes. Their importance has been shown in other cardiovascular diseases [33] and in association studies with the quantitative trait continuous carotid IMT [34]. Examination of RNA and proteins in a nonhypothesis driven manner, similar to GWAS for DNA, is also possible. Examination of the transcriptome is a relatively old technique via the use of microarrays, and it is this technology which actually led to the development of GWAS using DNA slides or cartridges rather than RNA and cDNA-based ones. More recently studies have been examining the possibility of ignoring the DNA level and trying to perform transcription profiling (examination of all the RNA's being produced at a specific time point). By examining changes in the level of transcription of a subset of RNAs and correlating these with changes in disease state, disease subtype or disease severity, we may be able to better understand how genetic differences influence disease processes [35].

nation of all the RNA's being produced at a specific time point). By examining changes in the level of transcription of a subset of RNAs and correlating these with changes in disease state, disease subtype or disease severity, we may be able to better understand how genetic differences influence disease processes [35]. There are reports that it is possible to differentiate between different stroke subtypes using this methodology, with expression levels of just 23 genes being able to differentiate between cardioembolic stroke and large vessel disease [36]. While such studies are not yet able to replace conventional investigative techniques for determining ischaemic stroke subtype, identification of a expression profiles may give novel insights into stroke pathogenesis, and perhaps identification of suitable biomarkers for monitoring risk reduction treatments. This technique has also been applied to associated phenotypes such as white matter hyperintensity in the brain, currently only detectable by MRI [37].

on of a expression profiles may give novel insights into stroke pathogenesis, and perhaps identification of suitable biomarkers for monitoring risk reduction treatments. This technique has also been applied to associated phenotypes such as white matter hyperintensity in the brain, currently only detectable by MRI [37]. 8. Conclusions Considerable evidence suggests genetic factors are important in ischaemic stroke risk. The advent of new techniques such as GWAS has contributed enormously to the understanding of the genetics of other complex disease and progress is just beginning to be made in stroke. For success large, well phenotyped case cohorts are required, and international collaborations are essential. NGS technology and techniques such as transcription profiling and proteomics will allow us to look for rarer variants in stroke cases and attempt to identify how these exert their effects at the molecular level, but whether these will be important remains to be determined. Box 1 Types of analysis for genetic investigation.

1. Introduction One of the gold standards of neuroprotectants against stroke in animal experiments [1, 2] induced mild (33 to 36°C) to moderate (28 to 32°C) hypothermia has been the focus of several clinical trials for the treatment of cerebral ischemia. In the past decade, prospective randomized controlled studies have demonstrated that induced hypothermia improves neurological function in patients suffering cardiac arrest from ventricular fibrillation [3] and reduces risk of death or disability in neonates following hypoxic-ischemic encephalopathy [4, 5]. However, the clinical translation of hypothermia for acute stroke treatment is still in its early stages. Many barriers remain, including onset time, duration, and depth of hypothermia [6]. In the process of extrapolating animal studies to human patients, significant gaps exist even between the design of laboratory experiments and clinical trials. For instance, many previous animal models used complete reperfusion [7–9], while most stroke patients suffer from permanent cerebral artery occlusion [10, 11]. Even with t-PA treatment, slightly less than one third of patients achieve complete reperfusion, one-third achieve partial reperfusion, and in the rest reperfusion is absent [11, 12]. Therefore, the ability to select animal stroke models that properly mimic clinical stroke is a critical step in evaluating the protective effects of induced hypothermia.

y less than one third of patients achieve complete reperfusion, one-third achieve partial reperfusion, and in the rest reperfusion is absent [11, 12]. Therefore, the ability to select animal stroke models that properly mimic clinical stroke is a critical step in evaluating the protective effects of induced hypothermia. Our laboratories have studied the protective effects of mild-to-moderate hypothermia for nearly two decades [6, 13–17]. Our recent hypothermia studies use a focal ischemic model with partial reperfusion in rats [16, 18, 19]; a model which is less frequently used in other laboratories. In this model, stroke is induced by bilateral common carotid artery (CCA) occlusion combined with permanent distal middle cerebral artery (MCA) occlusion [16, 20–22]. The bilateral CCAs are reopened 1 to 2 hours later while the distal MCA remains occluded [16, 19, 23, 24]. This technique therefore allows partial reperfusion [25, 26]. As discussed above, this model mimics many stroke patients who receive partial reperfusion, with or without t-PA treatment. However, to compare the protective effects of hypothermia in focal ischemia with partial reperfusion and complete reperfusion, we also used a model with transient three-vessel (bilateral CCAs and distal MCA) occlusion [18].

del mimics many stroke patients who receive partial reperfusion, with or without t-PA treatment. However, to compare the protective effects of hypothermia in focal ischemia with partial reperfusion and complete reperfusion, we also used a model with transient three-vessel (bilateral CCAs and distal MCA) occlusion [18]. Several excellent articles have reviewed the protective effects of hypothermia as function of onset time, duration, and depth of hypothermia, as well as its underlying protective mechanisms [27–30]. Particularly, van der Worp et al. have comprehensively reviewed past hypothermic studies [29], which either used temporary or permanent occlusion models. However, the protective affects of hypothermia in stroke models using partial reperfusion as described above have received significantly less attention. Therefore, this paper focuses mainly on our studies of the past several years on therapeutic time windows and the unique model of partial reperfusion.

cclusion models. However, the protective affects of hypothermia in stroke models using partial reperfusion as described above have received significantly less attention. Therefore, this paper focuses mainly on our studies of the past several years on therapeutic time windows and the unique model of partial reperfusion. 2. Intraischemic Moderate Hypothermia Offers Strong and Long-Term Protection in a Focal Ischemic Model with Partial Reperfusion In our first implementation of an ischemic model [16], we cauterized the distal MCA above the rhinal fissure and transiently occluded the bilateral CCAs for 1 hour. This model generates a well-delineated ischemic area limited to the cortex [20, 22]. Moderate hypothermia (30°C) monitored at the core body temperature was induced 10 minutes before ischemia onset and maintained for 1 hour after ischemia onset [16]. Although we did not directly monitor brain temperature, we previously observed a high correlation between rectal temperature and brain temperature in hypothermic rats [21]. We should add that because brain temperature in normothermic rats drops spontaneously during occlusion, core temperature may not accurately reflect brain temperature [2, 31]. Even so, we did not experimentally adjust any potential changes in brain temperature in order to minimize the introduction of possible artificial factors, which would likely exacerbate ischemic injury once the brain was heated. Our results showed that hypothermia reduced infarct size more than 80% compared with normothermia at 2 days after stroke (Figure 1(a)) [16]. Because some neuroprotectants offer transient protection, we also measured brain injury 2 months later and found similar protective effects at 60 days and 2 days (Figure 1(b)), suggesting that hypothermia decreases ischemic damage over the long term rather than merely delaying its emergence. This protective effect is further strengthened by the effects of hypothermia on behavioral deficits after stroke, which showed that hypothermia improved neurological functioning for up to 2 months [16].

, suggesting that hypothermia decreases ischemic damage over the long term rather than merely delaying its emergence. This protective effect is further strengthened by the effects of hypothermia on behavioral deficits after stroke, which showed that hypothermia improved neurological functioning for up to 2 months [16]. We then used this model to study the underlying protective mechanisms related to the PI3K/Akt cell signaling pathway [16]. The PI3K/Akt kinase pathway is known to promote neuron survival postischemia (reviewed by [32]) Figure 2. Akt activity is regulated by phosphorylation at Ser-473 and Thr-308 via upstream molecules, such as PDK1 and PTEN. While activated PDK1 phosphorylates Akt, activated PTEN dephosphorylates Akt. Activated Akt then blocks caspase/cytochrome c-mediated apoptosis by phosphorylating Akt substrates, such as FKHR and GSK3β. In our study, stroke resulted in transient increases in phosphorylated Akt (P-Akt) levels, but led to a reduction in phosphorylation levels of PTEN, PDK1, GSK3β, and FKHR [16]. However, in vitro Akt kinase assays showed that true Akt activity was decreased after stroke. Although hypothermia blocked the increase in P-Akt after stroke, it maintained true Akt activity. A functional role for this hypothermia-maintained activity is supported by the finding that the PI3K/Akt inhibitor, LY294004, enlarged infarct size in hypothermic animals. In addition, hypothermia attenuates a decrease in P-PTEN after stroke onset. Taken together, our results suggest that the PI3/Akt pathways play a critical role in the neuroprotection observed in intraischemic moderate hypothermia [16].

that the PI3K/Akt inhibitor, LY294004, enlarged infarct size in hypothermic animals. In addition, hypothermia attenuates a decrease in P-PTEN after stroke onset. Taken together, our results suggest that the PI3/Akt pathways play a critical role in the neuroprotection observed in intraischemic moderate hypothermia [16]. We also studied the potential roles of two critical components in the protein kinase C (PKC) pathway: δPKC [24] and εPKC [23]. δPKC is a kinase strongly implicated in executing ischemic damage while εPKC is neuroprotective [33]. We found that intraischemic hypothermia (30°C) blocks translocation of δPKC to the mitochondria and nucleus and attenuates δPKC cleavage [24], but it promotes εPKC activity, as evidenced by increased εPKC phosphorylation levels [23]. Therefore, our results suggest that both δPKC and εPKC may participate in the protective effects of intraischemic moderate hypothermia.

ocks translocation of δPKC to the mitochondria and nucleus and attenuates δPKC cleavage [24], but it promotes εPKC activity, as evidenced by increased εPKC phosphorylation levels [23]. Therefore, our results suggest that both δPKC and εPKC may participate in the protective effects of intraischemic moderate hypothermia. 3. Intraischemic Mild Hypothermia (33°C) Fails to Offer Protection in a More Severe Ischemic Model with Partial Reperfusion In our second study we compared the protective effects of mild (33°C) and moderate hypothermia (30°C) [19] either transiently induced during or after CCA occlusion or maintained during and after CCA occlusion. For stroke models, we extended the bilateral CCA occlusion period from 1 to 2 hours, while the distal MCA remained occluded (Figure 3) [19]. The hypothermic duration at both temperatures was either 2 hours during or after CCA occlusion or 4 hours during and after CCA occlusion. We found that 2 hours of mild hypothermia (33°C) induced either during or after CCA occlusion did not confer protection [19]. This was unexpected because our previous study showed that 2 hours of intraischemic hypothermia (33°C) reduced infarct size in a 2-hour MCA suture occlusion model in rats [14]. In addition, as van der Worp et al. [29] reviewed, previous studies have reported a substantial reduction in infarction even at 35°C, when hypothermia commenced before or at the start of MCA occlusion, with protective effects that were not clearly time dependent.

ize in a 2-hour MCA suture occlusion model in rats [14]. In addition, as van der Worp et al. [29] reviewed, previous studies have reported a substantial reduction in infarction even at 35°C, when hypothermia commenced before or at the start of MCA occlusion, with protective effects that were not clearly time dependent. In our study, however, 4 hours of mild hypothermia applied during and after CCA release slightly, but significantly, reduced infarct size by 22%. When we further reduced hypothermia from 33°C to 30°C, 2 hours of moderate hypothermia during CCA occlusion increased protection, significantly reducing infarct size by 46% (Figure 3). Nevertheless, 2 additional hours of moderate hypothermia (4 hours total) did not offer additional protection, suggesting a limited effect of prolonged moderate hypothermia applied during and after CCA release [19]. Using confocal microscopy and Western blotting, we found that when intraischemic hypothermia reduced infarct size, the subcellular translocation of cytochrome c and apoptosis-inducing factor (AIF) was blocked in the ischemic penumbra. However, when hypothermia (either intraischemic or delayed mild hypothermia) did not reduce infarct size, no effect was observed on these proapoptotic factors [19]. This suggests that inhibition of cytochrome c and AIF release corresponded to the protective effect of hypothermia.

) was blocked in the ischemic penumbra. However, when hypothermia (either intraischemic or delayed mild hypothermia) did not reduce infarct size, no effect was observed on these proapoptotic factors [19]. This suggests that inhibition of cytochrome c and AIF release corresponded to the protective effect of hypothermia. 4. Limited Therapeutic Time Windows of Moderate Hypothermia (30°C) in a Focal Ischemia with Complete Reperfusion After comparing the protective effects of both mild and moderate hypothermia in severe ischemic models with permanent distal MCA occlusion, we were not optimistic that mild hypothermia (33°C) could achieve protection. Thus, we focused on the therapeutic time window for moderate hypothermia (30°C) in a transient focal ischemic model with 1 hour of CCA and distal MCA occlusion, which allows complete reperfusion (Figure 4) [18]. Our aim was to determine the potential therapeutic time window for a brief moderate hypothermia in a less severe ischemic model. We found that 3 hours of moderate hypothermia started immediately after stroke onset spared almost all infarction (Figure 4(b)), and 3-hours of early moderate hypothermia induced 45 minutes after CCA occlusion markedly reduced infarction by more than 80%, whereas delayed hypothermia initiated 15 minutes after reperfusion did not prevent ischemic damage (Figure 4(b)) [18]. Together, these results suggest a very short therapeutic time window for a brief, moderate hypothermia.

hypothermia induced 45 minutes after CCA occlusion markedly reduced infarction by more than 80%, whereas delayed hypothermia initiated 15 minutes after reperfusion did not prevent ischemic damage (Figure 4(b)) [18]. Together, these results suggest a very short therapeutic time window for a brief, moderate hypothermia. Our study on therapeutic time windows is limited by the short 3-hour duration of hypothermia. It is highly likely that the delayed onset of hypothermia would have been protective if prolonged hypothermia had been used. For instance, Colbourne et al. found that prolonged hypothermia (24 hours of 33°C plus 24 hours of 35°C) started 2.5 hours after the onset of ischemia robustly reduced infarct volume and attenuated behavior deficits in a focal ischemia model with a 90-minute MCA occlusion in rats [34]. Clark et al. reported that hypothermia (33°C) lasting 12, 24, or 48 hours was required to reduce infarct size and improve functional outcomes when hypothermia was instituted 1 hour after permanent distal MCA and CCA occlusion, and prolonged hypothermia (24 or 48 hours) was better than shorter hypothermia (12 hours) [35]. Furthermore, delayed hypothermia beginning 1 hour after ischemia appears to require prolonged periods (12 to 24 hours) to generate protection even for global ischemia lasting just 5 minutes [36]. Therefore, the limited therapeutic effects of post-ischemic hypothermia in our studies may be specific to the experimental settings in our laboratory.

hermia beginning 1 hour after ischemia appears to require prolonged periods (12 to 24 hours) to generate protection even for global ischemia lasting just 5 minutes [36]. Therefore, the limited therapeutic effects of post-ischemic hypothermia in our studies may be specific to the experimental settings in our laboratory. Consistent with its protective effects, early hypothermia, but not delayed hypothermia, blocked TUNEL positive staining, a marker for apoptosis or cell death [18]. In addition, we found that early hypothermia attenuated the generation of superoxide compared with normothermia. However, both early and delayed hypothermia attenuated reductions in Mn-SOD protein levels and δPKC cleavage in the ischemic penumbra, suggesting that both Mn-SOD and δPKC cleavage may not be responsible for the differential protective effects of early and delayed hypothermia [18]. In addition, both early and delayed hypothermia preserved Akt phosphorylation. Nevertheless, only early hypothermia, but not delayed hypothermia, maintained PTEN phosphorylation (P-PTEN) [18], suggesting that P-PTEN may play a critical role in the protective effects of early hypothermia through the attenuation of ROS activity.

ion, both early and delayed hypothermia preserved Akt phosphorylation. Nevertheless, only early hypothermia, but not delayed hypothermia, maintained PTEN phosphorylation (P-PTEN) [18], suggesting that P-PTEN may play a critical role in the protective effects of early hypothermia through the attenuation of ROS activity. 5. Discussion As we have discussed, hypothermic studies performed in the laboratory have led to clinical investigations for cerebral ischemia. Significant enthusiasm for this approach still exists in the scientific community. A number of preliminary clinical trials (mostly phase I) to confirm the feasibility and safety of induced mild hypothermia for stroke patients have been completed, and several phase II clinical trials are currently in progress (http://clinicaltrials.gov/). However, whether mild-to-moderate hypothermia can be successfully translated clinically or, if successful, how long this will take has yet to be determined.

y of induced mild hypothermia for stroke patients have been completed, and several phase II clinical trials are currently in progress (http://clinicaltrials.gov/). However, whether mild-to-moderate hypothermia can be successfully translated clinically or, if successful, how long this will take has yet to be determined. The purpose of our basic research using animal models is to provide the rationale for clinical translation, although we cannot directly extrapolate settings from the laboratory to clinical trials. As discussed, our laboratory experiment is limited due to the short 3-hour duration of hypothermia, which contrasts to human clinical trials where hypothermia may last a few days. In addition, our study used infarct size as the criteria for evaluating the protective effects of hypothermia and not neurological function, as is often the case in clinical studies. Despite these limitations, our results serve as a warning of the persistent challenges we must confront as we seek to translate hypothermia to the clinic. First of all, the most strikingly disappointing results from our studies are the limited protective effects of hypothermia, including mild hypothermia, and the short therapeutic time window of moderate hypothermia. If these observations are true, successful clinical translation of induced hypothermia may prove to be more difficult than anticipated to achieve.

inting results from our studies are the limited protective effects of hypothermia, including mild hypothermia, and the short therapeutic time window of moderate hypothermia. If these observations are true, successful clinical translation of induced hypothermia may prove to be more difficult than anticipated to achieve. For example, we demonstrated that even intraischemic mild hypothermia (33°C) induced before ischemic onset failed to reduce infarct size in a focal ischemia model with permanent distal MCA occlusion and partial reperfusion upon bilateral CCA release. This model may be more severe than the model of MCA suture occlusion with reperfusion used by most laboratories, but we have no reason to believe it is more severe than strokes in humans. As previously discussed, many stroke patients suffer from permanent cerebral artery occlusion without reperfusion. To achieve protection, even our experimental ischemic models required reducing intraischemic hypothermia to 30°C or prolonging intraischemic mild hypothermia beyond CCA release. However, applying intraischemic hypothermia before stroke onset in clinical trials is nearly impossible, and inducing hypothermia in stroke patients beyond 33°C to 30°C is very difficult. Clinical trials often use mild rather than moderate hypothermia, and it takes significantly longer to reach the target temperature compared to experimental stroke in animal models.

re stroke onset in clinical trials is nearly impossible, and inducing hypothermia in stroke patients beyond 33°C to 30°C is very difficult. Clinical trials often use mild rather than moderate hypothermia, and it takes significantly longer to reach the target temperature compared to experimental stroke in animal models. Nevertheless, as we reviewed previously [6], other groups have shown that intraischemic mild hypothermia elicits protection even in permanent MCA occlusion models, in contrast to our recent studies. Our negative findings may simply reflect our specific setting and use of a unique model. Second, the therapeutic time window for moderate hypothermia is extremely narrow after stroke onset, even in the 1-hour transient focal ischemic model. To achieve protection, 3 hours of moderate hypothermia must be induced as early as 45 minutes after stroke onset; a 30-minute delay rendered the moderate hypothermia ineffective. Again, it is highly unlikely that most stroke patients can receive hypothermic treatment within 1 hour of stroke onset. In most clinical studies, mild-to-moderate hypothermia was initiated as late as 5 to 6 hours after stroke, and one to several hours were required to reach target temperatures [37, 38]. In addition, patients may not have reperfusion, or if there is reperfusion, it may occur at a very late stage.

1 hour of stroke onset. In most clinical studies, mild-to-moderate hypothermia was initiated as late as 5 to 6 hours after stroke, and one to several hours were required to reach target temperatures [37, 38]. In addition, patients may not have reperfusion, or if there is reperfusion, it may occur at a very late stage. Our studies on the underlying protective mechanisms may also offer some alternative clues or applications for clinical trials. For instance, we demonstrated that hypothermia reduces infarct size by preserving Akt activity and PTEN phosphorylation and by inhibiting ROS activity. If possible, pharmacological agents may be developed that improve Akt activity while inhibiting PTEN activity, or attenuating ROS production, and such pharmacological agents may be used in combination with induced hypothermia. In summary, despite confounding issues, laboratory studies have provided strong rationale for clinical application of hypothermia for acute stroke treatment. In clinical settings, a number of crucial variables need to be considered, including the onset time of hypothermia, its depth, and whether the strokes studied include reperfusion. Early reperfusion and rapid hypothermia initiation should be used to achieve maximal protection. Acknowledgments The authors wish to thank Ms. Cindy H. Samos for manuscript assistance. This study was supported by R01NS 064136 (H. Zhao) and R21057750 (H. Zhao), NINDS Grants R01 NS27292 (G. Steinberg).

In summary, despite confounding issues, laboratory studies have provided strong rationale for clinical application of hypothermia for acute stroke treatment. In clinical settings, a number of crucial variables need to be considered, including the onset time of hypothermia, its depth, and whether the strokes studied include reperfusion. Early reperfusion and rapid hypothermia initiation should be used to achieve maximal protection. Acknowledgments The authors wish to thank Ms. Cindy H. Samos for manuscript assistance. This study was supported by R01NS 064136 (H. Zhao) and R21057750 (H. Zhao), NINDS Grants R01 NS27292 (G. Steinberg). Figure 1 (Revised from [16]). Intraischemic moderate hypothermia (30°C) reduces infarct size in a focal ischemia with partial reperfusion. Focal ischemia was induced by 1 h of bilateral CCA occlusion and permanent dMCAo. Body core temperature was lowered to 30°C 10 min before stroke onset by spraying 70% alcohol on the rat body. (a) The upper panel shows representative infarcts stained with cresyl violet from rats euthanized 2 d after stroke. The pale area with asterisks represents the infarct region. Normothermic ischemia damaged the cortex ipsilateral to the occluded MCA, whereas hypothermia spared all or most of the injured cortex. Only a small lesion was observed in the presented section from a hypothermic rat. The bar graphs represent statistical analysis of infarct size 2 d after stroke. Two-way ANOVA (two factors, temperature and brain section level) was used to compare the effect of temperature on the infarct size at each level (data not shown) and on the mean of all 4 levels. Hypothermia (n = 7) reduced the mean infarct size by 80% compared with normothermia (n = 7; P = 0.001). (b) The upper panel shows representative sections stained with cresyl violet from animals surviving 2 months after stroke. Most of the cortex in the infracted hemisphere was lost in normothermic but not hypothermic rats. The lower panel of bar graphs shows infarct size 60 d after stroke. Hypothermia (n = 9) reduced infarct size 60 d after stroke compared with normothermia (n = 8; P = 0.001). # versus 37°C, P < 0.001.

2 months after stroke. Most of the cortex in the infracted hemisphere was lost in normothermic but not hypothermic rats. The lower panel of bar graphs shows infarct size 60 d after stroke. Hypothermia (n = 9) reduced infarct size 60 d after stroke compared with normothermia (n = 8; P = 0.001). # versus 37°C, P < 0.001. Figure 2 Diagram showing the major cascades that occur after stroke reviewed in this paper. AD: anoxic depolarization; AIF: apoptosis-inducing factor; BBB: blood brain barrier; CBF: cerebral blood flow; cyto c: cytochrome c; Fas L: Fas ligand; FKHR: Forkhead homologue in rhabdomyosarcoma; Glu: glutamate; GSK 3 β: glycogen synthase kinase 3β; MMP: matrix metalloprotease; NOS: nitric oxide synthesis; NO: nitric oxide; ONOO−: peroxynitrite; PI3K: phosphoinositide 3-kinase; PIP2: phosphatidyliositol-4,5-bisphosphate; PIP3: phosphatidyliositol-3,4,5-bisphosphate; PKC: protein kinase C; P-Akt: phosphorylated Akt; PTEN: phosphatase and tensin homologue deleted on chromosome 10; P-PDK1: phosphorylated phosphoinositide-dependent protein kinase-1; ROS: reactive oxygen species; RTK: receptor tyrosine kinase; VDCC: voltage-dependent calcium channel.

phatidyliositol-3,4,5-bisphosphate; PKC: protein kinase C; P-Akt: phosphorylated Akt; PTEN: phosphatase and tensin homologue deleted on chromosome 10; P-PDK1: phosphorylated phosphoinositide-dependent protein kinase-1; ROS: reactive oxygen species; RTK: receptor tyrosine kinase; VDCC: voltage-dependent calcium channel. Figure 3 (Revised from [19]) (a) Protocols for surgery and temperature management. Six groups of rats were studied. The distal MCA was occluded permanently. The black portion of the bar represents bilateral CCA occlusion (CCAo) for 2 h, and the gray portion indicates 2 h of temperature management after CCA release (CCAr), including 30°C, 33°C, and 37°C. Rats were allowed to survive for 48 h after stroke. (b) Photographs of representative infarct sections after cerebral ischemia from groups 1, 4, and 6. Permanent distal MCA occlusion plus 2 h of bilateral CCA occlusion caused an infarct in the ipsilateral cortex of the occluded MCA (left, group 1). A coronal section from Level 2 is presented. Four hours of mild hypothermia (center, group 4) mildly decreased infarct size. When the temperature was reduced to 30°C robust protection was observed (right, group 6). (c) Bar graph showing that hypothermia reduces infarct size after stroke only under certain conditions. A mean infarct size for each group was calculated as the sum of all 4 levels for each animal divided by the number of animals in each group. The infarct size did not differ among groups 1 through 3. However, the infarct in group 4 was reduced about 22% relative to group 1. When the temperature was decreased to 30°C (group 5) robust protection was observed; an additional 2 h of hypothermia in group 6 did not further reduce infarct size.

nimals in each group. The infarct size did not differ among groups 1 through 3. However, the infarct in group 4 was reduced about 22% relative to group 1. When the temperature was decreased to 30°C (group 5) robust protection was observed; an additional 2 h of hypothermia in group 6 did not further reduce infarct size. Figure 4 (revised from [18]) Limited therapeutic time windows for post-ischemic moderate hypothermia in a focal ischemia with complete reperfusion. (a) A diagram for experimental procedures comparing the protection of hypothermia. Rats were divided into 4 groups. Group 1, normothermia: body temperature was maintained at 37°C throughout the experiment. Group 2, intraischemic hypothermia: hypothermia was induced at ischemic onset and maintained for 3 h. Group 3, early hypothermia: body temperature was adjusted to 30°C 15 min before reperfusion and maintained for 3 h. Group 4, delayed hypothermia: body temperature was adjusted to 30°C 15 min after reperfusion and maintained for 3 h. (b) The upper panel shows representative infarcts stained by TTC. White areas are the infarct regions. The lower panel shows quantitation of infarct volumes. Values are mean ± S.E.M. (n = 8 per each group). ***P < 0.0001, versus normothermia.

1. Introduction Despite advances in magnetic resonance imaging, noncontrast computed tomography (NCCT) remains the most commonly used imaging modality in acute stroke. Early ischemic changes on NCCT, particularly those in the territory of the middle cerebral artery (MCA), have diagnostic and prognostic implications in patients receiving thrombolytic therapy [1, 2]. However, these changes are often subtle and as such may be of variable reliability [3–6]. The hyperdense middle cerebral artery sign (HMCAS) is a well-established marker of intraluminal MCA thrombus and was amongst the first NCCT changes described in acute ischemic stroke [7]. The presence of a HMCAS is associated with severe initial neurological deficits and poor outcomes following thrombolytic therapy with intravenous recombinant tissue plasminogen activator (rtPA) [8–10]. The persistence of a HMCAS following thrombolysis is also associated with a poor functional outcome [11]. More recently, the hyperdense internal carotid artery sign (HICAS) has been suggested as a common marker of terminal internal carotid artery thrombus [12]. The HICAS has been described as a hyperdensity in the distal segment of the internal carotid artery (ICA) seen on NCCT, which is indicative of thrombus within the supraclinoid segment of the distal ICA. In a previous study of 71 patients receiving intravenous and/or intra-arterial rtPA, a HICAS was present in nearly a quarter of patients and was associated with both a severe initial neurological deficit and a poor outcome [12].

on NCCT, which is indicative of thrombus within the supraclinoid segment of the distal ICA. In a previous study of 71 patients receiving intravenous and/or intra-arterial rtPA, a HICAS was present in nearly a quarter of patients and was associated with both a severe initial neurological deficit and a poor outcome [12]. We aimed to further investigate the prevalence of the HICAS in a larger, unselected cohort of patients undergoing intravenous thrombolysis. We also aimed to investigate the association of the HICAS with initial severity of neurological deficit and its role as prognostic marker in patients receiving intravenous rtPA. 2. Subjects and Methods Between December 2007 and May 2010, 123 consecutive patients with acute ischemic stroke were treated with intravenous rtPA at Aintree Stroke Centre. Demographic information, stroke risk factors, baseline neurological deficit, and functional outcome at discharge were documented prospectively. For the present study, 120 patients were included. 3 patients were excluded from analysis as they were initially imaged at other centres and as such their prethrombolysis NCCTs were not available for review. All patients received thrombolysis with intravenous rtPA 0.9 milligrams/kg administered in line with standard thrombolysis protocols. 2.1. Analysis of Radiological Data Each patient had a pretreatment NCCT scan and a second scan at 24 hours following thrombolysis. 30 patients were imaged with a single slice CT scanner, with a section thickness of 4 mm through the posterior fossa and 8 mm for the cerebral hemispheres.

2. Subjects and Methods Between December 2007 and May 2010, 123 consecutive patients with acute ischemic stroke were treated with intravenous rtPA at Aintree Stroke Centre. Demographic information, stroke risk factors, baseline neurological deficit, and functional outcome at discharge were documented prospectively. For the present study, 120 patients were included. 3 patients were excluded from analysis as they were initially imaged at other centres and as such their prethrombolysis NCCTs were not available for review. All patients received thrombolysis with intravenous rtPA 0.9 milligrams/kg administered in line with standard thrombolysis protocols. 2.1. Analysis of Radiological Data Each patient had a pretreatment NCCT scan and a second scan at 24 hours following thrombolysis. 30 patients were imaged with a single slice CT scanner, with a section thickness of 4 mm through the posterior fossa and 8 mm for the cerebral hemispheres. The remaining 90 patients were imaged by 4- and 16-slice scanners with a section thickness of 2.5 mm through the posterior fossa and 5 mm for the cerebral hemispheres. All 120 NCCT scans were reviewed retrospectively and independently by a radiologist (SB) and a stroke fellow (PF). Both investigators were blinded to all clinical information and inspected the prethrombolysis NCCT scans for the presence of a HICAS and/or a HMCAS. The HMCAS was defined as “an MCA denser than its contralateral counterpart” [13].

eviewed retrospectively and independently by a radiologist (SB) and a stroke fellow (PF). Both investigators were blinded to all clinical information and inspected the prethrombolysis NCCT scans for the presence of a HICAS and/or a HMCAS. The HMCAS was defined as “an MCA denser than its contralateral counterpart” [13]. The HICAS was defined as “A hyperdensity of the supraclinoid part of the ICA observed in the prepontine or premesencephalic cistern where the vessels form the Circle of Willis.” A HICAS was deemed to be present if the distal ICA was denser than its contralateral counterpart [12] (Figure 1). Both signs were rated as either present or absent. Disagreements were settled by a third, more experienced investigator (RD), who was also blinded to all clinical data. 2.2. Clinical Assessment Severity of neurological deficits at baseline and 24 hours after thrombolysis were assessed prospectively by using the National Institutes of Health Stroke Scale (NIHSS) conducted by clinicians certified in NIHSS scoring. Patients with NIHSS ≥10 at 24 hours were considered to have severe early postthrombolysis neurological deficits. Barthel scoring was undertaken at discharge as is routine practice in our unit. Poor outcome following thrombolysis was defined as a discharge Barthel score <15 or inpatient death. Haemorrhagic conversion following thrombolysis was defined as any intracranial haemorrhage on NCCT 24 hours after thrombolysis.

l deficits. Barthel scoring was undertaken at discharge as is routine practice in our unit. Poor outcome following thrombolysis was defined as a discharge Barthel score <15 or inpatient death. Haemorrhagic conversion following thrombolysis was defined as any intracranial haemorrhage on NCCT 24 hours after thrombolysis. 2.3. Statistical Analysis Data was analysed in GraphPad Prism 4.0 (GraphPad Software, San Diego, Calif, USA). The D'Agostino-Pearson omnibus test was used to test continuous data for normality. Paired/unpaired t-tests were used to compare normally distributed continuous variables with Mann-Whitney U tests used to compare nonparametrically distributed continuous data. Fisher's exact test was used to analyse categorical variables. Interobserver agreement for the HICAS and the HMCAS was assessed using Cohen's Kappa. Statistical significance was set at 0.05. 2.4. Ethical Review The project protocol was reviewed by the chair of the local Research Ethics Committee. 3. Results 120 patients were assessed. A HICAS was present in 3 patients (2.5%) and absent in 117 (97.5%). Table 1 demonstrates baseline characteristics and outcomes of patients with a HICAS and without a HICAS. In the 3 patients displaying a HICAS the mean maximum density of the affected supraclinoid ICA measured in the Hounsfield units was significantly higher than on the unaffected side. Mean maximum density of the hyperdense ICA 56.3 HU (95% CI 46.3 to 66.4), mean maximum density of the ICA on the unaffected side 28.3 HU (95% CI 26.9 to 29.7), P = 0.0067.

CAS the mean maximum density of the affected supraclinoid ICA measured in the Hounsfield units was significantly higher than on the unaffected side. Mean maximum density of the hyperdense ICA 56.3 HU (95% CI 46.3 to 66.4), mean maximum density of the ICA on the unaffected side 28.3 HU (95% CI 26.9 to 29.7), P = 0.0067. A HMCAS was present in 22 patients (18.3%). Patients with a HMCAS showed a nonsignificant trend towards displaying a HICAS (9.1% versus 1%), odds ratio (OR) 9.7 (95% CI 0.834 to 12.3), P = 0.086. In the 2 patients who displayed both HICAS and HMCAS, a continuous hyperdensity representing clot was seen between the terminal ICA and MCA. Table 2 demonstrates baseline characteristics and outcomes of patients with a HMCAS and without a HMCAS. In the patients displaying a HMCAS, the mean maximum density of the affected MCA measured in the Hounsfield units was significantly higher than on the unaffected side. Mean maximum density of the hyperdense MCA 46.3 HU (95% CI 44.0 to 48.6), mean maximum density of the MCA on the unaffected side 35.4 HU (95% CI 32.2 to 38.5), P < 0.0001. 3.1. Baseline Neurological Deficit and Haemorrhagic Conversion Patients with a HICAS had a significantly higher baseline mean NIHSS score (Mean NIHSS 21.0 95% CI 16.7 to 25.3) compared to those without this sign (Mean NIHSS 12.8 95% CI 11.7 to 13.9, P = 0.019). The presence of a HMCAS was also associated with significantly more severe prethrombolysis neurological deficits (Mean NIHSS 16.5 95% CI 14.3 to 18.7) when compared to patients without a HMCAS (Mean NIHSS 12.3 95% CI 11.1 to 13.4, P = 0.0025).

3.1. Baseline Neurological Deficit and Haemorrhagic Conversion Patients with a HICAS had a significantly higher baseline mean NIHSS score (Mean NIHSS 21.0 95% CI 16.7 to 25.3) compared to those without this sign (Mean NIHSS 12.8 95% CI 11.7 to 13.9, P = 0.019). The presence of a HMCAS was also associated with significantly more severe prethrombolysis neurological deficits (Mean NIHSS 16.5 95% CI 14.3 to 18.7) when compared to patients without a HMCAS (Mean NIHSS 12.3 95% CI 11.1 to 13.4, P = 0.0025). Haemorrhagic conversion occurred in 5% of patients, an incidence comparable to that seen in the SITS-MOST monitoring study [14]. No significant associations were observed between the presence of a HICAS (OR 2.45 95% CI 0.11 to 52.7, P = 1.0) or a HMCAS (OR 5.0 95% CI 0.94 to 26.7, P = 0.074) and intracranial haemorrhage following thrombolysis. 3.2. Short-Term and Long-Term Outcomes Following Thrombolysis At 24 hours following thrombolysis, severe neurological deficits (NIHSS score ≥10 points) were observed in 2 of 3 patients (66.6%) with a HICAS compared to 44 of 117 patients (37.1%) without a HICAS, OR 3.20 95% CI 0.28 to 36.7, P = 0.559. The presence of a HICAS on prethrombolysis NCCT was not significantly associated with a poor outcome at discharge (Barthel <15 or impatient death). Of the 3 patients with a HICAS, 2 (66%) had a poor outcome, compared with 45 (38.5%) of the 117 patients without HICAS, OR 3.20 95% CI 0.28 to 36.34, P = 0.560. The presence of a HICAS was not significantly associated with inpatient death (P = 1.0).

The presence of a HICAS on prethrombolysis NCCT was not significantly associated with a poor outcome at discharge (Barthel <15 or impatient death). Of the 3 patients with a HICAS, 2 (66%) had a poor outcome, compared with 45 (38.5%) of the 117 patients without HICAS, OR 3.20 95% CI 0.28 to 36.34, P = 0.560. The presence of a HICAS was not significantly associated with inpatient death (P = 1.0). The presence of a HMCAS on initial prethrombolysis NCCT was significantly associated with both a severe neurological deficit at 24 hours following thrombolysis and a poor outcome at discharge. A 24-hour NIHSS score of ≥10 was seen in 20 of 22 patients (90.1%) of patients with a HMCAS and 32 of 98 (32.7%) of those without a HMCAS, OR 20.6 95% CI 4.54 to 93.7, P < 0.0001. A poor outcome at discharge was observed in 14 of 22 (63.6%) patients with a HMCAS compared with 33 of 98 (33.7%) without a HMCAS, OR 3.45 95% CI 1.31 to 9.04, P = 0.015. The presence of a HMCAS was not significantly associated with inpatient death (P = 0.750). 3.3. Interobserver Agreement for the Hyperdense ICA and Hyperdense MCA Signs Interobserver agreement was excellent for the HMCAS-Cohen's Kappa 0.973 (95% CI 0.919 to 0.999) and fair for the HICAS-Cohen's Kappa 0.275 (95% CI 0.0 to 0.704).

The presence of a HMCAS was not significantly associated with inpatient death (P = 0.750). 3.3. Interobserver Agreement for the Hyperdense ICA and Hyperdense MCA Signs Interobserver agreement was excellent for the HMCAS-Cohen's Kappa 0.973 (95% CI 0.919 to 0.999) and fair for the HICAS-Cohen's Kappa 0.275 (95% CI 0.0 to 0.704). 4. Discussion In the first hours following acute ischaemic stroke, NCCT frequently demonstrates early parenchymal changes which may be preceded by hyperdense artery signs suggestive of intraluminal thrombus [15]. These early ischemic changes on NCCT are of prognostic significance in predicting functional outcome before thrombolysis is administered [1, 2]. Our study aimed to further investigate the prevalence and prognostic significance of the HICAS. Two previous studies have described distal ICA thrombus resulting in a hyperdense appearance of the terminal ICA on NCCT [12, 16]. Our study contains the largest series of patients to date and is the first study to include an unselected series of patients receiving intravenous thrombolysis with rtPA. A HICAS was present in 2.5% of the patients included in our study, this did not differ significantly from the 5.8% prevalence reported in the first paper to describe the HICAS [16] but does represent a significantly lower prevalence than the 24% reported in patients receiving thrombolysis by Ozdemir et al. (Fisher's exact test; P < 0.0001) [12].

of the patients included in our study, this did not differ significantly from the 5.8% prevalence reported in the first paper to describe the HICAS [16] but does represent a significantly lower prevalence than the 24% reported in patients receiving thrombolysis by Ozdemir et al. (Fisher's exact test; P < 0.0001) [12]. The lower prevalence of the HICAS observed in our series of patients undergoing thrombolysis may in part be explained by the NCCT imaging protocols employed. A quarter of the NCCT scans reviewed in our study used a thicker 8 mm slice rather than the 5 mm slices used by Ozdemir et al. [12]. A previous study has demonstrated that a reduction in slice thickness is associated with increased sensitivity in identifying hyperdense intracerebral arteries [16], as such the thicker slices used in some of the scans included in our study may have resulted in false negatives. However, even if these thicker 8 mm slice NCCT are excluded from analysis, the proportion of patients with a HICAS remains significantly lower than that described by Ozdemir et al. [12] and comparable to that observed in the first study to describe the HICAS [16]. Our data suggests the prevalence of the HICAS seen on standard prethrombolysis NCCT slices may be lower than that previously reported.

The lower prevalence of the HICAS observed in our series of patients undergoing thrombolysis may in part be explained by the NCCT imaging protocols employed. A quarter of the NCCT scans reviewed in our study used a thicker 8 mm slice rather than the 5 mm slices used by Ozdemir et al. [12]. A previous study has demonstrated that a reduction in slice thickness is associated with increased sensitivity in identifying hyperdense intracerebral arteries [16], as such the thicker slices used in some of the scans included in our study may have resulted in false negatives. However, even if these thicker 8 mm slice NCCT are excluded from analysis, the proportion of patients with a HICAS remains significantly lower than that described by Ozdemir et al. [12] and comparable to that observed in the first study to describe the HICAS [16]. Our data suggests the prevalence of the HICAS seen on standard prethrombolysis NCCT slices may be lower than that previously reported. Occlusion of the intracranial ICA is a potentially catastrophic event. When the distal intracranial ICA is acutely occluded patients are frequently refractory to intravenous thrombolysis and the outcome is often poor [17–19]. The significantly higher prethrombolysis NIHSS scores seen in patients with a HICAS in our study reflect the severe neurological deficits seen with terminal ICA thrombotic occlusion and are comparable to the findings of a previous study [12].

refractory to intravenous thrombolysis and the outcome is often poor [17–19]. The significantly higher prethrombolysis NIHSS scores seen in patients with a HICAS in our study reflect the severe neurological deficits seen with terminal ICA thrombotic occlusion and are comparable to the findings of a previous study [12]. Unlike the previous study undertaken by Ozdemir et al., we failed to demonstrate a significant association between the presence of a HICAS and severe early post thrombolysis neurological deficits or long term outcomes following thrombolysis [12]. Patients with a HICAS did display nonsignificant trends towards poor outcomes. Due to the low prevalence of the HICAS in our sample, our study lacked sufficient power to accurately determine the prognostic significance of the HICAS and the failure to demonstrate an association between the HICAS and poor outcomes in the present study may be attributable to a type II error. While 2 of the 3 patients with a HICAS failed to make any clinically significant improvement when treated with intravenous rt-PA, 1 of the 3 patients with a HICAS in our study showed an extremely good response to intravenous rt-PA, with an admission NIHSS of 22 decreasing to 8 at 24 hours post thrombolysis. This patients repeat CT scan at 24 hours post thrombolysis demonstrated resolution of the HICAS, suggestive of clot lysis and recanalisation. This patients unusually good response to peripheral rt-PA [17–19] may have contributed a suspected type II error.

ssion NIHSS of 22 decreasing to 8 at 24 hours post thrombolysis. This patients repeat CT scan at 24 hours post thrombolysis demonstrated resolution of the HICAS, suggestive of clot lysis and recanalisation. This patients unusually good response to peripheral rt-PA [17–19] may have contributed a suspected type II error. Patients with a HMCAS displayed significantly more severe baseline neurological deficits than those patients without this sign. The HMCAS was also associated with poor neurological and functional outcomes at 24 hours and at discharge. The significant associations observed between the HMCAS and both severe baseline neurological deficits and poor outcomes are in keeping with the findings of several previous studies [8–10]. Interobserver agreement was excellent for the HMCAS. Interobserver agreement for the HICAS was only fair; however, this result should be viewed with caution given the low prevalence of the HICAS in our study population, as reflected by the wide confidence intervals seen for Cohen's kappa. Our study has several limitations. While all clinical data including risk factors, demographics, and outcome following thrombolytic therapy were collected prospectively, prethrombolysis NCCTs were studied retrospectively.

Interobserver agreement was excellent for the HMCAS. Interobserver agreement for the HICAS was only fair; however, this result should be viewed with caution given the low prevalence of the HICAS in our study population, as reflected by the wide confidence intervals seen for Cohen's kappa. Our study has several limitations. While all clinical data including risk factors, demographics, and outcome following thrombolytic therapy were collected prospectively, prethrombolysis NCCTs were studied retrospectively. None of the patients included in our study underwent CT angiography prior to intravenous thrombolysis. As such, we are unable to validate the sensitivity and specificity of the HICAS in detecting terminal ICA thrombus against the gold standard of CT angiogram. Validation of presumed thrombotic occlusion against angiographic data may be of particular importance when investigating hyperdense artery signs as vessel calcification resulting in a false-positive HMCAS is well recognised [10, 15, 20]. We do, however note that both studies which have previously described the HICAS found no false positives when the HICAS was validated as a marker of terminal ICA thrombus using CT angiography [12, 16]. This suggests that the HICAS is less prone to false positives attributable to calcified vessels than the HMCAS.

, 20]. We do, however note that both studies which have previously described the HICAS found no false positives when the HICAS was validated as a marker of terminal ICA thrombus using CT angiography [12, 16]. This suggests that the HICAS is less prone to false positives attributable to calcified vessels than the HMCAS. A quarter of the patients included in our study were imaged using 8 mm through the cerebral hemispheres, with the remainder imaged using 5 mm sections. A previous study in nonthrombolysed acute stroke patients has shown that reduction of section thickness from 5 mm to 1 mm improved the sensitivity of detecting intraluminal ICA thrombus from 33% to 100% without reducing specificity. This may account for the low prevalence of the HICAS in our study and the low sensitivity reported in previous studies using 5 mm slices [12, 16]. Future studies using prethrombolysis thin slice NCCT through the course of the ICA as it traverses the prepontine, and premesencephalic cisterns should allow improved sensitivity in detecting distal ICA thrombus and may improve the predictive power of the HICAS as a prognostic marker.

tudies using 5 mm slices [12, 16]. Future studies using prethrombolysis thin slice NCCT through the course of the ICA as it traverses the prepontine, and premesencephalic cisterns should allow improved sensitivity in detecting distal ICA thrombus and may improve the predictive power of the HICAS as a prognostic marker. In summary, this study suggests the prevalence of the HICAS on standard prethrombolysis NCCT may be lower than that previously reported. The presence of a HICAS was associated with significantly more severe initial neurological deficits in keeping with acute terminal ICA occlusion. Larger studies, ideally using thin slice CT through the course of the terminal ICA, are required to further investigate the prevalence and prognostic significance of the HICAS in stroke patients receiving thrombolytic therapy. Conflict of Interests All authors report no conflict of interests to declare. Figure 1 (a) NCCT demonstrating HICAS (arrow). (b) CT Carotid Angiogram demonstrating corresponding terminal carotid occlusion (arrow). (c) NCCT demonstrating HMCAS (arrow). Table 1 Baseline characteristics and outcomes of patients with or without HICAS present. Patients with HICAS Patients without HICAS n = 3 n = 117 P

Conflict of Interests All authors report no conflict of interests to declare. Figure 1 (a) NCCT demonstrating HICAS (arrow). (b) CT Carotid Angiogram demonstrating corresponding terminal carotid occlusion (arrow). (c) NCCT demonstrating HMCAS (arrow). Table 1 Baseline characteristics and outcomes of patients with or without HICAS present. Patients with HICAS Patients without HICAS n = 3 n = 117 P Age 72.0 (65.4–78.6) 69.7 (67.5–71.9) 0.742 Female 2 (66.6%) 51 (43.6%) 1.0 Baseline NIHSS 21.0 (16.7–25.3) 12.8 (11.7–13.9) 0.019 Time to rtPA (minutes) 176 (37–315) 162 (151–173) 0.626 Atrial fibrillation 1 (33.3%) 27 (23.1%) 0.553 Hyperlipidaemia 2 (66.6%) 42 (35.9%) 0.554 Angina 0 (0%) 20 (17.1%) 1.0 Peripheral vascular disease 0 (0%) 6 (5.1%) 1.0 Hypertension 2 (66.6%) 72 (61.5%) 1.0 Myocardial infarction 0 (0%) 11 (9.4%) 1.0 Diabetes mellitus 0 (0%) 14 (11.9%) 1.0 Current smoker 1 (33.3%) 24 (20.5%) 0.507 Haemorrhagic conversion 0 (0%) 6 (5.1%) 1.0 24 hour NIHSS ≥10 2 (66.6%) 44 (37.1%) 0.559 inpatient death 0 (0%) 19 (16.2%) 1.0 Poor outcome 2 (66.6%) 45 (38.5%) 0.560 Values are mean (95% confidence interval) or n (%) as appropriate. Poor outcome defined as discharge Barthel score <15 or inpatient death. Table 2 Baseline characteristics and outcomes of patients with or without HMCAS present. Patients with HMCAS Patients without HMCAS n = 22 n = 98 P

Age 72.0 (65.4–78.6) 69.7 (67.5–71.9) 0.742 Female 2 (66.6%) 51 (43.6%) 1.0 Baseline NIHSS 21.0 (16.7–25.3) 12.8 (11.7–13.9) 0.019 Time to rtPA (minutes) 176 (37–315) 162 (151–173) 0.626 Atrial fibrillation 1 (33.3%) 27 (23.1%) 0.553 Hyperlipidaemia 2 (66.6%) 42 (35.9%) 0.554 Angina 0 (0%) 20 (17.1%) 1.0 Peripheral vascular disease 0 (0%) 6 (5.1%) 1.0 Hypertension 2 (66.6%) 72 (61.5%) 1.0 Myocardial infarction 0 (0%) 11 (9.4%) 1.0 Diabetes mellitus 0 (0%) 14 (11.9%) 1.0 Current smoker 1 (33.3%) 24 (20.5%) 0.507 Haemorrhagic conversion 0 (0%) 6 (5.1%) 1.0 24 hour NIHSS ≥10 2 (66.6%) 44 (37.1%) 0.559 inpatient death 0 (0%) 19 (16.2%) 1.0 Poor outcome 2 (66.6%) 45 (38.5%) 0.560 Values are mean (95% confidence interval) or n (%) as appropriate. Poor outcome defined as discharge Barthel score <15 or inpatient death. Table 2 Baseline characteristics and outcomes of patients with or without HMCAS present. Patients with HMCAS Patients without HMCAS n = 22 n = 98 P Age 69.9 (65.1–74.9) 69.7 (67.3–72.1) 0.935 Female 12 (54.5%) 41 (41.8%) 0.344 Baseline NIHSS 16.5 (14.3–18.7) 12.8 (11.1–13.4) 0.0025 Time to rtPA (minutes) 157 (135–180) 165 (153–178) 0.577 HICAS present 2 (9.1%) 1 (1.0%) 0.086 Atrial fibrillation 6 (27.3%) 22 (22.4%) 0.590 Hyperlipidaemia 9 (40.9%) 35 (35.7%) 0.635 Angina 5 (22.7%) 15 (15.3%) 0.526 Peripheral vascular disease 1 (4.5%) 5 (5.1%) 1.0 Hypertension 13 (59.1%) 61 (62.2%) 0.812 Myocardial infarction 1 (4.5%) 10 (10.2%) 0.687 Diabetes mellitus 3 (13.6%) 11 (11.2%) 0.720 Current smoker 6 (27.3%) 19 (19.4%) 0.397 Haemorrhagic Conversion 3 (13.6%) 3 (3.1%) 0.074 24 hour NIHSS ≥10 20 (90.1%) 32 (32.7%) <0.0001 Inpatient death 4 (18.1%) 15 (15.3%) 0.750 Poor outcome 14 (63.6%) 33 (33.7%) 0.015 Values are mean (95% confidence interval) or n (%) as appropriate.

tus 3 (13.6%) 11 (11.2%) 0.720 Current smoker 6 (27.3%) 19 (19.4%) 0.397 Haemorrhagic Conversion 3 (13.6%) 3 (3.1%) 0.074 24 hour NIHSS ≥10 20 (90.1%) 32 (32.7%) <0.0001 Inpatient death 4 (18.1%) 15 (15.3%) 0.750 Poor outcome 14 (63.6%) 33 (33.7%) 0.015 Values are mean (95% confidence interval) or n (%) as appropriate. Poor outcome defined as discharge Barthel score <15 or inpatient death.

1. Introduction Etiology of acute ischemic stroke (AIS) is known to significantly influence management, prognosis, and risk of recurrence. Certain stroke subtypes are associated with higher stroke severity at the time of presentation, which may account for the higher mortality seen. In 1993 the TOAST (Trail of ORG 10172 in Acute Stroke Treatment) investigators described a classification of AIS based on etiology, which is now the most commonly used etiological classification [1]. Comparison of clinical characteristics, functional outcomes, and mortality rates for specific ischemic stroke mechanisms may allow clinicians to identify those patients who are at higher risk and to evaluate treatment strategies more definitely. We conducted an observational study of all patients who presented to the emergency department (ED) with AIS and determined if ischemic stroke subtype (ISS) influences mortality even after correcting for stroke severity on initial presentation. 2. Methods This study was conducted at a tertiary care academic medical center, with an annual ED census of approximately 75,000 visits. The medical records of all patients with a discharge diagnosis of stroke or transient ischemic attack (TIA) or diagnoses which could be mistaken for stroke or TIA were screened (ICD-CM codes 433-437) between December 2001 and March 2004 to determine if the case met the criteria for diagnosis of ischemic stroke. This study was approved by the authors' institutional review board.

is of stroke or transient ischemic attack (TIA) or diagnoses which could be mistaken for stroke or TIA were screened (ICD-CM codes 433-437) between December 2001 and March 2004 to determine if the case met the criteria for diagnosis of ischemic stroke. This study was approved by the authors' institutional review board. The medical records for all patients were reviewed, and details of clinical history, demographic information, risk factor profile, neurological examination, brain imaging studies, and other diagnostic studies were abstracted. Followup was updated for the final data analysis using the date of the last service or dismissal available from registration databases. In addition, dates and causes of death were ascertained from the State of Minnesota Electronic Death Certificate Data; autopsy reports were also reviewed where available. The National Institutes of Health Stroke Scale (NIHSS) was calculated by physicians (LGS, RMG) certified in the NIHSS, based on previously validated methods [2, 3]. The scoring was derived from documentation of the neurological examination performed by the neurologist on call at the time of ED presentation. Stroke subtype was assigned to every patient by two independent physicians based on review of clinical history, neurological examination, and diagnostic studies in accordance with criteria outlined in the TOAST study. The TOAST classification includes the following categories: (1) large artery (LAD), (2) cardioembolic (CE), (3) small vessel (SAD), (4) other determined cause, and (5) cryptogenic.

ed on review of clinical history, neurological examination, and diagnostic studies in accordance with criteria outlined in the TOAST study. The TOAST classification includes the following categories: (1) large artery (LAD), (2) cardioembolic (CE), (3) small vessel (SAD), (4) other determined cause, and (5) cryptogenic. Patients defined as having large-artery atherosclerosis had imaging showing greater than 50% stenosis or occlusion of a major brain artery or branch cortical artery supplying the region of brain affected. A cardiac source of stroke was assigned to patients with at least one of the following predisposing factors (1) prosthetic valve, (2) significant mitral stenosis, (3) atrial fibrillation or flutter, (4) left atrial or ventricular thrombus, (5) myocardial infarction <6 months prior to stroke, (6) dilated cardiomyopathy, (7) akinetic/hypokinetic left ventricular segment, (8) atrial myxoma, (9) infective endocarditis, (10) sick-sinus syndrome, and (11) congestive heart failure, in the absence of significant ipsilateral arterial stenosis. Patients with small vessel occlusion had a clinical history of a classical lacunar syndrome with either no evidence of infarction on neuroimaging or less than 1.5 cm infarct in the corresponding subcortical or brainstem region. Patients with evidence of stroke of other more unusual etiologies such as vasculitis or hypercoagulable states were classified as stroke of other determined cause. Patients who did not meet the criteria for any of the above categories were defined as having cryptogenic stroke. This category was comprised of 3 distinct groups of patients (a) those that had more than one cause identified, (b) no cause identified despite extensive investigations, and (c) insufficient information obtained to identify a cause.

the criteria for any of the above categories were defined as having cryptogenic stroke. This category was comprised of 3 distinct groups of patients (a) those that had more than one cause identified, (b) no cause identified despite extensive investigations, and (c) insufficient information obtained to identify a cause. 3. Statistical Methods The primary outcome variable was mortality within 1 year, as estimated using the Kaplan-Meier method. For patients who died within 1 year, the duration of followup was calculated from the date of ED presentation to the date of death. The duration of followup for all remaining patients was censored at the date of last followup if within 1 year or at 366 days. Associations with survival were evaluated using indicator variables in Cox proportional hazards models. The associations were evaluated with and without adjusting for sex, age, and NIHSS (ln (score + 0.5) of NIHSS which was used in the model) and were summarized by calculating risk ratios (RRs) and 95% confidence intervals (CIs). All calculated P values were two sided, and P values less than 0.05 were considered statistically significant. Statistical analyses were performed using the SAS software package (SAS Institute, Inc, Cary, NC).

as used in the model) and were summarized by calculating risk ratios (RRs) and 95% confidence intervals (CIs). All calculated P values were two sided, and P values less than 0.05 were considered statistically significant. Statistical analyses were performed using the SAS software package (SAS Institute, Inc, Cary, NC). 4. Results The initial study population consisted of all 681 consecutive patients who presented to the ED with acute ischemic stroke. Among these 681 patients, 19 denied research authorization and were therefore excluded from further study in accordance with Minnesota Statute 144.335. For purposes of obtaining recent and consistent followup, this sample was further limited to the 500 patients who resided in the local county or the surrounding nine-county area at the time of the ED visit. Hence, during the period of study 500 eligible patients were identified. No patients were lost to followup. Two hundred and sixty one (52.2%) were male, and the mean age at presentation was 73.7 years (standard deviation, SD = 14.3, range 18 to 101 years). Magnetic resonance imaging or computed tomography (CT) of the brain was carried out in all patients with 60% of patients having both imaging modalities. Echocardiography was performed in 65% of patients, 92% of which were transesophageal echocardiography. 85% had vascular imaging with 65% of these having CT angiogram or magnetic resonance angiogram.

omputed tomography (CT) of the brain was carried out in all patients with 60% of patients having both imaging modalities. Echocardiography was performed in 65% of patients, 92% of which were transesophageal echocardiography. 85% had vascular imaging with 65% of these having CT angiogram or magnetic resonance angiogram. All 500 patients were assigned a subtype, large artery atherosclerosis 97 (19.4%), cardioembolic 144 (28.8%), small vessel disease 75 (15%), other determined cause 19 (3.8%), and unknown 165 (33%). The unknown category was further subdivided into: more than one cause identified 37 (7.4%), no cause identified 63 (12.6%), and those with insufficient investigations performed to identify a cause 65 (13.0%). Detailed information of the demographic characteristics by ISS is presented in Table 1.

and unknown 165 (33%). The unknown category was further subdivided into: more than one cause identified 37 (7.4%), no cause identified 63 (12.6%), and those with insufficient investigations performed to identify a cause 65 (13.0%). Detailed information of the demographic characteristics by ISS is presented in Table 1. One hundred and sixty patients died: 69 within the first 30 days, 27 within 31–90 days, 29 within 91–365 days, and 35 after 1 year. The estimated survival (±standard error) at 90 days, 180 days, and 1 year was 80.1% ± 1.8%, 77.5% ± 1.9%, and 73.5% ± 2.0%, respectively. Among the patients alive at last followup, the median followup was 1.8 years (interquartile range, 1.1–2.6 years). The lower 90-, 180-, and 360-day survival was seen in cardioembolic strokes (67.1%, 65.5%, and 58.2%, resp.), followed for cryptogenic strokes (78.0%, 75.3%, and 71.1%). Interestingly, when looking into the cryptogenic category, those with insufficient information to assign a stroke subtype had the lowest survival estimate (57.7% at 90 days, 56.1% at 180 days and 51.2% at 1 year). The survival estimates (±standard error) by TOAST category are presented in Table 2 and Figure 1.

71.1%). Interestingly, when looking into the cryptogenic category, those with insufficient information to assign a stroke subtype had the lowest survival estimate (57.7% at 90 days, 56.1% at 180 days and 51.2% at 1 year). The survival estimates (±standard error) by TOAST category are presented in Table 2 and Figure 1. In order to calculate risk ratios (RR) for comparing the survival within the first year across the TOAST categories, we built 3 models to adjust for different factors. These models are summarized in Table 3. Using the SAD group as the reference, patients with LAD, CE, or cryptogenic strokes had significantly poorer survival (Model 1). This association was still observed after adjusting for age and gender (Model 2). However, after adjusting for age and NIHSS (Model 3), only the difference between CE and SAD attained statistical significance, with a RR 3.4 (95% CI 1.2–9.6). Most deaths (60%) in our cohort were attributable to the acute stroke itself, confirming the results of others [4]. Other causes were respiratory distress/pneumonia (15%), cardiovascular (myocardial infarction and congestive heart failure) (7%), and renal failure (7%). We are unable to comment on whether there was a difference in tPA treatment or revascularization between subtypes as we did not have patient-specific data on which patients in this dataset as to who received t-PA by subtype. Overall, the number of patients who received t-PA during the study period was small and thus would likely have little effect on outcomes by subtype.

rence in tPA treatment or revascularization between subtypes as we did not have patient-specific data on which patients in this dataset as to who received t-PA by subtype. Overall, the number of patients who received t-PA during the study period was small and thus would likely have little effect on outcomes by subtype. 5. Discussion Cardioembolic stroke is known to have the worst prognosis amongst ischemic stroke subtypes, and this has been reported in the literature [4, 5]. However, in contrast to the current study, these studies have failed to show CE stroke as an independent predictor of mortality. Bang et al. showed that ischemic stroke subtype was a significant predictor of recurrent stroke after adjusting for potential confounders but did not find that it was an independent predictor of poor prognosis [6]. Sprigg et al. reported in their evaluation of the TAIST (Tinzaparin in Acute Ischemic Stroke Trial) that patients with small vessel occlusion had better outcome as compared to patients with large artery atherosclerosis or cardioembolic stroke [7]. Internationally there seems to be a similar trend. Recently Winter et al. reported from Marburg that patients with cardioembolic stroke had more severe clinical deficits on presentation, and a worse outcome, than the other stroke subtypes [8]. A community-based study from Brazil also reported the highest case fatality rate in strokes of undetermined etiology followed by cardioembolic stroke and large vessel atherothrombosis [9]. Lavados and colleagues from Chile reported a similar higher mortality for cardioembolic stroke when compared to small vessel disease [10].

nity-based study from Brazil also reported the highest case fatality rate in strokes of undetermined etiology followed by cardioembolic stroke and large vessel atherothrombosis [9]. Lavados and colleagues from Chile reported a similar higher mortality for cardioembolic stroke when compared to small vessel disease [10]. We have demonstrated an association between CE stroke and increased mortality, independent of age, gender, and NIHSS. It would appear likely that cardiac and other comorbidities most likely explain this finding. Cardiac conditions that predispose to stroke (such as extensive acute myocardial infarction, chronic myocardial injury with left ventricular aneurysm formation, valvular and nonvalvular atrial fibrillation) are themselves associated with increased mortality. It is not unexpected that such patients would carry a particularly poor prognosis after acute ischemic stroke. Similarly, conditions such as diabetes that may underlie such heart diseases may also increase risk for poor outcome following stroke.

r atrial fibrillation) are themselves associated with increased mortality. It is not unexpected that such patients would carry a particularly poor prognosis after acute ischemic stroke. Similarly, conditions such as diabetes that may underlie such heart diseases may also increase risk for poor outcome following stroke. The findings of this study clearly identify patients presenting with CE stroke as a particularly high risk group. How best to optimize therapy and ameliorate that risk for these patients, however, is uncertain. For example, whether stroke subtype may influence efficacy of established therapies such as thrombolytic therapy is unknown. It is probably reasonable to assume that embolic material in CE stroke is predominantly thrombus laden (rather than composed of plaque debris, as may pertain in aortic and carotid artery disease). If this assumption is indeed correct, then one could hypothesize that patients with CE stroke may be a subgroup that ought to derive greater benefit from timely administration of thrombolysis and may conceivably benefit from thrombolysis over an extended time window beyond the conventional three hours from symptom onset. Greater vigilance in respect of blood pressure control during the acute phase of stroke may be warranted. Heightened awareness of poor prognosis among these patients may also lead to more aggressive treatment of coexisting cardiac disorders.

n extended time window beyond the conventional three hours from symptom onset. Greater vigilance in respect of blood pressure control during the acute phase of stroke may be warranted. Heightened awareness of poor prognosis among these patients may also lead to more aggressive treatment of coexisting cardiac disorders. 6. Conclusion Cardioembolic ischemic stroke subtype determined by TOAST criteria predicts long-term mortality, even after adjusting for age and stroke severity. Further studies to define the precise nature of this increased risk will be required to guide the development of strategies which may improve outcome in this setting. Acknowledgments Dr. L. G. Stead was supported through a Mayo Foundation Emergency Medicine Research Career Development Award during the period of this research. This paper was made possible by Grant no. 1 UL1 RR024150 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and the NIH Roadmap for Medical Research. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Information on NCRR is available at http://www.ncrr.nih.gov/. Information on Reengineering the Clinical Research Enterprise can be obtained from http://nihroadmap.nih.gov/. Figure 1 Survival estimates by TOAST classification. Table 1 Summary of patient characteristics by TOAST classification.

Acknowledgments Dr. L. G. Stead was supported through a Mayo Foundation Emergency Medicine Research Career Development Award during the period of this research. This paper was made possible by Grant no. 1 UL1 RR024150 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and the NIH Roadmap for Medical Research. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Information on NCRR is available at http://www.ncrr.nih.gov/. Information on Reengineering the Clinical Research Enterprise can be obtained from http://nihroadmap.nih.gov/. Figure 1 Survival estimates by TOAST classification. Table 1 Summary of patient characteristics by TOAST classification. Large vessel (N = 97) Cardioembolic (N = 144) Small vessel (N = 75) Other (N = 19) Multiple causes (N = 37) No cause identified (N = 63) Insufficient info (N = 65) Male gender 67 (69.1%) 66 (45.8%) 39 (52%) 10 (52.6%) 21 (56.8%) 32 (50.8%) 26 (40%) Age Mean (SD) 70.7 (12.22) 78.4 (11.81) 72.6 (13.50) 50.7 (19.71) 73.8 (9.90) 66.6 (16.78) 82.3 (8.32) Range 39–91 41–101 44–94 18–82 39–90 25–89 59–98 NIHSS Mean (SD) 7.0 (6.40) 10.1 (9.06) 3.8 (2.71) 5.7 (5.77) 8.1 (8.92) 6.3 (7.14) 13.4 (11.26) Median 4.0 7.0 3.0 5.0 4.0 4.0 9.5 Q1, Q3 3.0, 10.0 3.0, 15.0 2.0, 5.0 1.0, 8.0 2.0, 8.0 1.0, 8.0 4.0, 20.0 Prior MI 14 (14.4%) 16 (11.1%) 11 (14.7%) 1 (5.3%) 13 (35.1%) 8 (12.7%) 9 (13.8%) CHF 7 (7.2%) 46 (31.9%) 6 (8%) 0 (0%) 12 (32.4%) 2 (3.2%) 10 (15.4%) Coronary artery disease 25 (25.8%) 40 (27.8%) 19 (25.3%) 3 (15.8%) 18 (48.6%) 13 (20.6%) 16 (24.6%) Atrial fibrillation 8 (8.2%) 89 (61.8%) 3 (4%) 2 (10.5%) 15 (40.5%) 3 (4.8%) 6 (9.2%) Prior stroke 22 (22.7%) 35 (24.3%) 13 (17.3%) 2 (10.5%) 11 (29.7%) 15 (23.8%) 19 (29.2%) Prior TIA 19 (19.6%) 20 (13.9%) 14 (18.7%) 0 (0%) 8 (21.6%) 9 (14.3%) 12 (18.5%) HTN 75 (77.3%) 110 (76.4%) 61 (81.3%) 8 (42.1%) 32 (86.5%) 48 (76.2%) 51 (78.5%) Hyperlipidemia 58 (59.8%) 56 (38.9%) 43 (57.3%) 3 (15.8%) 18 (48.6%) 31 (49.2%) 23 (35.4%) Diabetes 28 (%) 35 (%) 30 (%) 2 (%) 9 (%) 18 (%) 13 (%) Smoker Never 32 (33%) 79 (54.9%) 44 (58.7%) 10 (52.6%) 20 (54.1%) 23 (36.5%) 30 (46.2%) Active 24 (24.7%) 14 (9.7%) 13 (17.3%) 4 (21.1%) 2 (5.4%) 16 (25.4%) 6 (9.2%) Former 40 (41.2%) 44 (30.6%) 17 (22.7%) 5 (26.3%) 12 (32.4%) 23 (36.5%) 25 (38.5%) Table 2 Survival estimates within the first year.

) 9 (%) 18 (%) 13 (%) Smoker Never 32 (33%) 79 (54.9%) 44 (58.7%) 10 (52.6%) 20 (54.1%) 23 (36.5%) 30 (46.2%) Active 24 (24.7%) 14 (9.7%) 13 (17.3%) 4 (21.1%) 2 (5.4%) 16 (25.4%) 6 (9.2%) Former 40 (41.2%) 44 (30.6%) 17 (22.7%) 5 (26.3%) 12 (32.4%) 23 (36.5%) 25 (38.5%) Table 2 Survival estimates within the first year. TOAST N Events Survival at 90 days (SE) Survival at 180 days (SE) Survival at 360 days (SE) Large artery 97 16 87.1% (0.04) 82.4% (0.04) 82.4% (0.04) Cardioembolic 144 57 67.1% (0.04) 65.5% (0.04) 58.2% (0.04) Small vessels 75 4 98.6% (0.01) 97.1% (0.02) 94.2% (0.03) Other 19 3 89.5% (0.07) 84.2% (0.08) 84.2% (0.08) Cryptogenic 165 45 78.0% (0.03) 75.3% (0.03) 71.1% (0.04) More than one cause 37 7 85.5% (0.06) 82.3% (0.07) 79.2% (0.07) No cause 63 7 94.8% (0.03) 91.2% (0.04) 87.4% (0.05) Insufficient info 65 31 57.7% (0.06) 56.1% (0.06) 51.2% (0.06) Table 3 Risk ratios of mortality. Factors Model 1 Model 2 Model 3 included RR (95% CI) RR (95% CI) RR (95% CI) P value P value P value TOAST LAD 3.5 (1.2–10.4) P = 0.027 4.0 (1.3–11.9) P = 0.014 2.1 (0.7–6.4) P = 0.18 CE 9.4 (3.4–25.9) P < 0.001 7.8 (2.8–21.4) P < 0.001 3.4 (1.2–9.6) P = 0.020 SAD Reference Reference Reference Reference Other 3.1 (0.7–13.9) P = 0.14 9.0 (2.0–41.3) P = 0.005 4.7 (1.0–21.6) P = 0.05 Cryptogenic 18.9 (2.2–17.1) P < 0.001 5.9 (2.1–16.3) P < 0.001 2.5 (0.9–7.0) P = 0.094 Female n/a 0.9 (0.6–1.3) P = 0.48 n/a Age* n/a 1.8 (1.5–2.2) P < 0.001 1.6 (1.3–1.9) P < 0.001 Ln(NIHSS)** n/a n/a 3.6 (2.8–4.7) P < 0.001 *Risk ratio per 10 year increase in age. **Risk per a doubling in NIHSS score.

1. Introduction Stroke is an important cause of mortality and morbidity worldwide [1]. Despite recent advances in antithrombotic treatment, poststroke disability has significant economic and social burden. As brain has limited capacity to regenerate, there is the need to develop therapeutic strategies to enhance neuroprotection and repair. Autologous stem-cell transplantation has been tried but has limited due to unproven efficacy and lack of available facility widespread [2].

has significant economic and social burden. As brain has limited capacity to regenerate, there is the need to develop therapeutic strategies to enhance neuroprotection and repair. Autologous stem-cell transplantation has been tried but has limited due to unproven efficacy and lack of available facility widespread [2]. Currently, few treatments exist for acute stroke, comprising mainly aspirin and thrombolytic drugs which have poor availability in the developing countries and very narrow time window for its intervention. A clear need exists to identify new drugs. Granulocyte colony stimulating-factor (G-CSF) is a cytokine that acts on hematopoietic stem (CD34+) cells and stimulates proliferation, maturation, and survival of the neutrophilic granulocyte lineage. It is widely employed to mobilize bone marrow stem cells in patients with leukaemia treated with bone marrow transplantation and chemotherapy-induced neutropenia for last two decades [3]. Since Schäbitz et al. [4] observed infarct size-reducing capabilities of G-CSF in animal stroke model, a number of preclinical investigations were initiated to explore its neuroprotective abilities. In later experimental studies of cerebral ischemia, G-CSF was found to be neuroprotective via different mechanisms, including mobilization of haemopoietic stem cells, antiapoptosis, neuronal differentiation, angiogenesis, and anti-inflammation [5, 6]. These properties are particularly significant in view of apoptosis, and inflammation has implication in the pathophysiology of cerebral ischemic injury. In virtue of the above properties, it was speculated that G-CSF not only inhibits neuron death, but also generates new neuronal tissue formation. The observation of G-CSF's effect on mobilization of stem cells from the bone marrow initiated explorations of its potential benefit in stroke with the assumption that mobilized stem cells may home into the injured brain.

lated that G-CSF not only inhibits neuron death, but also generates new neuronal tissue formation. The observation of G-CSF's effect on mobilization of stem cells from the bone marrow initiated explorations of its potential benefit in stroke with the assumption that mobilized stem cells may home into the injured brain. Meta-analysis from the animal studies suggested that G-CSF both reduces infarct size and enhances functional recovery, and its effect is presumably dose dependent [7].

lated that G-CSF not only inhibits neuron death, but also generates new neuronal tissue formation. The observation of G-CSF's effect on mobilization of stem cells from the bone marrow initiated explorations of its potential benefit in stroke with the assumption that mobilized stem cells may home into the injured brain. Meta-analysis from the animal studies suggested that G-CSF both reduces infarct size and enhances functional recovery, and its effect is presumably dose dependent [7]. Three small clinical trials investigated the safety and feasibility and efficacy of stem cell mobilization by G-CSF in 7, 24, and 44 patients at different doses of G-CSF with acute ischemic stroke patients, respectively [8–10]. In all studies, G-CSF therapy appeared to be safe and reasonably well tolerated. Summary of G-CSF published studies in stroke patients are given in Table 1. There are several trials of G-CSF therapy in stroke ongoing across the world. Results of these trials will be helpful in knowing the efficacy of G-CSF therapy in stroke. Building on preclinical and clinical data suggesting functional and survival benefit using granulocyte colony-stimulating factor (G-CSF) in this fashion, we undertook a single centre, randomized, open-label pilot trial in patients with acute ischemic stroke. Moreover, the therapy is less invasive, relatively inexpensive (compared to rt-PA), ethically acceptable, and has long therapeutic window. The aim of the present study was to assess the safety and efficacy of G-CSFs at 10 μg/kg G-CSF in patients with acute ischemic stroke and to assess the effect on circulating stem cell and blood cell counts.

less invasive, relatively inexpensive (compared to rt-PA), ethically acceptable, and has long therapeutic window. The aim of the present study was to assess the safety and efficacy of G-CSFs at 10 μg/kg G-CSF in patients with acute ischemic stroke and to assess the effect on circulating stem cell and blood cell counts. 2. Methods 2.1. Participants All patients with acute ischemic stroke attending the neurology services at All India Institute of Medical Sciences, New Delhi, between January 2008 and May 2008, were screened for eligibility of this study. Patients with stroke (defined as rapidly developing clinical symptoms and/or signs of focal loss of cerebral function, with symptoms lasting more than 24 hours with no apparent cause other than that of vascular origin) were considered eligible if they fulfilled all of the following: age between 30 and 75 years, within seventh day from onset, computed tomography and/or magnetic resonance imaging scan of the brain showing no haematoma, and relevant lesions within the middle cerebral artery territory, Glasgow coma scale (GCS) score above eight (eye and motor score of more than six in patients with aphasia), Barthel index (BI) score of 55 or less, National Institute of health stroke Scale (NIHSS) score between 7 and 20, and inability to walk unaided or raise upper limb by 90 degree and clinically stable. A patient were defined as stable when they had normal respiration, was afebrile, had blood pressure less than mean arterial pressure of 125mmHg (but no hypotension defined as systolic BP <90 mmHg), and had fasting venous blood sugar level less than 200 mg% along with normal serum urea and electrolytes.

inically stable. A patient were defined as stable when they had normal respiration, was afebrile, had blood pressure less than mean arterial pressure of 125mmHg (but no hypotension defined as systolic BP <90 mmHg), and had fasting venous blood sugar level less than 200 mg% along with normal serum urea and electrolytes. Patients meeting the above criteria were excluded from the study if they had any one of the following: lacunar syndrome, intracranial pathologies (e.g., tumor and infection), intubation, comorbidity likely to limit survival to less than three years, for example, malignant diseases, hepatic or renal failure, prestroke disability leading to dependence on others for activities of daily living, haematological dysfunction (a history of major bleeding requiring blood transfusion or of leukopenia thrombocytopenia), inaccessibility for followup, pregnancy or unwillingness to provide written informed consent (by self or next of kin). The study was approved by the Institute Ethics Committee of AIIMS.

daily living, haematological dysfunction (a history of major bleeding requiring blood transfusion or of leukopenia thrombocytopenia), inaccessibility for followup, pregnancy or unwillingness to provide written informed consent (by self or next of kin). The study was approved by the Institute Ethics Committee of AIIMS. 2.2. Study Design This was a 12-month duration, randomized, open-label, parallel-group study. Eligible consenting subjects were randomly assigned in a 1 : 1 ratio to G-CSF therapy for five days along with conventional management or conventional management alone. Patients were randomly allocated to one of the two groups, by use of a computer-generated simple randomization table. The randomized allocation of groups was performed by a blinded, independent coordinator not related to patient care in the study via telephonic call. Subsequent to random allocation to groups, intervention was not blinded.

ly allocated to one of the two groups, by use of a computer-generated simple randomization table. The randomized allocation of groups was performed by a blinded, independent coordinator not related to patient care in the study via telephonic call. Subsequent to random allocation to groups, intervention was not blinded. All patients were evaluated according to a protocol that included demographic data, medical history, stroke risk factors, and neurological examination. To determine stroke severity, we used the BI (scores range from 0 to 100, with lower scores indicating increasing severity) as an index of functional recovery and NIHSS score (scores range from 0 to 42, with higher scores indicating increasing severity) as an index of neurological deficit. After all of the data had been recorded, patients were randomized to receive either subcutaneous human recombinant G-CSF (filgastrim, Grafeel, India) 10 μg/kg subcutaneously administered daily for five days along with conventional treatment or conventional treatment alone. Intervention was given within two hours of randomization. We assessed safety of subcutaneous G-CSF infusion by recording the development of immediate or delayed reactions. Immediate reactions included allergic reactions (tachycardia, fever, skin eruption, and leukocytosis). Leukocyte counts were measured on day one, three, five, and seven from blood samples. One week after the initiation of therapy, patients were discharged unless clinically warranted.

of immediate or delayed reactions. Immediate reactions included allergic reactions (tachycardia, fever, skin eruption, and leukocytosis). Leukocyte counts were measured on day one, three, five, and seven from blood samples. One week after the initiation of therapy, patients were discharged unless clinically warranted. After discharge, all of the patients were followed up at one month; modified Rankin scale (scores range from zero to six, with higher scores indicating increasing severity) as an index of functional recovery, along with BI and NIHSS, was recorded. Adverse events elicited included bone pain, headache, liver dysfunction, myocardial infarction, recurrence of stroke, and peripheral arterial thromboses. Subsequently, all of the patients were followed up at six and twelve months in the outpatient department, and neurological functions were assessed using all three scales. The 12-month scores from the BI, NIHSS, and mRS were used to assess treatment efficacy. Improvement was defined as the percentage change in mean group scores between baseline and 12 months. To evaluate tumor formation as a delayed complication, we performed a regular physical examination including visual inspection of skin and oral mucosa, a follow-up magnetic resonance imaging brain at one month and six-months, and whole body FDG-positron emission tomography at the end of 12 months.

seline and 12 months. To evaluate tumor formation as a delayed complication, we performed a regular physical examination including visual inspection of skin and oral mucosa, a follow-up magnetic resonance imaging brain at one month and six-months, and whole body FDG-positron emission tomography at the end of 12 months. 2.3. Statistical Analysis Analysis was done after completion of six-month followup. The primary analyses of efficacy and safety were performed on the intention to treat analysis. This included all patients who were randomized to receive treatment. Baseline characteristics and differences between G-CSF and control groups with different outcome measures were analyzed using Mann-Whitney U-tests. Data was analyzed using the SPSS statistical package, version 17.0 (SPSS, Chicago, IL, USA).

analysis. This included all patients who were randomized to receive treatment. Baseline characteristics and differences between G-CSF and control groups with different outcome measures were analyzed using Mann-Whitney U-tests. Data was analyzed using the SPSS statistical package, version 17.0 (SPSS, Chicago, IL, USA). 3. Results Between January 2008 and May 2008, a total of 19 patients with acute ischemic stroke were screened for eligibility (Figure 1). Of those 19 patients, nine were excluded, as four had intracranial bleed, three had improved Barthel index, and two refusals to consent by caregivers. All ten consecutive patients who were found eligible were randomly assigned to G-CSF or control. At baseline, five patients were in G-CSF group and five patients were in control group. In G-CSF group, all patients except one completed 5-day course of G-CSF therapy. One patient had clinical deterioration on day three of G-CSF therapy, so intervention was withheld, and patient died on eighth day after randomization. None of the patients showed deterioration in NIHSS, BI, or mRS during the followup. No patient developed liver or renal dysfunction. PET scan at one year did not show any evidence of tumor formation. As per protocol numbers of patients completed trial were four in G-CSF arm and five in control arm. There were no losses to followup over the study period. Baseline characterises of individual patients is summarized in Table 2. There was no significant difference in baseline characteristics between the two groups (Table 3).

r protocol numbers of patients completed trial were four in G-CSF arm and five in control arm. There were no losses to followup over the study period. Baseline characterises of individual patients is summarized in Table 2. There was no significant difference in baseline characteristics between the two groups (Table 3). Improvement in BI, NIHSS, and mRS did not differ significantly between the G-CSF and control groups, Figures 2, 3, and 4. Although a trend of higher improvement of BI score is seen in the intervention group, the difference did not achieve statistical significance. Rise in maximum leukocyte count at hospital stay from baseline and clinical outcome score in individual patients wise assessed at different time interval is given in Table 4. There was statistically significant rise in the mean leukocyte count and alkaline phosphatase levels in the intervention arm as compared with baseline and to control arm (P < 0.005 and 0.01, resp.). Rise in mean leukocyte is represented in Figure 5. Rise in peripheral blood CD-34 count did not differ significantly between the G-CSF and control groups.

nt rise in the mean leukocyte count and alkaline phosphatase levels in the intervention arm as compared with baseline and to control arm (P < 0.005 and 0.01, resp.). Rise in mean leukocyte is represented in Figure 5. Rise in peripheral blood CD-34 count did not differ significantly between the G-CSF and control groups. G-CSF therapy was reasonably well tolerated. Of the five patients receiving G-CSF, one reported mild bone pain that lasted one day and subsided spontaneously. One patient in G-CSF arm had deep venous thrombosis in left lower limb that required hospitalisation and subsided with treatment. There were no aggravations of stroke symptoms during the course of therapy and hospital stay. There was no aggravation of limb weakness, speech impairment, or sensory impairment during the 12-month followup in either study group. No severe adverse effects were seen in any of patients receiving G-CSF therapy arm or control arm. 4. Discussion This is the first preliminary randomized controlled study to explore safety and preliminary efficacy of G-CSF in patients with stroke from India. In accordance with previous studies, we found G-CSF therapy safe and well tolerated in five patients with acute stroke [8–10]. We failed to find effectiveness of G-CSF in improving neurologic outcome in patients with acute ischemic stroke. With only five patients in each arm, the pilot study was not designed to have the power to detect outcome differences between the groups. Two points deserve some comments.

4. Discussion This is the first preliminary randomized controlled study to explore safety and preliminary efficacy of G-CSF in patients with stroke from India. In accordance with previous studies, we found G-CSF therapy safe and well tolerated in five patients with acute stroke [8–10]. We failed to find effectiveness of G-CSF in improving neurologic outcome in patients with acute ischemic stroke. With only five patients in each arm, the pilot study was not designed to have the power to detect outcome differences between the groups. Two points deserve some comments. Optimal dose of G-CSF: several studies have been completed at different doses of G-CSF. (Table 1.) Our study suggest that G-CSF at dose of administered 10 μg/kg daily for five days appeared to be safe and reasonably well tolerated, and there was higher trend of improvement in Barthel index score in intervention group compared to control. Previous studies [10, 11] have used 10 μg/kg daily for five days protocol. To allow comparison of our results with the published studies and in future to facilitate meta-analyses, we have followed the same protocol. Several studies are ongoing with different doses of G-CSF in stroke and results of all studies will be helpful to determine the dose-response gradient of G-CSF. CD-34+ cells in peripheral blood increases significantly after G-CSF injection of 10 μg/kg for consecutive five days [10]. There was dose-dependent beneficial effect observed in treatment with patient with DWI lesion >14–17 cm3 [8]. Our study provide basis for the second trial with the dose of 10 μg/kg daily for five days. In view of similar findings from other G-CSF trials [8–10] in stroke patients, G-CSF therapy appeared safe and reasonably well-tolerated. Our data also suggest that rise in the leukocyte and alkaline phosphatase at this dose is not challenging in acute stage of stroke. There is clear need for identification of optimal dose of the G-CSF at which its effects is highest in improving functional outcome in stroke. More studies are needed to determine the optimal dose of G-CSF.

est that rise in the leukocyte and alkaline phosphatase at this dose is not challenging in acute stage of stroke. There is clear need for identification of optimal dose of the G-CSF at which its effects is highest in improving functional outcome in stroke. More studies are needed to determine the optimal dose of G-CSF. Timing of G-CSF injection: a therapeutics time window for intervention is a major promise of G-CSF therapy. Time of intervention used in clinical studies is summarised in Table 1. At present-time treatment for stroke, particularly thrombolytic therapy with tissue plasminogen activator is challenging because of its short time window of efficacy therefore, there is a clear need for novel and effective treatment options, with a longer time window. Shyu et al. [9] tested within seven days of onset of stroke and found there were consistent trend towards improvement in neurological functional recovery in G-CSF group. CD34+ stem cells also effectively mobilized and appeared to be safe and well tolerated after G-CSF injection when treatment is delayed for month in ischemic stroke patients [10]. Wider therapeutic window would be a significant achievement for stroke, since patients often does not reach hospital—nor is the disease often diagnosed until later than 3 hours after onset. Schäbitz et al. [8] 2010 (AXIS trial) tested within 12 hours of onset of stroke and found it is well tolerated and more effective with higher doses in patients with larger lesion volume. Timing of GCSF administration is likely to influence its neuroprotective effects. Mechanism of action of G-CSF may primarily on neurons; it is likely that the earlier treatment may have more potent neuroprotective effect. Our study suggests that treatment with G-CSF within seven days of onset of stroke appeared to be safe and reasonably well tolerated.

ikely to influence its neuroprotective effects. Mechanism of action of G-CSF may primarily on neurons; it is likely that the earlier treatment may have more potent neuroprotective effect. Our study suggests that treatment with G-CSF within seven days of onset of stroke appeared to be safe and reasonably well tolerated. Based on the findings of the pilot study, Drug-Controller-General of India has approved our multicentric, phase I/II safety and efficacy, randomized controlled trial of G-CSF with 200 sample size of patients. Our planned clinical trial is to establish safety and explore efficacy of G-CSF therapy in acute stroke patients. The protocol is under consideration for funding in the Department of Biotechnology, Government of India. Limitation of Study The study is with small number of sample size; however, other studies are ongoing and published in international journals with small number of sample sizes. In conclusion, G-CSF administered 10 μg/kg daily for five days appeared to be safe and well tolerated in five patients aged 35–75 years with acute ischemic stroke in accordance with other published G-CSF trials in stroke patients. However, in view of limited sample size, the results of the current study must be interpreted with caution. Further, large, adequately powered, multicenter randomized placebo-controlled, blinded trials are needed to test the efficacy of G-CSF in patients with acute ischemic stroke. Figure 1 Enrolment, randomization, and analysis of patients.

In conclusion, G-CSF administered 10 μg/kg daily for five days appeared to be safe and well tolerated in five patients aged 35–75 years with acute ischemic stroke in accordance with other published G-CSF trials in stroke patients. However, in view of limited sample size, the results of the current study must be interpreted with caution. Further, large, adequately powered, multicenter randomized placebo-controlled, blinded trials are needed to test the efficacy of G-CSF in patients with acute ischemic stroke. Figure 1 Enrolment, randomization, and analysis of patients. Figure 2 Mean Barthel index scale score at baseline, one month and six months of the intervention and control group. 1; baseline, 2; one month, 3; six months, BI scores range from 0 to 100, with lower scores indicating increasing severity. Figure 3 Mean National Institute of Health scale score, at baseline, one month and six months of the intervention and control group. 1; baseline, 2; one month, 3; six months, NIHSS scores range from 0 to 42, with higher scores indicating increasing severity. Figure 4 Mean modified Rankin scale score at one month and six months of the intervention and control group. 1; one month, 2; six months, mRs score range from 0 to 6, with higher scores indicating increasing severity. Figure 5 Mean Leukocyte count of intervention and control groups at Baseline, Day 1, Day 3, and Day 5. Table 1 Summary of published studies of G-CSF therapy in stroke patients.

Figure 4 Mean modified Rankin scale score at one month and six months of the intervention and control group. 1; one month, 2; six months, mRs score range from 0 to 6, with higher scores indicating increasing severity. Figure 5 Mean Leukocyte count of intervention and control groups at Baseline, Day 1, Day 3, and Day 5. Table 1 Summary of published studies of G-CSF therapy in stroke patients. Author (year) Trial design/phase G-CSF regimen Time after stroke Patients (intervention/control) Comments Floel et al. (2011) Randomized controlled trial 10 μg/kg s/c for 10 days 4 months after 21 Intervention 20 Placebo Feasibility and safe and reasonable tolerable in chronic stroke patients Schäbitz et al. (2010) Randomized, placebo-controlled 30 μg/kg, 90 μg/kg, 135 μg/kg, 180 μg/kg Within 12 hours 14/ Placebo 8/30 μg/kg 7/90 μg/kg 8/135 μg/kg 7/180 μg/kg Well tolerated even in higher doses and Treatment effect in patients with higher volume of lesion size (˃14–17 cm3) at baseline Shyu et al. (2006) Single blind controlled/pilot 15 μg/kg/day s/c for 5 days Within 7 days 7 Intervention 15 μg/kg/day s/c for 5 days 3 Control No thrombotic complications, and improved outcome in G-CSF group NIHSS 59% in G-CSF group, 36% in controls group BI 120% in G-CSF group, and 60% in controls group Sprigg et al. (2006) Double-blind placebo-controlled/pilot Dose escalation 1–10 μg/kg s/c for 1 or 5 days 7–30 days 12/Placebo 4/1 μg/kg (single dose) 4/3 μg/kg (single dose) 4/10 μg/kg (single dose) 4/1 μg/kg (five dose) 4/3 μg/kg (five dose) 4/10 μg/kg (five dose) No difference in SAEs although non significant increase in infection rates in active group Significant increase in CD-34+ with 10 μg/kg (five dose) at day five Zhang (2006) Double-blind placebo-controlled/pilot 2  μg/kg/day s/c for 5 days Within 7 days 15  Intervention 30  Control No difference in adverse events reported and significant reduction in NIHSS Table 2 Patient's characteristics.

es in active group Significant increase in CD-34+ with 10 μg/kg (five dose) at day five Zhang (2006) Double-blind placebo-controlled/pilot 2  μg/kg/day s/c for 5 days Within 7 days 15  Intervention 30  Control No difference in adverse events reported and significant reduction in NIHSS Table 2 Patient's characteristics. Case no. 1 2 3 4 5 6 7 8 9 10 Allocation Control G-CSF Control G-CSF G-CSF G-CSF Control Control Control G-CSF Days b/w onset of stroke and randomization 3 4 7 4 4 3 6 2 1 5 Age/sex 56 45 35 45 40 55 55 65 30 38 Territory and side Rt MCA Rt MCA Lt MCA Lt MCA Rt MCA Both MCA & ACA Rt MCA Lt MCA Lt MCA Rt MCA Previous stroke − − − − − − − + − − GCS baseline 15.00 15.00 15.00 14.00 15.00 14.00 15.00 11.00 15.00 15.00 Hypertension + − − − − + − − − − Diabetes − − − − − − − − − − Dyslipidemia − + − − − + − − − − Smoker − − − − − + − + − − Table 3 Summary of patient characteristics and efficacy outcomes (difference between six-month and baseline scores). Intervention n = 5 Control n = 5 P-value Age in years* 44 ± 6.5 (38 to 55) 48 ± 14.9 (30 to 65) 0.63 GCS score* 14.7 ± 5 (14 to 15) 14.2 ± 1.7 (11 to 15) 0.65 Days b/w onset of stroke and randomization* 4 ± 2.44 (1 to 7) 4 ± 0.7 (3 to 5) 0.87 NIHSS score∗† 14 ± 3.9 (8 to 19) 11.0 ± 3.39 ( 7 to 15) 0.23 Mean difference of NIHSS from six months to baseline −7.0 −6.75 0.90 BI score∗‡ 25 ± 17.67 (5 to 45) 32 ± 21.09 (15 to 55) 0.58 Mean difference of BI from Six months to baseline 51.25 44 0.59 Mean difference of mRS from six months to one month −0.5 −0.4 0.79 *Figures represent mean ± SD (range) or numbers.

to 15) 0.23 Mean difference of NIHSS from six months to baseline −7.0 −6.75 0.90 BI score∗‡ 25 ± 17.67 (5 to 45) 32 ± 21.09 (15 to 55) 0.58 Mean difference of BI from Six months to baseline 51.25 44 0.59 Mean difference of mRS from six months to one month −0.5 −0.4 0.79 *Figures represent mean ± SD (range) or numbers. †NIHSS scores range from 0 to 42, with higher scores indicating increasing severity. ‡BI scores range from 0 to 100, with lower scores indicating increasing severity. §mRs score range from 0 to 6, with higher scores indicating increasing severity. Table 4 Leukocyte counts at baseline and maximum count during hospital stay and clinical score during baseline one month, six months, and 12 months. Total leukocyte count Stroke scale scores at baseline, one month/six months and 12 months Patient no. Baseline Maximum during hospital stay NIHSS BI mRS G-CSF group 1 13400 40800 14/10/9 10/35/65 4/4 2 12400 — 19/−/− 5/−/− −/− 3 7500 35400 14/9/6 45/80/85 3/3 4 7600 39200 15/7/3 25/90/95 3/2 5 8400 33400 8/9/5 40/60/80 4/3 Control group 1 7000 7000 13/10/5 15/35/55 4/4 2 13500 13500 12/10/4 55/60/75 4/3 3 8500 10400 15/12/− 20/30/55 4/4 4 7300 7300 7/2/2 55/100/100 2/1 5 10100 10100 8/4/2 15/90/95 2/2 NIHSS: National Institutes of Health stroke scale, BI: Barthel index, mRS: modified ranking scale NIHSS score range from 0 to 42 (lower score represents better outcome and higher score represents worse outcome) BI score range from 0 to 100 (Higher score represents the better outcome and lower score represents the worse outcome)

Control group 1 7000 7000 13/10/5 15/35/55 4/4 2 13500 13500 12/10/4 55/60/75 4/3 3 8500 10400 15/12/− 20/30/55 4/4 4 7300 7300 7/2/2 55/100/100 2/1 5 10100 10100 8/4/2 15/90/95 2/2 NIHSS: National Institutes of Health stroke scale, BI: Barthel index, mRS: modified ranking scale NIHSS score range from 0 to 42 (lower score represents better outcome and higher score represents worse outcome) BI score range from 0 to 100 (Higher score represents the better outcome and lower score represents the worse outcome) mRS score range from 0 to 6 (lower score represents better outcome and higher score represents worse outcome).

1. Introduction Stroke is the third cause of mortality and one of most frequent causes of long-term neurological disability. Well-established risk factors for stroke include increasing age, hypertension, diabetes mellitus, cigarette smoking, obesity, heart disease, atrial fibrillation and sedentary [1, 2]. However, a significant number of patients experience stroke in the absence of any risk factors; a hypothesis is that many risk factors have not been recognized yet, including genetic risk factors. The role of genetics has been evidenced through studies on twins and family history. Twin studies have shown that monozygotic twins are 1.6 more likely to be concordant for stroke than dizygotic twins [3]. Family history of stroke is a well-defined risk factor (OR 1.76 95% CI 1.7–1.9) [3].

cluding genetic risk factors. The role of genetics has been evidenced through studies on twins and family history. Twin studies have shown that monozygotic twins are 1.6 more likely to be concordant for stroke than dizygotic twins [3]. Family history of stroke is a well-defined risk factor (OR 1.76 95% CI 1.7–1.9) [3]. Given these data, genetic studies have increasingly been performed with the objective of revealing the genetic basis of cerebrovascular diseases. Genetic studies have been proposed to (1) reveal the pathogenetic basis of stroke, which might become a therapeutic target for new drugs, (2) optimize risk assessment, (3) identify populations requiring more aggressive therapeutic strategies, and (4) choose the optimal drug therapy by assessing the risk/benefit ratio based on genetic characteristics [4]. The latter application has been extensively studied in pharmacogenetic studies [5–7]. Recently, genetic studies have moved to “pharmacogenomic” that involve a genome-wide association approach which scans the entire genome looking through thousands of genetic variants; these hypothesis-free studies have the aim of discovering novel genes associated with a specific disease. This review has the aim of reporting on the latest developments regarding pharmacogenetics and pharmacogenomics of stroke, focusing on the most commonly used drugs in the acute phase, for primary and secondary prevention.

ypothesis-free studies have the aim of discovering novel genes associated with a specific disease. This review has the aim of reporting on the latest developments regarding pharmacogenetics and pharmacogenomics of stroke, focusing on the most commonly used drugs in the acute phase, for primary and secondary prevention. 2. Methods This review was planned using key words such as “pharmacogenetics” or “pharmacogenomics” and “stroke” to search literature. These words were combined with “antihypertensive agents,” “statins,” “hydroxymethylglutaryl-CoA Reductase Inhibitors,” “tissue plasminogen activator,” “anticoagulants,” “vitamin K antagonist,” “antiplatelets,” “cyclooxygenase Inhibitors,” “aspirin,” “clopidogrel,” and “acetil salicylic acid/dipyridamole.” The following electronic databases were searched: MEDLINE (1995-June 11 2011) and EMBASE (1995-June 11 2011). One of the researchers (SA) read all the abstracts and selected all articles that included either “stroke” as outcome in primary prevention studies or as the target population in acute stroke treatment or secondary prevention studies. If any doubt was raised on an article's relevance, a second opinion was formulated by VC. 3. Results In this section, pharmacogenetic studies involving drugs currently used for ischemic stroke (prevention or acute phase therapy) are reviewed.

The following electronic databases were searched: MEDLINE (1995-June 11 2011) and EMBASE (1995-June 11 2011). One of the researchers (SA) read all the abstracts and selected all articles that included either “stroke” as outcome in primary prevention studies or as the target population in acute stroke treatment or secondary prevention studies. If any doubt was raised on an article's relevance, a second opinion was formulated by VC. 3. Results In this section, pharmacogenetic studies involving drugs currently used for ischemic stroke (prevention or acute phase therapy) are reviewed. 3.1. Antihypertensive Agents Hypertension is the most common stroke risk factor [41]. β1 and β2 adrenergic receptor (AR) plays a major role in cardiac disease; their codifying genes have been associated with response to antihypertensive drugs. β1-AR gene interacted with beta-blocker (BB) therapy. Stroke risk has been shown to be higher in rs#2429511 carriers treated with BB (OR: 1.24, 95% CI: 1.03–1.50). On the contrary, BB therapy did not interact with β2-AR gene variants on the risks of ischemic stroke (Table 1) [14]. A large randomised trial on treated hypertensive patients, enrolled to add either verapamil SR or trandolapril (International Verapamil SR-Trandolapril Study, INVEST study), focused on the genetic component of hypertension (INVEST-GENES) (Table 1) [8, 9, 17, 18, 20]. One of the papers derived from this study examined the polymorphism of α-adducin (ADD1) Gly460Trp and race. The authors chose this polymorphism because α-adducin, a cytoskeleton protein related with sodium sensitivity and diuretics efficacy, has been linked to essential hypertension [42]. The results did not evidence any diuretic-genotype interaction [20]. On the contrary, a population-based case control study on the same polymorphism found that diuretics protected ADD1 460 Trp carriers from combined nonfatal MI/nonfatal stroke outcome. Other antihypertensive agents (e.g., beta blockers, ACE inhibitors, and calcium-channel blocker) did not show the same effect [19].

20]. On the contrary, a population-based case control study on the same polymorphism found that diuretics protected ADD1 460 Trp carriers from combined nonfatal MI/nonfatal stroke outcome. Other antihypertensive agents (e.g., beta blockers, ACE inhibitors, and calcium-channel blocker) did not show the same effect [19]. The randomised INVEST-GENES study also investigated the relation between subunit β1 of the gene that encodes for a conductance calcium and voltage-dependent potassium channel (KCNMB1) genotype and response to calcium antagonists. The results showed that carriers of the Leu 110 polymorphism have a reduced risk of combined death, MI, and stroke when assuming verapamil SR to treat hypertension [9]. In addition, the same research group focused on G-protein-coupled receptor kinases (GRKs), receptors involved in beta-adrenergic signalling. GRK2 SNPs (rs1894111 G > A) and GRK5 Gln41Leu polymorphism were investigated in patients treated with atenolol or hydrochlorothiazide. The authors concluded that GRK 41Leu variant did not interact with any of the studied treatment regarding a combined cardiovascular outcome including death, MI, and stroke [8]. Finally, Pacawnosky investigated for an association between nitric oxide synthase (NOS 3) polymorphism [18], beta-adrenergic receptor gene (ADRB1 and ADRB2) [17], and response to different antihypertensive agents. The first study focused on two NOS 3 polymorphisms since nitric oxide regulates vascular tone and is associated with many cardiac diseases [43]; no outcome or drug interaction was associated with genotype [18]. Also the second study did not evidence any genotype-drug interaction on stroke [17].

antihypertensive agents. The first study focused on two NOS 3 polymorphisms since nitric oxide regulates vascular tone and is associated with many cardiac diseases [43]; no outcome or drug interaction was associated with genotype [18]. Also the second study did not evidence any genotype-drug interaction on stroke [17]. A population-based prospective cohort study focused on the renin-angiotensin system which is affected by ACE-inhibitors and BB (Table 1) [15, 16]. Neither of the studies observed any interaction between drug use and genotype when stroke was considered as outcome [15, 16]. The genetics of hypertension-associated treatment (GenHAT) study investigated the ACE insertion/deletion (ACE I/D) polymorphism in a large population of hypertensive patients with one or more cardiovascular risk factors. This randomised study did not report any association between treatment, genotype, and primary or secondary outcomes [11].

ension-associated treatment (GenHAT) study investigated the ACE insertion/deletion (ACE I/D) polymorphism in a large population of hypertensive patients with one or more cardiovascular risk factors. This randomised study did not report any association between treatment, genotype, and primary or secondary outcomes [11]. The same result was replicated in a more articulated investigation on the ACE gene and 12 other polymorphisms (ADD1 Gly460Trp, β1AR Gly389Arg, β2AR Arg16Gly, β2AR Gln27Glu, β3AR Trp64Arg, AGT Met235Thr, Aldosterone synthase promoter C-344T, Type 1 angiotensinogen receptor A1166C, bradykinin 2 receptor I/D, CYP2C9∗2 versus CYP2C9∗1, CYP2C9∗3 versus CYP2C9∗1, G protein β3-subunit C825T) [10]. This study was the product of the randomised LIFE (Losartan Intervention for Endpoint reduction in Hypertension) study trial, which included patients with hypertension and left ventricular hypertrophy treated with losartan versus atenolol. The authors did not evidence any genetic-drug interaction on different outcomes such as blood pressure and heart rate control, composite adverse cardiovascular outcome, cardiovascular death, MI, and stroke; in fact, they concluded that the clinical superiority of losartan in 25% stroke reduction compared to atenolol was not explained by these susceptibility genes [10].

action on different outcomes such as blood pressure and heart rate control, composite adverse cardiovascular outcome, cardiovascular death, MI, and stroke; in fact, they concluded that the clinical superiority of losartan in 25% stroke reduction compared to atenolol was not explained by these susceptibility genes [10]. A role in modulating antihypertensive agents has been suggested for the gene which codes for the precursor of atrial natriuretic polypeptide (NPPA gene). The polymorphism of this gene was studied by the GenHAT study [12]. The objective was to demonstrate that minor NPPA alleles in the T2238C or G664A variants had lower rates of primary outcome events compared with common allele homozygotes, if treated with diuretics. Subjects randomly receiving amlodipine, chlorthalidone, lisinopril, or doxazosin were included in a genetic for treatment interaction analysis. Carriers of the minor C allele had more favourable stroke outcome when taking diuretics, whereas TT allele carriers had better stroke outcome when receiving a calcium channel blocker [12]. GenHAT [13] also showed that stroke risk was higher on lisinopril versus amlodipine in common GG homozygotes of fibrinogen beta (FGB) gene, which codes for a polypeptide of the coagulation factor fibrinogen. On the contrary, variant A allele carriers on lisinopril had slightly lower stroke risk. Finally, a pharmacogenetic study on perindopril failed to demonstrate a role for ACE I/D polymorphism on stroke [21].

GG homozygotes of fibrinogen beta (FGB) gene, which codes for a polypeptide of the coagulation factor fibrinogen. On the contrary, variant A allele carriers on lisinopril had slightly lower stroke risk. Finally, a pharmacogenetic study on perindopril failed to demonstrate a role for ACE I/D polymorphism on stroke [21]. 3.2. Statins The most currently used drugs for hypercholesterolemia are statins; although very effective, they induce a significant rate of adverse events such as myopathies and abnormal transaminase levels. Recent pharmacogenetics data has contributed to better understanding statin pharmacokinetics and pharmacodynamic variability [44]. Pharmacogenetic and dynamic properties have been extensively studied, but only few studies included stroke as outcome (Table 2) [22–25].

such as myopathies and abnormal transaminase levels. Recent pharmacogenetics data has contributed to better understanding statin pharmacokinetics and pharmacodynamic variability [44]. Pharmacogenetic and dynamic properties have been extensively studied, but only few studies included stroke as outcome (Table 2) [22–25]. A population-based cohort study focused on apolipoprotein E, a protein involved in lipid clearance rate and conversion together with the production of triglycerides and very low-density lipoprotein. The Apo E gene encodes for three alleles: E2, E3, and E4 [45]. The results did not show gene-statin interaction with stroke; stroke risk was reduced independently of Apo E genotype in statin users [24]. The same author examined the effect of ACE I/D polymorphism on stroke using the Gen-HAT data. None of the outcomes evidenced significant ACE I/D-pravastatin interaction [25]. The randomised heart protection study focused on Kinesin family member 6 (KIF) gene, whose variant has been associated with reduced coronary events [46]. The authors did not find any significant interaction between the studied polymorphism KIF Trp719Arg and simvastatin use for any of the outcomes, including stroke [23]. The only study that yielded positive results was a case-control study that involved patient with MI and stroke. The authors focused on six genes that have been associated to statin treatment response: ATP-binding cassette subfamily B (ABCB1) gene that encodes for a drug transporter involved in statins metabolism; CETP, human hepatic lipase gene (LIPC) and low density lipoprotein receptor (LDLR), genes related to lipid metabolism; HMGCR, the target protein of statins; NOS3, a key gene implicated in maintaining the endothelium, which in turn mediates several effects of statins [22]. The authors found 5 polymorphisms (one in CETP and 1 in LIPC) that had significant interactions with statins on stroke outcome [22], the highest significance level was found in the CETP SNPs (rs5883), which was associated with stroke risk in simvastatin users. No gene level interactions were found for stroke [22].

22]. The authors found 5 polymorphisms (one in CETP and 1 in LIPC) that had significant interactions with statins on stroke outcome [22], the highest significance level was found in the CETP SNPs (rs5883), which was associated with stroke risk in simvastatin users. No gene level interactions were found for stroke [22]. 3.3. Tissue Plasminogen Activator Recombinant tissue plasminogen activator (rTPA) is the only licensed drug to treat ischemic stroke in the acute phase (within 3–4.5 hours from onset). This drug is administered to treat ischemic stroke and restore blood flow to the brain [47, 48]. The clinical benefit of rTPA is counterbalanced by a higher risk of hemorrhagic complications; 2–10% of patients develops symptomatic hemorrhagic transformations and 40% asymptomatic hemorrhagic events [49–51]. The functional role of rt-PA is to convert plasminogen into plasmin, which has fibrinolytic activity. The higher activity of the enzyme produces hyperfibrinolysis and consequently bleeding, whereas lower activity causes hypofibrinolysis and, as a consequence, thrombosis or embolism [52]. Genetic association studies have sought to investigate genetic profiles correlated with clinical and pathophysiological rt-PA response (Table 3). Broderick et al. [26, 53] examined the role of the ApoE phenotypes and reported that rt-PA efficacy was greater in acute stroke patients with an ApoE E2 phenotype (OR: 6.4; 95%, CI: 2.7–15.3), whereas the outcome of placebo-treated patients with or without Apo E E2 did not differ [26]. Conversely, a Spanish group did not report on any association with Apo E genotype and hemorrhagic risk and recanalisation rate after thrombolytic treatment [27]. The same group explored the hypothesis that matrix metalloproteinase-9 gene (MMP-9), which codes for proteins associated with blood-brain barrier disruption, was associated with hemorrhagic transformation in rTPA-treated patients. However, the authors did not find any association between a MMP-9 C-1562T common polymorphism and hemorrhagic risk [32]. On the other hand, the authors reported that thrombolytic intervention yielded middle cerebral artery (MCA) recanalisation associated with DD homozygosis of ACE I/D polymorphism; this has been linked to procoagulant factors including PAI-1, fibrinogen's levels as well as Factors VII and X activities [29].

ic risk [32]. On the other hand, the authors reported that thrombolytic intervention yielded middle cerebral artery (MCA) recanalisation associated with DD homozygosis of ACE I/D polymorphism; this has been linked to procoagulant factors including PAI-1, fibrinogen's levels as well as Factors VII and X activities [29]. Another study of the same group has identified V34L factor XII polymorphism as a predictor of outcome with rTPA treatment; good outcome was associated with VV genotype and low fibrinogen levels, while a higher risk of inefficacy of thrombolytic therapy and mortality was found with L34 genotype and high fibrinogen levels [31]. In addition, Fernandez-Cadenas and colleagues studied the influence of two genes coding for fibrinolysis inhibitors, thrombin-activatable fibrinolysis inhibitor (TAFI), and plasminogen activator inhibitor-1 (PAI-1) genes. They demonstrated that TAFI Thr325Ile polymorphism predicted the absence of recanalisation with t-PA infusion. On the contrary, PAI-1 4 G/5 G polymorphism did not influence recanalisation rate. However, the combination of these two polymorphisms doubled the risk of negative response to therapy [30]. A recent study using a candidate gene approach has explored the association of 263 SNPs and recanalisation rate in TPA-treated patients; cluster of differentiation 40 (CD40) 1C > T and matrix Gla protein (MGP)-7A > G polymorphism were both associated with reocclusion although only the latter was associated with neurological worsening at 24 h [28]. This may be due to the role of CD40 in thrombosis and inflammation [54], while MGP gene might have a protective role in atherosclerosis [55]. To date, GWAs has not been performed on human subjects.

sm were both associated with reocclusion although only the latter was associated with neurological worsening at 24 h [28]. This may be due to the role of CD40 in thrombosis and inflammation [54], while MGP gene might have a protective role in atherosclerosis [55]. To date, GWAs has not been performed on human subjects. 3.4. Anticoagulants Anticoagulation is first-line treatment for cardioembolic stroke. Although these drugs are effective in almost 60% of cases, the hemorrhagic risk is double and even higher in the first period of therapy [1]. Recent acquisition on pharmacogenetics of warfarin has been suggested to be able to predict the optimal initial dosage of warfarin using a genotype-guided approach (Table 4). This approach promises to adequately prevent stroke and to minimize hemorrhagic risk. Several candidate gene studies have mainly focused on cytochrome P450 (CYP) and vitamin K epoxide reductase complex subunit 1 (VKORC1) [56, 57]. Cytocrome P 450 metabolises in the liver S-warfarin by CYP2C9 and R-warfarin by the CYP1A1, CYP1A2, and CYP3A4; these enzymes affect warfarin kinetics, and several SNPs of CYP450 have been correlated with its sensitivity [58]. The VKORC1 enzyme converts the epoxide into reduced vitamin K; however warfarin inhibits this reaction. As a consequence, the physiologic role of vitamin K, which produces γ-carboxylation of several coagulation factors (prothrombin, factor VII, IX, and X), is inhibited.

0 have been correlated with its sensitivity [58]. The VKORC1 enzyme converts the epoxide into reduced vitamin K; however warfarin inhibits this reaction. As a consequence, the physiologic role of vitamin K, which produces γ-carboxylation of several coagulation factors (prothrombin, factor VII, IX, and X), is inhibited. Several groups have studied the role of VKORC1 in warfarin/acenocoumarol dose finding, dose maintenance, and bleeding risk associated with these drugs [33, 34, 59–61]. Only two studies have focused on patients receiving vitamin K antagonist following cardioembolic stroke. One found that the time and cumulative dosage of phenprocoumon needed to achieve target 2-3 INR ratio were associated with the presence of the VKORC1 C283 + C837T (rs2359612) polymorphism. Carriers of TT genotype needed shorter time to achieve target INR ratio (3.2 days) compared to CC carriers (6.5 days) [33]. The second paper evaluated the roles of VKORC1, gamma-glutamyl carboxylase (GGCX), calumenin (CALU), and cytochrome P450 2C9 (CYP2C9) in warfarin maintenance dose on Japanese stroke sufferers. Of the twelve SNPs analysed, the authors found that the 1639G > A, 3730G > A VKORC1 genotypes; the 8016G > A GGCX genotype, and the 42613A > C CYP2C9 genotype were associated with dose maintenance. Thus, the variation in warfarin dose was explained for 33.3% by age, sex, weight, and three genetic polymorphisms (VKORC1-1639G > A, CYP2C9 42613A > C, GGCX 8016G > A). The importance of these loci has been recently confirmed using genome-wide association studies in acenocoumarol-treated patients [62, 63]. These studies found that the SNPs with the highest significance level were located in chromosome (cr.) 16 (rs10871454 and rs9923231) linked to VKORC1 and cr. 10 (rs4086116 and rs105791) linked with CYP2C9 gene. After adjusting for these two SNPs, two other polymorphisms reached significant association with acenocoumarol: rs2108622 within CYP4F2 gene on cr.19 and rs1995891 within CYP2C18 on cr. 10 [62, 63].

omosome (cr.) 16 (rs10871454 and rs9923231) linked to VKORC1 and cr. 10 (rs4086116 and rs105791) linked with CYP2C9 gene. After adjusting for these two SNPs, two other polymorphisms reached significant association with acenocoumarol: rs2108622 within CYP4F2 gene on cr.19 and rs1995891 within CYP2C18 on cr. 10 [62, 63]. 3.5. Antiplatelets Antiplatelet drugs are commonly used treatment for ischemic noncardioembolic stroke [1]. 3.5.1. Aspirin Aspirin is the more commonly used drug of this class, and its efficacy ranges between 13% and 25%. Its physiological role is to acetylate serine residue 530 in the active site of cyclooxygenase-1 (COX-1), sterically inhibiting the metabolism of arachidonic acid and consequently reducing thromboxane A2 (TxB2), which activates platelets. Numerous studies have investigated the genetic basis associated with recurrence of ischemic event in aspirin-treated patients (e.g., aspirin failure) (Table 4). COX-1 C50T allele has been correlated with a higher level of 11-dehydro-TxB2, both before and after aspirin; however, the haplotype studied did not confirm a genetic basis for aspirin failure [64, 65]. In addition, this polymorphism is not associated with a higher risk of stroke [39].

3.5.1. Aspirin Aspirin is the more commonly used drug of this class, and its efficacy ranges between 13% and 25%. Its physiological role is to acetylate serine residue 530 in the active site of cyclooxygenase-1 (COX-1), sterically inhibiting the metabolism of arachidonic acid and consequently reducing thromboxane A2 (TxB2), which activates platelets. Numerous studies have investigated the genetic basis associated with recurrence of ischemic event in aspirin-treated patients (e.g., aspirin failure) (Table 4). COX-1 C50T allele has been correlated with a higher level of 11-dehydro-TxB2, both before and after aspirin; however, the haplotype studied did not confirm a genetic basis for aspirin failure [64, 65]. In addition, this polymorphism is not associated with a higher risk of stroke [39]. An interesting study compared the COX-gene sequence of patients with recurrent stroke (at least with two episodes) on aspirin and healthy subjects. The study found fourteen SNPs, and half of these lead to amino-acid substitutions; however, none of these variations was located near the COX catalytic site, thus this genetic polymorphisms could not explain the failure to respond to aspirin in this population of stroke patients [40].

) on aspirin and healthy subjects. The study found fourteen SNPs, and half of these lead to amino-acid substitutions; however, none of these variations was located near the COX catalytic site, thus this genetic polymorphisms could not explain the failure to respond to aspirin in this population of stroke patients [40]. 3.5.2. Clopidogrel Clopidogrel is an oral, thienopyridine antiplatelet drug that irreversibly inhibits the P2Y12 subtype of ADP receptor, which has a major role in platelet aggregation. Clopidogrel has proven to be less effective in carriers of CYP2C19-reduced function allele [37, 38, 66]. These data have been confirmed in a recent meta-analysis that pooled 9 randomised trails for acute coronary syndrome or percutaneous coronary intervention; either homozygosis or heterozygosis carriers experience higher risk of stroke (Table 4) [35]. This could be caused by a relative reduction in the active metabolite of the drug, or by an insufficient inhibition of platelet aggregation. At a clinical level, CYP2C19 allele carriers have major adverse cardiovascular events, including stroke [35]. The TRITON TIMI 38 study on patients with acute coronary syndrome treated with PCI following clopidogrel versus another thienopyridine, “prasugrel,” explored the role of ABCB1, a glycoprotein that might affect clopidogrel transport and metabolism. The polymorphism on ABCB1 3435C → T was correlated to a significant increase in adverse outcome including cardiovascular death, MI, or stroke (P = 0.0064). Specifically, TT homozygote patients had a 72% increased risk of the primary endpoint compared with CT/CC individuals [36]. This result might be a consequence of the absolute reduction in maximum platelet aggregation that has been evidenced in healthy subjects enrolled in the same study [36]. Furthermore, the PLATO study explored the same polymorphisms in noncoronary artery bypass graft patients on clopidogrel versus ticagrelor, a novel ADP receptor blocker that does not need hepatic activation, and so is not influenced by CYP2C19 alleles. Patients on ticagrelor were less likely to experience stroke independently of CYP2C19 or ABCB1 genotype. In addition, no specific genotype-drug interaction was associated with any major bleeding risk [37]. Finally, an important GWA study has been performed on a healthy Amish population and found a positive association with clopidogrel response measured by ADP platelet aggregation percentage and 10q24 region (Table 4).

pe. In addition, no specific genotype-drug interaction was associated with any major bleeding risk [37]. Finally, an important GWA study has been performed on a healthy Amish population and found a positive association with clopidogrel response measured by ADP platelet aggregation percentage and 10q24 region (Table 4). This region contains CYP2C19∗2 genotype, which accounts for approximately 12% of the variation in clopidogrel response [38]. In addition, this study found a relevant association between this CYP2C19∗2 variant and event-free survival of adverse cardiovascular outcome in an independent population of 227 patients that underwent percutaneous coronary intervention [38]. 4. Conclusions Pharmacogenetics of stroke is a promising approach for optimizing treatment strategies aimed at decreasing stroke incidence and recurrence. Many candidate gene studies have examined the roles of polymorphisms on stroke treatment, and some of these have been replicated in GWA studies. However, few studies have considered stroke as an independent outcome, probably due to the relatively small number of events in the trails. Antihypertensive agents are the most extensively studied drug class. Some polymorphisms have been consistently identified but results remain controversial, probably due to differences in study designs and methods, small sample sizes, and short durations of follow-up [67]. Statins and stroke have failed to find any interaction with most of the studied polymorphism. In addition, GWAs that consider stroke as an outcome are not available.

Antihypertensive agents are the most extensively studied drug class. Some polymorphisms have been consistently identified but results remain controversial, probably due to differences in study designs and methods, small sample sizes, and short durations of follow-up [67]. Statins and stroke have failed to find any interaction with most of the studied polymorphism. In addition, GWAs that consider stroke as an outcome are not available. Tissue plasminogen activator has been investigated only in small studies on acute intravenous thrombolysis; thus, the pharmacogenetic data need to be reproduced in larger trails, and GWAs should be planned on humans in order to move forward in this field. Interestingly, a GWA study on ischemic rats has shown the genes regulated by rTPA treatment where different from the ones involved in ischemic stroke. In addition, gene expression profiles differed when reperfusion was or was not achieved [68]. If these results were to be replicated on humans, blood plasma could be used to monitor gene expression profiles, which are diversely associated with stroke and rtPA vessel recanalisation.

the ones involved in ischemic stroke. In addition, gene expression profiles differed when reperfusion was or was not achieved [68]. If these results were to be replicated on humans, blood plasma could be used to monitor gene expression profiles, which are diversely associated with stroke and rtPA vessel recanalisation. Anticoagulants dose variability has been consistently reported to be explained by CYP2C9 and VKORC1 for the 33% and up to the 58% when adding clinical information [69]. For this reason, the United States Food and Drug Administration suggests testing for these two polymorphisms in order to achieve stable dose and to avoid major hemorrhagic events. Although the pharmacogenetic approach on warfarin is feasible in clinical practice [70]; its use in improving outcome (i.e., shorter time to achieve range INR, more stable dosing, greater percentage of time in therapeutic range, and lesser major bleeding events) over the classical clinical approach has been proven in only small samples [71, 72]; for this reason, larger studies (GIFT, COAG, and EU-PACT) are ongoing to demonstrate the usefulness of the genetic approach and clinical algorithms (see http://www.clinicaltrials.gov/) on outcome improvement. Unfortunately, genotype-guided warfarin dosing has not been demonstrated to be cost effective [73]. Finally, in the near future, vitamin K antagonists could be gradually replaced in many indications with the newer anticoagulants (e.g., Dabigatran, Apixaban, and Rivaroxaban), which do not require monitoring and dose adjustment [74–76].

enotype-guided warfarin dosing has not been demonstrated to be cost effective [73]. Finally, in the near future, vitamin K antagonists could be gradually replaced in many indications with the newer anticoagulants (e.g., Dabigatran, Apixaban, and Rivaroxaban), which do not require monitoring and dose adjustment [74–76]. Antiaggregants: genetic studies on aspirin failure in recurrent stroke patients have been unsuccessful in finding their genetic determinant. Several polymorphisms have been linked to poor clinical response to clopidogrel, but to date, no study has proven the usefulness of the pharmacogenetic approach with clopidogrel in improving outcome. For this reason, the ACCF/AHA [77] disagreed with the FDA regarding their decision to add a warning on clopidogrel label recommending genetic testing when prescribing it for the first time. However, several studies, focusing on coronary disease, not on stroke, are ongoing: GeCCO, RAPID GENE, TARGET PCI, and GIANT (see http://www.clinicaltrials.gov/).

7] disagreed with the FDA regarding their decision to add a warning on clopidogrel label recommending genetic testing when prescribing it for the first time. However, several studies, focusing on coronary disease, not on stroke, are ongoing: GeCCO, RAPID GENE, TARGET PCI, and GIANT (see http://www.clinicaltrials.gov/). The future availability and low cost of technology will allow for the screening of a large number of genetic determinants. This will lead to the description of polymorphisms that affect drug pharmacokinetics and dynamics in each given patient. Furthermore, this information will optimize the efficacy/toxicity ratio. Although promising, the results of pharmacogenetic studies need to be confirmed in prospective randomised trials of comparative effectiveness, comparing the classical clinical and the genotype-guided approach, before being used in clinical settings. Furthermore, no study has explored yet the clinical usefulness of the genetic approach in reducing adverse events. For these reasons, although promising, the genotype-guided approach for drug prescriptions is not routinely recommended [56]. Acknowledgments The authors would like to gratefully thank Dr. Stephanie Anne-Carine Debette and an unknown reviewer for critical revision of the manuscript, as well as Thomas Kilcline for writing assistance. Table 1 Antihypertensive agents.

The future availability and low cost of technology will allow for the screening of a large number of genetic determinants. This will lead to the description of polymorphisms that affect drug pharmacokinetics and dynamics in each given patient. Furthermore, this information will optimize the efficacy/toxicity ratio. Although promising, the results of pharmacogenetic studies need to be confirmed in prospective randomised trials of comparative effectiveness, comparing the classical clinical and the genotype-guided approach, before being used in clinical settings. Furthermore, no study has explored yet the clinical usefulness of the genetic approach in reducing adverse events. For these reasons, although promising, the genotype-guided approach for drug prescriptions is not routinely recommended [56]. Acknowledgments The authors would like to gratefully thank Dr. Stephanie Anne-Carine Debette and an unknown reviewer for critical revision of the manuscript, as well as Thomas Kilcline for writing assistance. Table 1 Antihypertensive agents. Name Outcome Gene and variant Sample size/drugs used Effect estimates and significance levels INVEST-GENES [8] Death/MI or stroke GRK2 SNPs (rs1894111 G > A) GRK5 Gln41Leu 48/Verapamil SR, atenolol GRK5 41Leu decreased the risk of adverse cardiovascular outcome adjusted independently of treatment (OR 0.535, 95% CI: 0.313–0.951)

ome Gene and variant Sample size/drugs used Effect estimates and significance levels INVEST-GENES [8] Death/MI or stroke GRK2 SNPs (rs1894111 G > A) GRK5 Gln41Leu 48/Verapamil SR, atenolol GRK5 41Leu decreased the risk of adverse cardiovascular outcome adjusted independently of treatment (OR 0.535, 95% CI: 0.313–0.951) INVEST-GENES [9] Death/MI or stroke KCNMB1 Glu65Lys KCNMB1 Val110Leu 5979 with HTN/Verapamil SR, atenolol KCNMB1 110Leu had reduced risk of composite outcome (HR 0.68 (95% CI 0.47–0.998); this effect was higher in Verapamil SR (HR 0.587, 95% CI 0.33–1.04) than atenolol-treated patients (HR 0.946, 95% CI 0.56–1.59) LIFE substudy [10] Cardiovascular events 13 polymorphisms (angiotensin-converting enzyme I/D, α-adducin Gly460Trp, β1-adrenergic receptor Gly389Arg, β2-adrenergic receptor Arg16Gly, β2-adrenergic receptor Gln27Glu, β3-adrenergic receptor Trp64Arg, angiotensinogen Met235Thr, aldosterone synthase promoter C-344T, type 1 angiotensinogen receptor A1166C, bradykinin 2 receptor I/D, CYP2C9∗2 versus CYP2C9∗1,  CYP2C9∗3 versus CYP2C9∗1, G protein β3-subunit C825T) 3503/Losartan, atenolol No significant genotype-drug interaction on the outcome GEN-HAT [11] Primary: fatal CHD/nonfatal MI. Secondary: stroke, all-cause mortality, combined CHD, and combined cardiovascular disease ACE I/D 37,939/chlorthalidone, amlodipine, lisinopril, or doxazosin No significant association with the outcome was reported; no significant gene-drug interaction was found

LIFE substudy [10] Cardiovascular events 13 polymorphisms (angiotensin-converting enzyme I/D, α-adducin Gly460Trp, β1-adrenergic receptor Gly389Arg, β2-adrenergic receptor Arg16Gly, β2-adrenergic receptor Gln27Glu, β3-adrenergic receptor Trp64Arg, angiotensinogen Met235Thr, aldosterone synthase promoter C-344T, type 1 angiotensinogen receptor A1166C, bradykinin 2 receptor I/D, CYP2C9∗2 versus CYP2C9∗1,  CYP2C9∗3 versus CYP2C9∗1, G protein β3-subunit C825T) 3503/Losartan, atenolol No significant genotype-drug interaction on the outcome GEN-HAT [11] Primary: fatal CHD/nonfatal MI. Secondary: stroke, all-cause mortality, combined CHD, and combined cardiovascular disease ACE I/D 37,939/chlorthalidone, amlodipine, lisinopril, or doxazosin No significant association with the outcome was reported; no significant gene-drug interaction was found GEN-HAT [12] Primary: fatal CHD/nonfatal MI. Secondary: stroke, all-cause mortality, combined CHD, and 6-mos systolic and diastolic BP changes NPPA SNP T2238C (rs5065) NPPA SNP G664A (rs5063) 38,462 with HTN/chlorthalidone, amlodipine, lisinopril, or doxazosin NPPA T2238C TT variant x  “chlorthalidone versus amlodipine” interaction was significantly associated stroke (HR 1.09 95% CI 0.95–1.26). NPPA T2238C TT variant x “chlorthalidone versus amlodipine + lisinopril” interaction was significantly associated with stroke (HR 1.09 95% CI 0.95–1.26). NPPA T2238C CC variant x “chlorthalidone versus amlodipine” interaction was significantly associated with stroke (HR 1.18 95% CI 0.72–1.90). Either NPPA T2238C variant or NPPA G664A was not significantly associated with stroke and “chlorthalidone versus lisinopril,” “chlorthalidone versus doxazosin”

95% CI 0.95–1.26). NPPA T2238C CC variant x “chlorthalidone versus amlodipine” interaction was significantly associated with stroke (HR 1.18 95% CI 0.72–1.90). Either NPPA T2238C variant or NPPA G664A was not significantly associated with stroke and “chlorthalidone versus lisinopril,” “chlorthalidone versus doxazosin” GEN-HAT [13] Primary: fatal CHD/nonfatal MI. Secondary: stroke, heart failure, all-cause mortality, end-stage renal disease FGB G455A 30 076 with HTN/chlorthalidone, amlodipine, lisinopril Common GG homozygotes had higher stroke risk on lisinopril versus amlodipine (HR 1.38, P < 0.001); variant A allele carriers had slightly lower risk on lisinopril versus amlodipine (HR 0.96, P value for interaction = 0.03) Lemaitre et al. [14] MI, ischemic stroke ADRB1 (Seven SNPs plus haplotype), ADRB2 (five SNPs plus haplotypes) 938 cases with MI or stroke/beta blocker beta1-AR gene variation and beta-blocker use showed a positive interaction on ischemic stroke risk (P = 0.04). Homozygosis or heterozygosis for rs#2429511 variant was associated with higher MI/stroke combined risk in beta-blocker users (OR 1.24 95% CI 1.03–1.50). No interaction of ADRB2 with beta-blocker use and outcomes Rotterdam study [15] MI, stroke AGT (M235T) 4097 with HTN/ACEI, BB No significant gene-drug interaction was found on stroke Rotterdam study [16] MI, stroke AGTR1 (C573T) ACE (I/D) 4097 with HTN/ACEI, BB No significant AGTR1 and ACE I/D interaction on stroke risk with ACEI or BB

Lemaitre et al. [14] MI, ischemic stroke ADRB1 (Seven SNPs plus haplotype), ADRB2 (five SNPs plus haplotypes) 938 cases with MI or stroke/beta blocker beta1-AR gene variation and beta-blocker use showed a positive interaction on ischemic stroke risk (P = 0.04). Homozygosis or heterozygosis for rs#2429511 variant was associated with higher MI/stroke combined risk in beta-blocker users (OR 1.24 95% CI 1.03–1.50). No interaction of ADRB2 with beta-blocker use and outcomes Rotterdam study [15] MI, stroke AGT (M235T) 4097 with HTN/ACEI, BB No significant gene-drug interaction was found on stroke Rotterdam study [16] MI, stroke AGTR1 (C573T) ACE (I/D) 4097 with HTN/ACEI, BB No significant AGTR1 and ACE I/D interaction on stroke risk with ACEI or BB INVEST-GENES [17] Death/nonfatal MI/nonfatal stroke ADRB1 (Ser49GLy, Arg389Gly) and ADRB2 (Gly16Arg, Gln27Glu, 523 C > A) 5,895 CAD patients/Verapamil SR, atenolol No association between any haplotype and treatment on stroke INVEST-GENES [18] Death/nonfatal MI/nonfatal stroke NOS3-786T > C (rs2070744), NOS3 Glu298 > Asp (rs1799983) 258 death/MI/stroke versus 774 control No genetic interaction with drugs and composite outcome Psaty et al. [19] MI/nonfatal stroke ADD1 (Gly460Trp) Cases versus controls ADD1 Trp460 variant had lower stroke risk on diuretics (OR, 0.49; 95% CI, 0.32–0.77). The point estimate of diuretic-adducin interaction was SI 0.45 (95% CI 0.26–0.79) for the combined outcome MI and stroke; separate analyses yielded similar results: MI (SI 0.41 95% CI 0.21–0.80) and stroke (SI 0.53 95% CI 0.24–1.19)

s ADD1 Trp460 variant had lower stroke risk on diuretics (OR, 0.49; 95% CI, 0.32–0.77). The point estimate of diuretic-adducin interaction was SI 0.45 (95% CI 0.26–0.79) for the combined outcome MI and stroke; separate analyses yielded similar results: MI (SI 0.41 95% CI 0.21–0.80) and stroke (SI 0.53 95% CI 0.24–1.19) INVEST-GENES [20] Death/nonfatal MI/nonfatal stroke ADD1 Gly460Trp 5,979 CAD patients/Verapamil SR, atenolol ADD1 Trp460 black carriers had higher combined outcome risk (aHR 2.62, 95% CI 1.23–5.58), compared to whites (aHR 1.24 95% CI 0.90–1.71) and Hispanics (aHR 1.43 95% CI 0.86–2.39). No significant interaction between the ADD1 polymorphism and diuretic use for either primary outcome or secondary outcomes PROGRESS [21] Fatal and nonfatal stroke (ischemic or hemorrhagic), nonfatal MI/coronary death, composite nonfatal stroke/nonfatal MI/vascular death, all-cause mortality, dementia, and cognitive decline ACE I/D 5688 with stroke or TIA/perindopril No associations between ACE genotypes and cerebrovascular disease history or cardiovascular risk factors was demonstrated. The ACE genotype was not associated with the long-term risks of stroke. The ACE genotype did not modify the relative benefits of perindopril over placebo Table 2 Statins.

stroke or TIA/perindopril No associations between ACE genotypes and cerebrovascular disease history or cardiovascular risk factors was demonstrated. The ACE genotype was not associated with the long-term risks of stroke. The ACE genotype did not modify the relative benefits of perindopril over placebo Table 2 Statins. Author's name/study name Outcome Gene (variant) Sample size/drug Effect estimates and findings Hindorff et al. [22] Nonfatal MI/nonfatal stroke ABCB1, CETP, HMGCR, LDLR, LIPC, NOS3 865 with MI, 368 with stroke and 2686 controls/statins No gene-statin interactions for stroke. 5 SNP-statin interactions on stroke (one CETP, four LIPC); no gene level association for stroke; SNP level association: two SNPs (one CETP, one LDLR) for stroke. The highest significance was found for stroke in CETP rs5883 carriers on simvastatin (OR 3.60 95% CI 1.22–7.70) Heart protection study [23] Major coronary event (coronary death or nonfatal MI), major vascular event (major coronary event plus revascularization or stroke) KIF6 Trp719Arg polymorphism (rs20455) on vascular risk and response to statin therapy in from of the heart protection study 18,348 participants/simvastatin No significant gene-statin interaction with any of the outcome, including stroke Rotterdam study [24] Death, MI, stroke Apo E (E2, E3, E4) 7983 older than 55 yo/statins No significant gene-statin interaction with any of the outcome. Statins reduce stroke risk (aOR 0.50 95% CI 0.28–0.91) independently of Apo E genotype

Heart protection study [23] Major coronary event (coronary death or nonfatal MI), major vascular event (major coronary event plus revascularization or stroke) KIF6 Trp719Arg polymorphism (rs20455) on vascular risk and response to statin therapy in from of the heart protection study 18,348 participants/simvastatin No significant gene-statin interaction with any of the outcome, including stroke Rotterdam study [24] Death, MI, stroke Apo E (E2, E3, E4) 7983 older than 55 yo/statins No significant gene-statin interaction with any of the outcome. Statins reduce stroke risk (aOR 0.50 95% CI 0.28–0.91) independently of Apo E genotype GenHAT [25] Primary outcome: all-cause mortality, secondary outcomes (fatal CHD and nonfatal MI, CVD mortality, CHD, stroke, other CVD, non-CVD mortality, stroke, and heart failure) ACE (I/D) 9467/pravastatin No significant gene-statin interaction with any of the outcome Table 3 Tissue plasminogen activator. Author's name/study name Outcome Gene (Variant) Sample Size/drug Effect estimates and findings Broderick et al. 2001 [26] Favourable outcome (NIHSS of 0 or 1, Barthel Index of 95 or 100, Modified Rankin Scale of 0 or 1, and a Glasgow Outcome Scale of 1.) ApoE (E2, E3, E4) 409 ischemic stroke/rTPA versus PB within 3 hours ApoE E2 phenotype-rt-PA interaction was associated with good outcome at 3 months (OR: 6.4; 95% CI: 2.7–15.3). Apo E4 phenotype not related to favorable 3 month outcome, response to t-PA, 3-month mortality, or risk of intracerebral hemorrhage

ale of 1.) ApoE (E2, E3, E4) 409 ischemic stroke/rTPA versus PB within 3 hours ApoE E2 phenotype-rt-PA interaction was associated with good outcome at 3 months (OR: 6.4; 95% CI: 2.7–15.3). Apo E4 phenotype not related to favorable 3 month outcome, response to t-PA, 3-month mortality, or risk of intracerebral hemorrhage Fernández-Cadenas et al. [27] Recanalization rate, NIHSS at 48 hours and mRS score at 3 months, heamorrhagic transformation ApoE (E2, E3, E4) 77 ischemic stroke/rTPA within 3 hours No significant association of ApoE genotype and the studied outcome Del Río Espínola et al. [28] Reocclusion rate 236 SNPs form candidate genes for vascular risk factor 222 ischemic stroke/rTPA rs1883832 SNP from CD40 gene (OR: 0.077; 95% CI: 0.009–0.66) and rs1800801 SNP from the MGP gene (OR 15.25; 95% CI: 2.23–104.46) were independently associated with reocclusion after adjustment for clinical predictors Fernández-Cadenas et al. [29] Recanalization ACE (I/D) 96 ischemic stroke/rTPA within 3 hours ACE DD homozygosis was significantly associated with recanalization rate following rTPA (OR: 4.3 95% CI: 1.35–13.49). No relation was found between ACE I/D polymorphism and symptomatic hemorrhagic complications. No association between ACE genotypes and Factor VII or Factor X activities

chemic stroke/rTPA within 3 hours ACE DD homozygosis was significantly associated with recanalization rate following rTPA (OR: 4.3 95% CI: 1.35–13.49). No relation was found between ACE I/D polymorphism and symptomatic hemorrhagic complications. No association between ACE genotypes and Factor VII or Factor X activities Fernández-Cadenas et al. [30] Recanalization PAI-1 4G/5G TAFI (Thr325Ile) 139 with ischemic stroke/TPA within 3 hours PAI-1 4 G/5 G was not associated with recanalization. TAFI Thr325Ile polymorphism was associated with recanalization resistance (OR 5.6 95% CI 1.2–20). Combination of TAFI and PAI-1 polymorphisms double the risk of absence of recanalization (OR: 11.1; 95% CI: 1.4–89.8) González-Conejero et al. [31] Death, recanalization Factor XIII (FXIII) V34L 200 with ischemic stroke/TPA within 3 hours FXIII 34 L carriers had higher death risk than V/V (OR 2.50 95% CI 1.00–7.06); high fibrinogen levels higher risk than lower levels (OR 2.72 95% CI 1.01–7.44); FXIII 34L and high fibrinogen level higher risk than FXII V and low fibrinogen (OR 5.74 95% CI 1.51–11.56). No difference in recanalization rate Montaner et al. [32] Hemorrhagic transformation MMP9 (C1562T) 61 with ischemic stroke/TPA within 3 hours The polymorphism studied does not increase hemorrhagic risk Table 4 Anticoagulants and antiplatelets.

González-Conejero et al. [31] Death, recanalization Factor XIII (FXIII) V34L 200 with ischemic stroke/TPA within 3 hours FXIII 34 L carriers had higher death risk than V/V (OR 2.50 95% CI 1.00–7.06); high fibrinogen levels higher risk than lower levels (OR 2.72 95% CI 1.01–7.44); FXIII 34L and high fibrinogen level higher risk than FXII V and low fibrinogen (OR 5.74 95% CI 1.51–11.56). No difference in recanalization rate Montaner et al. [32] Hemorrhagic transformation MMP9 (C1562T) 61 with ischemic stroke/TPA within 3 hours The polymorphism studied does not increase hemorrhagic risk Table 4 Anticoagulants and antiplatelets. Author's name/study name Outcome Gene (Variant) Sample size/drug Effect estimates and findings Anticoagulants Arnold et al. [33] Dose finding VKORC1 C283 + 837C → T (rs2359612) 49 with cerebrovascular disease/phenprocoumon VKORC1 TT carriers reached an INR of 2-3 after a mean time of 3.2 days (n = 5), CT carriers after 4.4 days (n = 27), and CC carriers after 6.5 days (n = 15) Kimura et al. [34] Warfarin maintenance dose (VKORC1), gamma-glutamyl carboxylase (GGCX), calumenin (CALU), and cytochrome P450 2C9 (CYP2C9) 93 Japanese on stable anticoagulation therapy 1639 G > A (P = 0.004) and 3730 G > A genotypes (P = 0.006) in VKORC1, the 8016 G > A genotype in GGCX (P = 0.022), and the 42613 A > C genotype in CYP2C9 (P = 0.015) were associated with effective warfarin dose Antiplatelets Meta-analysis of 9 different studies (CLARITY TIMI 28, EXCLESIOR, TRITION TIMI 38, AFIJI, FASSTS-MI, RECLOSE, ISAR, CLEAR PLATELETS, Intermountain) [35] Composite outcome (cardiovascular death/MI/stroke) and stent thrombosis CYP2C19/1 or 2 reduced function alleles (∗2, ∗3, ∗4, ∗5, ∗6, ∗7, and ∗8) 9685 patients (91% had PCI, 54% had ACS)/clopidogrel Carriers of 1 (HR 1.55; 95% CI, 1.11–2.17) or 2 (HR 1.76; 95% CI, 1.24–2.50) reduced-function CYP2C19 alleles had higher risk of composite outcome events TRITON-TIMI 38 [36] Composite outcome (cardiovascular death/MI/ischemic stroke) CYP2C19/1 or 2 reduced function alleles (∗2, ∗3, ∗4, ∗5, ∗6, ∗7, and ∗8) ABCB1/3435C → T 2932 patients with ACS undergoing PCI/clopidogrel versus prasugrel TT homozygotes of ABCB1 genotype had increased risk of the composite outcome compared to CT or CC carriers (HR 1.72, 95% CI 1.22–2.44). Carriers of a CYP2C19 reduced-function allele only (Kaplan-Meier event rate 11.5%), ABCB1 3435 TT homozygotes only (Kaplan-Meier event rate 12.6%), or both (Kaplan-Meier event rate 13.6%) had increased risk of composite outcome (pooled HR 1.97, 95% CI 1.38–2.82).

mpared to CT or CC carriers (HR 1.72, 95% CI 1.22–2.44). Carriers of a CYP2C19 reduced-function allele only (Kaplan-Meier event rate 11.5%), ABCB1 3435 TT homozygotes only (Kaplan-Meier event rate 12.6%), or both (Kaplan-Meier event rate 13.6%) had increased risk of composite outcome (pooled HR 1.97, 95% CI 1.38–2.82). No significant genotype-prasugrel interaction was reported PLATO [37] Composite outcome (cardiovascular death/MI/stroke) CYP2C19/1 or 2 reduced function alleles ABCB1/3435C → T 10285 patients with ACS undergoing non-CABG/clopidogrel versus ticagrelor Either with (HR 0.77, 95% CI 0.60–0.99) and without (0.86, 0.74–1.01, P = 0.0608) any CYP2C19 reduced-function alleles patients on ticagrelor experienced lower risk of composite outcome compared to patients on clopidogrel (interaction P = 0.46). Independently of ABCB1 genotype, patients on ticagrelor had lower risk of the composite outcome compared to clopidogrel users (interaction P = 0.39; HR 0.71, 95% CI 0.55–0.92). No significant interaction was found on treatment and genotype regarding major bleeding PAPI study and Mount Sinai study [38] Composite outcome (cardiovascular death, MI, ischemic stroke, stent thrombosis, unplanned target vessel revascularization, unplanned nontarget vessel revascularization, hospitalization for coronary ischemia) GWA 429 white healthy Amish individuals/clopidogrel; results replicated in 227 undergoing PCI 13 SNPs in 10q24 region, where CYP2C18–CYP2C19–CYP2C9–CYP2C8 gene cluster is found, were associated with reduced response to clopidogrel. CYP2C19∗2 allele carriers were at higher risk for composite outcome (adjusted HR 2.42 95% CI 1.18–4.99) Clappers et al. [39] Composite outcome (cardiovascular death/MI/stroke) COX-1/C50T 496 admitted to Coronary Care Unit for different reasons/aspirin No interaction was found on genotype and aspirin for the composite outcome Hillarp et al. [40] n.a. COX-1/C116T, del 137–142, C144T, G6841A, G7331C, A7742A, C10427A, C10608A, del 10675A, G12254A, T12378C, G19187A, C19242T, G19255A 68 with recurrent stroke/ASA 14 variants of the Cox-1 gene were identified and 7 involved amino acid substitutions of the Cox-1 molecule.

for the composite outcome Hillarp et al. [40] n.a. COX-1/C116T, del 137–142, C144T, G6841A, G7331C, A7742A, C10427A, C10608A, del 10675A, G12254A, T12378C, G19187A, C19242T, G19255A 68 with recurrent stroke/ASA 14 variants of the Cox-1 gene were identified and 7 involved amino acid substitutions of the Cox-1 molecule. None of the mutations were located near the catalytic site ABCB1: ATP-binding cassette subfamily B, ACEI: angiotensin convertin enzyme inhibitors, ACE I/D: angiotensin convertin enzyme insertion/deletion, ACS: acute coronary syndrome, ADD1: α-adducin, ADRB: β-adrenergic receptor, AGT: angiotensinogen, AGTR1: angiotensin receptor II type 1, APO E: apolipoprotein E, BP: blood pressure, CABG: coronary artery bypass graft, CAD: coronary heart disease, CD: cluster of differentiation, CEPT: cholesteryl ester transfer protein, CHD: coronary artery disease, COX: cyclooxygenase, CVD: cerebrovascular disease, CYP: cytochrome P, FGB: fibrinogen beta, GRK: G-protein-coupled receptor kinase, GWA: genome-wide association, HMG-CoR: hydroxyl-methylcoenzyme A reductase, HR: hazard ratio, HTN: hypertension, KCNMB: conductance calcium and voltage-dependent potassium channel, KIN 6: kinesin family member 6, LDLR: low-density lipoprotein receptor, LIPC: human hepatic lipase, MGP: matrix Gla protein, MI: myocardial infarction, MMP: matrix metalloproteinase, NIHSS: National Institute of Health stroke scale, NOS: nitric oxide synthase, NPPA: atrial natriuetic polypeptide precursor, OR: odds ratio, PAI: plasminogen activator inhibitor, PCI: percutaneous coronary intervention, SI: sinergy index, TAFI: thrombin-activable fibrinolysis inhibitor, verapamil SR: verapamil-sustained release, VKORC1: vitamin K epoxide reductase complex subunit 1.

1. Introduction The introduction of intravenous recombinant tissue plasminogen activator (rtPA) has revolutionized the management of acute ischemic stroke (AIS). Treatment with rtPA has been shown to improve patients' outcomes at 3 months; however, its effectiveness decreased with time from the onset of stroke symptoms [1, 2]. Many stroke patients eligible for thrombolysis were not treated appropriately because of delayed presentation to the hospital or delayed examinations and management in the hospital. Although delays are mainly caused by patients themselves [3], it should be possible to minimize the in-hospital delay. According to the recommendations made by the National Institute of Neurological Disorders and Stroke, a patient with AIS should receive rtPA within 60 minutes of arrival at the emergency department (ED) [4]. A pilot study to address the quality of acute stroke care in 4 states of the US found that less than 20% of the patients treated with intravenous rtPA received it within 60 minutes of arrival [5]. A quasi-experimental trial (The Stroke Practice Improvement Network) to improve adherence to stroke performance measures concluded that the implementation of site-specific interventions did not increase the proportion of delivery of thrombolytic therapy within one hour of hospital arrival during the 6-month intervention period [6].

imental trial (The Stroke Practice Improvement Network) to improve adherence to stroke performance measures concluded that the implementation of site-specific interventions did not increase the proportion of delivery of thrombolytic therapy within one hour of hospital arrival during the 6-month intervention period [6]. In Taiwan, only a minority of stroke patients are treated with rtPA. A nationwide study (Taiwan Stroke Registry) showed that 10.42% of AIS patients arriving within 2 hours of onset were treated with rtPA [7]. Although a study indicated that the adoption of less restrictive exclusion criteria for rtPA significantly increased the number of patients eligible for thrombolysis, there were still only 6.3% of patients who arrived within 3 hours of stroke onset received thrombolytic therapy [8]. Insufficient time to complete required studies was a main reason for exclusion from rtPA. Thus, this study was aimed to determine if the modification of protocol shortened the in-hospital delay and facilitated thrombolytic therapy.

f patients who arrived within 3 hours of stroke onset received thrombolytic therapy [8]. Insufficient time to complete required studies was a main reason for exclusion from rtPA. Thus, this study was aimed to determine if the modification of protocol shortened the in-hospital delay and facilitated thrombolytic therapy. 2. Materials and Methods This was a before-and-after study to investigate the effectiveness of implementation of a new thrombolysis protocol. Our institution is a 1000-bed community hospital serving a city and its adjoining rural area of around 500,000 inhabitants in southern Taiwan. The study population consisted of all AIS patients directly presenting to the ED within 3 hours of stroke onset in the one-year period from October 2009 to September 2010 (Period II) after the implementation of the new thrombolysis protocol. The major modification is that a nurse practitioner (NP) was designated to coordinate the newly designed parallel pathway for candidate patients. The control group comprised those patients who presented in the two-year period from October 2007 to September 2009 (Period I). The number of neurologists (five) on the acute stroke team and the number of computed tomography (CT) scanners (two) in the study hospital did not change during these two periods.

te patients. The control group comprised those patients who presented in the two-year period from October 2007 to September 2009 (Period I). The number of neurologists (five) on the acute stroke team and the number of computed tomography (CT) scanners (two) in the study hospital did not change during these two periods. A standardized data abstraction form has been used in the registration of stroke patients in our institute since September 2006. We recorded the demographics, clinical characteristics, laboratory findings, radiological characteristics, and medications before and during hospitalization of the patients. The stroke severity was recorded on presentation by National Institute of Health Stroke Scale (NIHSS). The exact time of arrival at ED, evaluation by neurologists, receiving CT scans, and onset of thrombolysis were collected prospectively by a trained study nurse. The details in Taiwan Stroke Registry with the similar design had been described elsewhere [7]. The outcome recorded in this study included mortality and functional status at discharge, presented by modified Rankin Scale (mRS) score. The status at 3 months after discharge was obtained by the study nurse from the medical record or personal/telephone interview. The eligibility of thrombolysis for each patient was reviewed retrospectively by two senior neurologists according to the exclusion criteria set by the Department of Health and Bureau of National Health Insurance in Taiwan [8]. Every patient had a follow-up head CT scan 24 hours after thrombolytic therapy. To evaluate the safety of thrombolysis, we defined symptomatic intracranial hemorrhage (SICH) as any hemorrhage plus a neurological deterioration of 4 or more points on the NIHSS [9]. The data collection had been approved by the Institutional Review Board.

d a follow-up head CT scan 24 hours after thrombolytic therapy. To evaluate the safety of thrombolysis, we defined symptomatic intracranial hemorrhage (SICH) as any hemorrhage plus a neurological deterioration of 4 or more points on the NIHSS [9]. The data collection had been approved by the Institutional Review Board. By analyzing our original thrombolysis protocol designed in 2007 (Figure 1(a)), we divided the door-to-needle time (DNT) into three steps: from ED arrival to CT scanning, from CT scanning to neurology evaluation, and from neurology evaluation to start of thrombolysis. The median time of obtaining a CT scan had been within 25 minutes before this study [10]. The major in-hospital delays occurred in the latter two steps. The prior protocol was an inefficient sequential algorithm (Figure 1(a)). Therefore, we implemented a parallel protocol (Figure 1(b)) to minimize the delays, and ED NPs were incorporated as coordinators into the acute stroke team to collaborate with the physicians and other departments.

ccurred in the latter two steps. The prior protocol was an inefficient sequential algorithm (Figure 1(a)). Therefore, we implemented a parallel protocol (Figure 1(b)) to minimize the delays, and ED NPs were incorporated as coordinators into the acute stroke team to collaborate with the physicians and other departments. We have a total of five NPs working morning and evening shifts in the ED. The ED NPs help take care of all emergency patients. However, once a patient was suspected to have AIS at the triage desk, an NP was assigned to this patient until rtPA was administered or the diagnosis was proven otherwise. If the patient was a candidate for thrombolysis based on the screening criteria, the designated NP would soon notify the on-call neurologist by telephone before the patient was sent for noncontrast CT scanning. In addition, the NP coordinated the patient care, including initial assessment of NIHSS and evaluation of suitability for thrombolysis, collections of CT images and results of laboratory tests, and preliminary explanation regarding the benefits and risks of thrombolytic therapy to the patients and/or their family. Hence multiple tasks can be done in parallel by the collaboration of the ED physician, the NP, the on-call neurologist, and other departments.

llections of CT images and results of laboratory tests, and preliminary explanation regarding the benefits and risks of thrombolytic therapy to the patients and/or their family. Hence multiple tasks can be done in parallel by the collaboration of the ED physician, the NP, the on-call neurologist, and other departments. While the neurologist was evaluating a candidate patient for thrombolytic therapy, rtPA was brought to the bedside unmixed pending further treatment decision making. If the patient was determined eligible for thrombolysis, rtPA was administered immediately after informed written consent was obtained from the patient or next of kin. If thrombolysis was not indicated, the drug box was returned unopened to the pharmacy. To investigate the effectiveness of the new protocol, we assessed time intervals between ED arrival and actions including: CT scan, reports of blood tests, neurology evaluation, and thrombolysis. To monitor the efficiency of thrombolysis over time, we computed the running median of DNTs at 3-month intervals in the control and study periods. The patients with mRS 0-1 were considered as having a favorable functional outcome.

tions including: CT scan, reports of blood tests, neurology evaluation, and thrombolysis. To monitor the efficiency of thrombolysis over time, we computed the running median of DNTs at 3-month intervals in the control and study periods. The patients with mRS 0-1 were considered as having a favorable functional outcome. Median values and interquartile ranges of the time intervals were used for descriptive statistics because of their nonnormal distributions. Comparison of median values was done with the Mann-Whitney test. Student's t-test was used to evaluate differences in continuous variables with normal distribution. Chi-square test or Fisher's exact test was used as appropriate to compare categorical data. A value of P < 0.05 was regarded as significant. All statistical analyses were performed using Windows SPSS version 15.0 (SPSS Inc., Chicago, ILL, USA).

d to evaluate differences in continuous variables with normal distribution. Chi-square test or Fisher's exact test was used as appropriate to compare categorical data. A value of P < 0.05 was regarded as significant. All statistical analyses were performed using Windows SPSS version 15.0 (SPSS Inc., Chicago, ILL, USA). 3. Results In Period I, a total of 1062 AIS patients were admitted, with 338 patients arriving within 3 hours. They were examined using the original thrombolysis protocol. Of these 338 patients, 52 (15.4%) patients were eligible for thrombolysis. In Period II, 586 patients with AIS were admitted and 139 of them arrived within 3 hours. Twenty (14.4%) patients were indicated to have thrombolysis. Common reasons for exclusion from treatment included age over 80 years, minor or rapidly improving stroke, severe stroke, history of both diabetes and prior ischemic stroke, and elevated blood pressure (Figure 2). During Period I, one patient refused thrombolysis and 11 patients were either considered ineligible by ED doctors or were unable to complete CT and laboratory studies in time. During Period II, in addition to the 20 eligible patients, one patient who met the exclusion criteria because of age was treated as per family request. Therefore, for patients within 3 hours of onset, the treatment rate increased from 11.8% (40/338) to 15.1% (21/139) between Periods I and II (Table 1). There was no significant difference in the proportion of eligible patients.

one patient who met the exclusion criteria because of age was treated as per family request. Therefore, for patients within 3 hours of onset, the treatment rate increased from 11.8% (40/338) to 15.1% (21/139) between Periods I and II (Table 1). There was no significant difference in the proportion of eligible patients. For all the patients with AIS directly presenting to the ED within 3 hours of stroke onset, the median time from arrival to CT examination decreased significantly from 29 to 20 minutes. At the same time, the median time from arrival to neurology evaluation was reduced considerably from 61 to 43 minutes (Table 1). For those patients treated with rtPA, the implementation of the new protocol significantly reduced the door-to-neurology evaluation time from 46 to 37 minutes and the DNT from 68.5 to 58 minutes (Table 2). The 3-month running median of DNTs also decreased from 104 to 40 minutes over the 3-year period (Figure 3). The onset-to-needle time was not changed despite longer onset-to-door time during Period II, reflecting that we administered rtPA to more patients with delayed presentation of more than 2 hours after onset (19% in Period II versus none in Period I). This factor also contributes to a nonsignificant increase in the proportion of thrombolysed patients. The time from arrival to report of prothrombin time/partial thromboplastin time, remained around 50 minutes. Baseline NIHSS scores and the proportion of patients with favorable outcome after 3 months did not change between the two periods.

ributes to a nonsignificant increase in the proportion of thrombolysed patients. The time from arrival to report of prothrombin time/partial thromboplastin time, remained around 50 minutes. Baseline NIHSS scores and the proportion of patients with favorable outcome after 3 months did not change between the two periods. 4. Discussion We demonstrated the significant effect on shortening DNT of rtPA administration in this before-and-after comparison in the same hospital. The major changes between the two periods are the implementation of the parallel protocol and the introduction of NPs as coordinators in ED. Since intravenous rtPA should be administered within the narrow 3-hour time window, a substantial proportion of patients who arrived early did not receive thrombolytic therapy owing to in-hospital delays including: delayed physician evaluation, neurologic consultation, neuroimaging, and laboratory tests [11]. In-hospital delays can be shortened through the organization of a stroke team, the development of stroke pathways, and the training of ED personnel [12]. Although recent studies proved that rtPA is effective up to 4.5 hours after stroke onset [13], its effectiveness decays with the time between onset and treatment [2]. Therefore, every effort should be taken to hasten the start of the treatment, and the target treatment with rtPA should be within one hour of patient's arrival in the ED [14].

s proved that rtPA is effective up to 4.5 hours after stroke onset [13], its effectiveness decays with the time between onset and treatment [2]. Therefore, every effort should be taken to hasten the start of the treatment, and the target treatment with rtPA should be within one hour of patient's arrival in the ED [14]. By using a parallel strategy to overcome the in-hospital delays, our revised protocol effectively reduced the median time of DNT below the recommended 60 minutes. Rapid identification of potential candidates for thrombolysis is the paramount first step in order that neurology evaluation, CT scans, and laboratory studies can be arranged immediately upon ED arrival. However, this poses a challenge to busy ED physicians. In Period II, an NP was soon assigned to a thrombolytic candidate upon hospital arrival. After preliminary screening, the NP notified the neurologist who made the decision of thrombolysis. This practice could prevent a busy ED physician from excluding patients who actually qualify for treatment because of delayed examinations or unfamiliarity with rtPA eligibility criteria, as we have shown that 21% (11/52) of eligible patients were not treated in Period I but all eligible patients were treated in Period II. In one study, although the agreement for determination of rtPA eligibility was good between ED attendings and stroke neurologists, 18% of thrombolysis candidates were still designated as ineligible by ED attendings and the proportion was even higher for ED residents [15].

ll eligible patients were treated in Period II. In one study, although the agreement for determination of rtPA eligibility was good between ED attendings and stroke neurologists, 18% of thrombolysis candidates were still designated as ineligible by ED attendings and the proportion was even higher for ED residents [15]. Physicians who are not neurologists tend not to give patients thrombolytic treatment [16]. ED physicians usually did not administer rtPA until a neurologist was called in for consultation, considering the high rate of SICH among the Chinese-Taiwanese [17]. Because of the limited personnel and funding resources in a community hospital setting, neurologists are not available on a 24/7 basis at our institution. Since a team approach is important for the successful implementation of a stroke protocol [18] and NP care has been shown to increase compliance with clinical practice guidelines [19], we incorporated ED NPs into our acute stroke tame. The Calgary Stroke Program has demonstrated that the use of stroke NPs reduced the DNT from 90 to 60 minutes and the door-to-CT time from 60 to 30 minutes [20]. Nonetheless, the role of NPs in our stroke team is different from that in the Calgary Stroke Program in two aspects. First, the NPs are not dedicated stroke NPs and they also carry out routine jobs in the ED. Second, they coordinate and facilitate the whole care process, but not as autonomous ED care providers [21]. The obvious drawbacks are as follows: the neurologists still need to assess the patients in person, and the NPs have to defer their work when a stroke patient arrives. However, this has the advantage of not increasing direct hospital costs.

acilitate the whole care process, but not as autonomous ED care providers [21]. The obvious drawbacks are as follows: the neurologists still need to assess the patients in person, and the NPs have to defer their work when a stroke patient arrives. However, this has the advantage of not increasing direct hospital costs. Thrombolytic therapy inevitably carries the risk of SICH, especially when there is any deviation from the preset criteria [22, 23]. Hence stroke patients must be checked against a lengthy list of exclusion criteria which require a detailed time-consuming history taking. For this purpose, the NP helped complete the exclusion checklist while the neurologist was on the way to the bedside. It could be a concern if shortening of DNT might cause some patients to be thrombolysed without fulfilling strict criteria. In the present study, the reduction in DNT was not associated with an increased proportion of SICH and all the thrombolysed patients were eligible except one patient aged over 80 years. Furthermore, our revised protocol may prevent inappropriate use of rtPA because the exclusion criteria were checked by both the NP and the neurologist. Another concern regarding our practice is that we do not wait for formal CT interpretation by radiologists. Such practice seemed to be safe and did not increase the risks of SICH [24].

revised protocol may prevent inappropriate use of rtPA because the exclusion criteria were checked by both the NP and the neurologist. Another concern regarding our practice is that we do not wait for formal CT interpretation by radiologists. Such practice seemed to be safe and did not increase the risks of SICH [24]. The introduction of a computerized in-hospital alert system has significantly reduced the time intervals from ED arrival to evaluation steps and treatment [25]. However, hospitals have to be equipped with computerized network systems and have to develop the computer program. By adoption of a parallel algorithm and recruitment of NPs into the acute stroke team, our thrombolysis protocol has been a success similar to the computerized system.

evaluation steps and treatment [25]. However, hospitals have to be equipped with computerized network systems and have to develop the computer program. By adoption of a parallel algorithm and recruitment of NPs into the acute stroke team, our thrombolysis protocol has been a success similar to the computerized system. Although the new protocol has shortened the DNT below 60 minutes, it is far from satisfactory because ultraearly thrombolysis (treatment within 70 minutes of stroke onset) resulted in a much higher likelihood of good outcomes for patients with moderate and severe strokes [26]. A recent study has demonstrated that the median DNT could be decreased to only 20 minutes in a university hospital setting [27]. In the present study, the time from arrival to availability of coagulation tests was not improved significantly. Therefore the time spent in the central laboratory blood analysis will be a main limiting factor for further reduction of DNT. One approach is to proceed to treatment pending the results of prothrombin time or partial thromboplastin time unless there is a clinical reason to expect abnormal results of these tests [24]. Another approach is to use point-of-care devices for measurements of international normalized ratio for patients taking oral anticoagulants or when information regarding anticoagulation is unavailable. The use of point-of-care device (Coaguchek XS; Roche, Switzerland) saved an average of 28 minutes in one study [28].

. Another approach is to use point-of-care devices for measurements of international normalized ratio for patients taking oral anticoagulants or when information regarding anticoagulation is unavailable. The use of point-of-care device (Coaguchek XS; Roche, Switzerland) saved an average of 28 minutes in one study [28]. Despite the successful shortening of DNT in this study, the percentage of stroke patients treated with rtPA is still small. To overcome this problem, community programs to educate patients to seek treatment sooner after a stroke should be an integral component of stroke care. In addition, an effective prehospital stroke code system should be established. Prehospital notification by EMS not only shortened the prehospital delay [29], but also reduced DNT [30, 31]. The prenotification ensures that the CT is ready and available for the arriving stroke patient and makes it possible for the stroke neurologist to already be at the ED when the patient arrives. The combined effect of shortening prehospital delay and in-hospital delay would further decrease the treatment time from symptom onset, resulting in improved patients' outcomes.

d available for the arriving stroke patient and makes it possible for the stroke neurologist to already be at the ED when the patient arrives. The combined effect of shortening prehospital delay and in-hospital delay would further decrease the treatment time from symptom onset, resulting in improved patients' outcomes. This study does have several limitations. First, we did not have statistical power to detect a difference in the rates of thrombolysis between the two periods. However, we did demonstrate that shortening of in-hospital delays might increase the number of thrombolysed patients, especially for those who arrived more than two hours after onset. Inclusion of more patients with late arrivals might explain the lack of a decrease in onset-to-needle time in the study period. Second, the improvement in DNT might be partly due to the continuing education and training of the medical staff. Third, we could not show an improvement in the 3-month outcomes of patients. It takes many factors to achieve favorable functional outcomes, including shortened onset-to-needle time. The combined effect of decreased prehospital and in-hospital delays can be explored in future studies. 5. Conclusions It is possible to shorten the time intervals of stroke management by assigning an NP as an ED coordinator of the parallel thrombolysis protocol to overcome the specific sources of delays in a community hospital setting with limited resources and faculty. Our successful model may help to promote the efficient use of NPs in team-based care for acute stroke patients.

f stroke management by assigning an NP as an ED coordinator of the parallel thrombolysis protocol to overcome the specific sources of delays in a community hospital setting with limited resources and faculty. Our successful model may help to promote the efficient use of NPs in team-based care for acute stroke patients. Acknowledgments The authors are very grateful to Eric E. Smith for his helpful comments on an earlier draft. The authors also thank Darren Wu for help in polishing the English of the paper. Figure 1 The original thrombolysis protocol based on a sequential algorithm (a) and the new thrombolysis protocol based on a parallel algorithm (b). Figure 2 Clinical results before (a) and after (b) the implementation of the new thrombolysis protocol. Figure 3 Time series of the three-month running median of door-to-needle times. Table 1 Patients with acute ischemic stroke directly presenting to the emergency department within 3 hours of stroke onset. 2007/10–2009/9 (n = 338) 2009/10–2010/9 (n = 139) P value Age, mean ± SD: y 69.1 ± 12.5   71.9 ± 12.5 0.029 Female, n (%) 136 (40.2) 59 (42.4) 0.656 Time from onset to, median (IQR): min Arrival 65 (34–108) 66 (36–117) 0.217 Time from arrival to, median (IQR): min CT scan 29 (19–50) 20 (13–38) <0.001 Neurology evaluation 61 (40–96) 43 (31–61) <0.001 Eligible for rtPA, n (%) 52 (15.4) 20 (14.4) 0.782 Treated with rtPA, n (%) 40 (11.8) 21 (15.1) 0.331 CT: computed tomography; IQR: interquartile range; SD: standard deviation; rtPA: recombinant tissue plasminogen activator. Table 2 Patients treated with thrombolytic therapy.

2007/10–2009/9 (n = 338) 2009/10–2010/9 (n = 139) P value Age, mean ± SD: y 69.1 ± 12.5   71.9 ± 12.5 0.029 Female, n (%) 136 (40.2) 59 (42.4) 0.656 Time from onset to, median (IQR): min Arrival 65 (34–108) 66 (36–117) 0.217 Time from arrival to, median (IQR): min CT scan 29 (19–50) 20 (13–38) <0.001 Neurology evaluation 61 (40–96) 43 (31–61) <0.001 Eligible for rtPA, n (%) 52 (15.4) 20 (14.4) 0.782 Treated with rtPA, n (%) 40 (11.8) 21 (15.1) 0.331 CT: computed tomography; IQR: interquartile range; SD: standard deviation; rtPA: recombinant tissue plasminogen activator. Table 2 Patients treated with thrombolytic therapy. 2007/10–2009/9 (n = 40) 2009/10–2010/9 (n = 21) P value Age, mean ± SD: y 65.6 ± 12.1 71.3 ± 13.3 0.095 Female, n (%) 12 (30.0) 9 (42.9) 0.315 Pretreatment NIHSS, median 16 18 0.451 Time from onset to, median (IQR): min Arrival 38.5 (22–73) 54 (27–103) 0.151 Thrombolysis 112.5 (95–137) 121 (88–158) 0.796 Arrival between 2 and 3 hours, n (%) 0 (0) 4 (19.0) 0.012 Time from arrival to, median (IQR): min CT scan 16.5 (12–23) 14 (11–19) 0.248 PT/PTT 52 (46–58) 48 (39–60) 0.288 Neurology evaluation 46 (32–63) 37 (28–43) 0.026 Thrombolysis 68.5 (57–83) 58 (54–69) 0.035 ICU admission 133.5 (95–152) 116 (94–143) 0.230 mRS 0-1, n (%) 14 (35.0) 6 (28.6) 0.611 SICH, n (%) 5 (12.5) 2 (9.5) 1.000a aFisher's exact test.

2007/10–2009/9 (n = 40) 2009/10–2010/9 (n = 21) P value Age, mean ± SD: y 65.6 ± 12.1 71.3 ± 13.3 0.095 Female, n (%) 12 (30.0) 9 (42.9) 0.315 Pretreatment NIHSS, median 16 18 0.451 Time from onset to, median (IQR): min Arrival 38.5 (22–73) 54 (27–103) 0.151 Thrombolysis 112.5 (95–137) 121 (88–158) 0.796 Arrival between 2 and 3 hours, n (%) 0 (0) 4 (19.0) 0.012 Time from arrival to, median (IQR): min CT scan 16.5 (12–23) 14 (11–19) 0.248 PT/PTT 52 (46–58) 48 (39–60) 0.288 Neurology evaluation 46 (32–63) 37 (28–43) 0.026 Thrombolysis 68.5 (57–83) 58 (54–69) 0.035 ICU admission 133.5 (95–152) 116 (94–143) 0.230 mRS 0-1, n (%) 14 (35.0) 6 (28.6) 0.611 SICH, n (%) 5 (12.5) 2 (9.5) 1.000a aFisher's exact test. CT: computed tomography; ICU: intensive care unit; IQR: interquartile range; mRS: modified Rankin Scale; NIHSS: National Institutes of Health Stroke Scale; PT: prothrombin time; PTT: partial thromboplastin time; SD: standard deviation; SICH: symptomatic intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator.

Study of stroke among young adults and children has been a relatively neglected area until recently. Stroke in young adults is often considered to be rare, but this misconception is colored by the high incidence of stroke in old people. Approximately 5% of all strokes occur in people younger than 45 years of age, another 5% occur in those 45 to 50 years of age, and 1/4 occur in working aged individuals. Although stroke mortality is lower among the young, their risk to die from their stroke is almost 100 times compared to their nonstroke age counterparts, whereas the same ratio is only 4-fold in the elderly. Similarly, stroke morbidities are lower in the young, but these patients live with their neurological deficits much longer and many have to give up their work and social life. Stroke care in young people is especially demanding because it often affects work, education, and close family to a large extent and because of expected long survival. The study of stroke among young people is important for several reasons. The etiology of stroke is much more diverse in the young compared to old patients. This has therapeutical consequences and may affect outcome both in the short and the long term. Risk factors for stroke differ between young and old patients and may indicate separate approaches as to secondary preventive treatment. Stroke in young adults provides an opportunity to study stroke in general because of less comorbidity than in old patients. This may disclose mechanisms also relevant to older patients.

sk factors for stroke differ between young and old patients and may indicate separate approaches as to secondary preventive treatment. Stroke in young adults provides an opportunity to study stroke in general because of less comorbidity than in old patients. This may disclose mechanisms also relevant to older patients. Stroke in children is rare and is associated with unique challenges. Diagnosis is often delayed because symptoms may be subtle and unspecific. Furthermore, etiology and risk factors in children with stroke differ from young adults with stroke. Once almost neglected, now stroke in children and young adults is under intense research. Started with single-center small studies and shifted to several multicenter collaborations, scientists establish firmly many facades of stroke in children and in the young, including epidemiologic, etiologic, genetic, and prognostic features. Describing risk factor profiles has led to improved treatments. Evidence-based treatments have started to emerge, for example, in Sickle cell disease. New etiologic classifications and one international guideline for childhood stroke have appeared. Few textbooks have been published or are under preparation, and an international meeting dedicated to young stroke is planned.

atments. Evidence-based treatments have started to emerge, for example, in Sickle cell disease. New etiologic classifications and one international guideline for childhood stroke have appeared. Few textbooks have been published or are under preparation, and an international meeting dedicated to young stroke is planned. This special issue is one of the numerous efforts in disseminating state-of-the-art information on the field covering a broad range of important topics as to stroke in children and young adults. It includes two case reports, two research articles, three clinical articles and 15 reviews. Seven articles deal with stroke in children, and 15 articles with stroke in young adults. Several review articles in this special issue stress the importance of extensive investigations in children with stroke including MRI to disclose the underlying etiology and risk factors. Rare causes including diabetic ketoacidosis-associated stroke, cardiac diseases, vascular abnormalities such as Moyamoya disease, but also more general causes such as dissection must be considered. A clinical study concludes that correct prophylaxis reduces the rate of recurrence in children with stroke. Other review articles disclose that more than half of the surviving children have long-term neurological sequels. A research article reports impaired cognitive development and impaired performance as to writing, reading, and arithmetic in children with stroke. A review article reports that the annual incidence of stroke in adults under 45 years ranges between 8.7 and 21 per 100,000. Several review articles show that etiology of cerebral infarction in young adults is varied. Most cerebral infarcts in the elderly are caused by conventional etiologies, for example, large-artery atherosclerotic disease, cerebral embolism (mainly atrial fibrillation), and small-vessel disease, and only 10% are caused by rare etiologies or cause remains undetermined. Conversely, one-fourth of cerebral infarcts in children and young adults are caused by unconventional etiologies and roughly one-third remains undetermined even after a complete workup. Where atrial fibrillation is a common cause of cerebral infarction in old patients, structural heart diseases including patent foramen ovale are frequent in young adults. However, a review article points out that atherosclerosis, which is a common cause of cerebral infarction in old patients, may be threatening even to the young adults.

llation is a common cause of cerebral infarction in old patients, structural heart diseases including patent foramen ovale are frequent in young adults. However, a review article points out that atherosclerosis, which is a common cause of cerebral infarction in old patients, may be threatening even to the young adults. Genetic causes of stroke are more common in young adults than in old adults with stroke. Several review articles report on genetic thrombophilic disorders, genetic connective tissue diseases such as Ehlers-Danlos syndrome, and Fabry disease which is an X-linked lysosomal storage disorder. Stroke in developing countries is associated with etiologies which are uncommon in industrialized countries. Thus, a review article highlights the importance of cardioembolic stroke due to endocarditis in India. Stroke in pregnancy and the puerperium represents unique challenges. Two review articles address this rare but important topic. Previous studies have disclosed a link between migraine and cerebral infarction. However, this association is poorly understood. A review article presents several important issues which must be considered in this regard including patent foramen ovale, migraine specific drugs, and genetic components.

ut important topic. Previous studies have disclosed a link between migraine and cerebral infarction. However, this association is poorly understood. A review article presents several important issues which must be considered in this regard including patent foramen ovale, migraine specific drugs, and genetic components. Psychological adjustment is an important concern after stroke in young adults. A review article addresses this issue with particular consideration on service provision and return to work. A clinical article report that there was no difference as to ischemic stroke severity on admission and one week after stroke onset between patients younger than 50 years and patients older than 50 years. Because of long expected survival, information on long-term outcome after stroke in young adults is important. A review article provides a summary of long-term outcome both as to mortality, recurrent vascular events, and function. We hope that the present special issue provides important information on stroke in children and young adults and to be of help both as to treatment of our patients and in stimulating further research. Halvor Naess Turgut Tatlisumak Janika Kõrv

1. Where Are We? The classical molecular targets for stroke include those involved in oedema/inflammation control, axonal regeneration/plasticity, neurogenesis/angiogenesis, and events that support recovery. For decades, old targets for stroke were based on observations of molecular and cellular changes after stroke. Numerous inflammatory markers, growth-associated proteins, cell cycle proteins, NMDA receptors, molecules involved in synaptic plasticity, dendritic branching, neural sprouting or extracellular matrix remodelling were key targets. The field of neuroprotection generated consistent preclinical findings of mechanisms of cell death but these failed to be translated into clinical therapies. Many clinical trials were carried out using doses that were already known to be ineffective in preclinical trials, or employing time delays outside the established therapeutic window. Some trials were based on preclinical data showing relatively weak effects or those that were only established in one limited model. Similar problems may occur in the field of neural repair without careful work on the key points associated with clinical translation [1]. The effective delivery of neural repair strategies is another major issue in recovery after stroke. Several growth factors and cytokines have been shown to mediate neurogenesis and angiogenesis [2]. However these are pleiotrophic molecules with likely multiorgan effects when delivered systemically. The fine tuning of approaches that combine the modulation of the inhibitory environment together with the promotion of intrinsic axonal outgrowth needs further experimental work before combined therapeutic strategies will be transferable to clinic trials. It is likely that only when some answers have been found to these issues will our therapeutic efforts meet our expectations [3]. Selective delivery systems, or more selective small molecules, will need to be developed to minimize side-effects in a neural repair therapeutic.

tegies will be transferable to clinic trials. It is likely that only when some answers have been found to these issues will our therapeutic efforts meet our expectations [3]. Selective delivery systems, or more selective small molecules, will need to be developed to minimize side-effects in a neural repair therapeutic. Nanomedicine is probably opening new opportunities in this field as it may provide opportunities to deliver larger quantities of drugs with the additional possibility to target therapeutics to specific brain regions (superparamagnetic particles) and deliver to specific cell types following antibody-mediated endocytosis [4].

edicine is probably opening new opportunities in this field as it may provide opportunities to deliver larger quantities of drugs with the additional possibility to target therapeutics to specific brain regions (superparamagnetic particles) and deliver to specific cell types following antibody-mediated endocytosis [4]. Stroke is a clinically heterogeneous disease, with infarcts commonly occurring in different tissue compartments (white matter and gray matter) and brain regions (basal ganglia, cortex, thalamus, brainstem), and occurs most often in aged individuals. Combinatorial treatments require much greater work in pharmacological and toxicological testing. Further, treatments that promote anatomical rewiring will need to be administered in combination with behavioural activity to help “stamp in” patterns of brain rewiring that are adaptive and to avoid the formation of maladaptive patterns of wiring. A promising experimental treatment will need, at the very least, to be tested in several different rodent stroke models and aged animals. Despite these issues, it is becoming clear that the partial recovery that is commonly seen after stroke is associated with a reorganization of brain circuitry, and those methods that can safely and effectively enhance this reorganization could potentially have great clinical value [5]. It is important to remember that all stroke patients exhibit some degree of functional recovery. This process occurs in a matter of days, continues most dramatically for the first month in upper and lower extremity motor function [6] and for up to a year in language and other cognitive modalities [7, 8]. This recovery is only partial, leading to the tremendous long-term personal and financial burdens of this disease [9, 10]. What mediates natural neural repair in stroke and what are the pharmacological targets to promote improved recovery? Many of these processes of structural and physiological change after stroke have been correlated with recovery but the causal mechanisms of neural repair in stroke have not been defined. Axonal sprouting from the cortex contralateral to an infarct into the cervical spinal cord and brainstem ipsilateral to the infarct correlates with recovery of forelimb use [11, 12]. Neurogenesis after stroke is associated with functional recovery, in that blocking mitotic activity after stroke reduces cognitive recovery [13]. The degree of angiogenesis after stroke in humans is correlated with the level of recovery [14].

ipsilateral to the infarct correlates with recovery of forelimb use [11, 12]. Neurogenesis after stroke is associated with functional recovery, in that blocking mitotic activity after stroke reduces cognitive recovery [13]. The degree of angiogenesis after stroke in humans is correlated with the level of recovery [14]. Stem cell, growth factor, and cytokine therapies that promote functional recovery correlate with increases in angiogenesis and neurogenesis near the infarct [15, 16].

ipsilateral to the infarct correlates with recovery of forelimb use [11, 12]. Neurogenesis after stroke is associated with functional recovery, in that blocking mitotic activity after stroke reduces cognitive recovery [13]. The degree of angiogenesis after stroke in humans is correlated with the level of recovery [14]. Stem cell, growth factor, and cytokine therapies that promote functional recovery correlate with increases in angiogenesis and neurogenesis near the infarct [15, 16]. In recent years, the field of neural repair in stroke has identified cellular systems of reorganization and possible new molecular mechanisms. However, conceptual barriers now limit the generation of clinically useful agents. First, it is not clear what the causal mechanisms of neural repair are in stroke. Second, adequate delivery systems for neural repair drugs failed and need to be determined for candidate molecules. Third, ad hoc applications of existing pharmacological agents that enhance attention, mood, or arousal to stroke were unsuccessful. New approaches that specifically harness the molecular systems of learning and memory provide a new avenue for stroke repair drugs. Fourth, combinatorial treatments for neural repair need to be considered for clinical therapies. Finally, neural repair therapies have as a goal altering brain connections, that is, rewiring cognitive maps and active neural networks. These actions may also trigger a unique set of “neural repair side effects” that need to be considered in planning clinical trials [17]. Future research will be needed to address the above limitations in this field and problems in translation from the basic science to poststroke clinics.

aps and active neural networks. These actions may also trigger a unique set of “neural repair side effects” that need to be considered in planning clinical trials [17]. Future research will be needed to address the above limitations in this field and problems in translation from the basic science to poststroke clinics. 2. Heading towards the XXII Century The Whole Human Genome Project (HGP) early in the XXI century ushered in a wave of optimism and anticipation that new therapies and even cures for many diseases would soon be forthcoming [18]. Aside from impressive progress in reducing the costs of genotyping [19], the promise offered by the HGP has been largely unrealized, particularly in relation to stroke [20]. More than 100 Genome-Wide Association studies [21] made possible with the new information provided by the HGP have yielded many interesting findings about the genetics of stroke-related brain injury, but all have generally fallen far short of identifying a genetic basis for vulnerability to cerebral ischemia [22]. With the notable exception of monogenic diseases, genome-wide association studies (GWAS) have generally not been an efficient strategy to elucidate the genetic mechanisms of disease, particularly in complex pathologies such as ischemic cerebral injury [23]. These studies have taught us that most disease pathologies, including those associated with cerebral ischemia, are polygenic and involve highly variable contributions from the genes involved. Such findings raise the important question: why are the genetic components of complex diseases so variable? Polygenomic pathology as well as individual gene variability contributes to stroke as described in many GWAS. Long before the HGP was completed, it was recognized that genetic factors were not the only, or perhaps even not the most important, determinants of responses to some diseases. It was recognized early on that “epigenetic” factors were major players in the aetiology and progression of many diseases [24].

d in many GWAS. Long before the HGP was completed, it was recognized that genetic factors were not the only, or perhaps even not the most important, determinants of responses to some diseases. It was recognized early on that “epigenetic” factors were major players in the aetiology and progression of many diseases [24]. Following completion of the HGP, understanding of epigenetic mechanisms has expanded rapidly, and it is now recognized that epigenetic regulation involves three main categories of mechanisms [25, 26], that is, DNA methylation that attenuates gene expression; enzymes that add and remove acetyl groups to lysine residues in histone proteins and thereby facilitate or inhibit their dissociation for DNA with subsequent increases or decreases in gene expression, respectively; and the pathways that regulate the synthesis and action of micro-RNAs (miRNAs) that regulate mRNA translation. MicroRNAs represent the best-characterized subclass of ncRNAs. Together, these epigenetic mechanisms convert environmental conditions and physiological stresses into long-term changes in gene expression and translation. In contrast to DNA methylation and histone modification, the main function of miRNAs is associated mainly with message translation rather than with gene transcription. The miRNA molecules directly bind mRNA and either retard or accelerate its degradation. In addition, miRNA binding to mRNA can block message translation [27]. The sequences coding for miRNAs often arise from intronic DNA and regulate the gene products coded by adjacent exons. More than 1,000 unique sequences of miRNA have been identified, and together these regulate approximately 30% of all mammalian genes [27]. A single miRNA can help regulate multiple different gene products, and a single gene product can be regulated by multiple different miRNAs. As such, miRNAs play key roles in many cellular functions and are particularly important in cardiovascular biology [28–33]. Expression and action of miRNAs change with development and in response to nutritional stress [34]. The actions of specific miRNA molecules can be inhibited by reverse-sense antagomirs, and these have proven useful in many studies of miRNA function [35, 36].

are particularly important in cardiovascular biology [28–33]. Expression and action of miRNAs change with development and in response to nutritional stress [34]. The actions of specific miRNA molecules can be inhibited by reverse-sense antagomirs, and these have proven useful in many studies of miRNA function [35, 36]. 3. The RNA Machine RNAs are an integral component of chromosomes and contribute to their structural organization [37, 38]. It is now becoming apparent that chromatin architecture and epigenetic memory are regulated by RNA-directed processes where, although the exact mechanisms are yet to be understood, involve the recruitment of histone modifying complexes and DNA methyltransferases to specific loci [39]. Whereas long nonprotein coding RNAs (ncRNAs) have been classically implicated in the regulation of dosage compensation and genomic imprinting in animals [40], they seem to play a much broader role in the epigenetic control of developmental trajectories [39]. For example, ncRNAs may repress gene expression and be associated with complex epigenetic phenomena [41, 42]. Small ncRNAs have been consistently linked with heterochromatin formation via the process of RNA interference (RNAi). Higher-level nuclear organization and chromosome dynamics are also regulated by ncRNAs in a variety of systems. These findings reveal RNA-based mechanistic links between these processes in mitosis. The RNAi pathway along with directed histone modifications also regulates the organization of the nucleolus [43, 44]. In mammals, transcription of long ncRNAs contributes to various processes including T cell receptor recombination [42], maintenance of telomeres [45, 46], X-chromosome pairing required for dosage compensation [47], and inactive X-chromosome perinucleolar localization [48]. The functional organization of chromatin can also be regulated by ncRNAs derived from repetitive elements. Given the abundance of transcribed repetitive sequences, this may represent a genome-wide strategy for the control of chromatin domains that may be conserved throughout eukaryotes. Moreover, such observations and others suggest that a large portion of the genome may in fact be functionally active and that transposon-derived sequences may not be reliable indices of the rate of neutral evolution [49].

me-wide strategy for the control of chromatin domains that may be conserved throughout eukaryotes. Moreover, such observations and others suggest that a large portion of the genome may in fact be functionally active and that transposon-derived sequences may not be reliable indices of the rate of neutral evolution [49]. 4. A World of Noncoding RNAs The examples above provide proof-of-principle that RNA can regulate gene expression at many levels and by using a wide array of mechanisms. The ENCODE project showed that at least 93% of analyzed human genome nucleotides are transcribed in different cells, with similar findings in mice and other eukaryotes, which indicate that there may be a vast reservoir of biologically meaningful RNAs that could greatly exceed the ~1.2% encoding proteins. A fraction of RNAs with short open reading frames (ORFs) potentially encodes peptides but on the other side of the ledger many currently annotated ORFs are not conserved and may be false, which could reduce the number of protein-coding genes in the human genome. There has been debate about whether these ncRNAs are (in the main) functional or simply noise. In some cases, it may be the transcript or merely the act of transcription, or both, that are relevant. Nevertheless, many observations indicate that substantial numbers of ncRNAs are intrinsically functional. These include the fact that many loci produce spliced (and alternatively spliced) transcripts that are developmentally regulated. A large fraction of ncRNAs are expressed in specific regions of the brain, exhibiting precise cellular locations. Some mark new domains within the cell, which means that ncRNAs are also set to have a major impact in cell biology. Comparative analyses indicate that ncRNA promoters are, on average, more conserved than those of protein-coding genes and that ncRNA sequences, secondary structures, and splice site motifs have been subject to purifying selection. Moreover, many ncRNAs are evolving quickly, and some have undergone recent positive selection, as exemplified by HAR1 RNA expressed in the human brain, which contains the sequence conserved in mammals that most rapidly diverged after the human-chimpanzee separation. Although the need for large-scale approaches to explore the function of ncRNAs is evident, a glance at the genome browser will show noncoding expressed sequence tags associated with most genes of interest that may have regulatory functions.

ed in mammals that most rapidly diverged after the human-chimpanzee separation. Although the need for large-scale approaches to explore the function of ncRNAs is evident, a glance at the genome browser will show noncoding expressed sequence tags associated with most genes of interest that may have regulatory functions. ncRNAs are already being identified as markers for cancer and associated with other complex diseases such as coronary disease, diabetes, and Alzheimer's. The elucidation of their function may significantly contribute to the understanding and treatment of such conditions. It may also transform our understanding of the genetic programming of multicellular organisms, particularly as it appears that regulation dominates the information content of complex systems [49].

er's. The elucidation of their function may significantly contribute to the understanding and treatment of such conditions. It may also transform our understanding of the genetic programming of multicellular organisms, particularly as it appears that regulation dominates the information content of complex systems [49]. 5. RNA-Based Epigenetic Mechanisms Implicated in Stroke The predisposition to and the development of cerebrovascular diseases involves the dynamic interplay between environmental and intrinsic vascular, systemic, and CNS risk factors. Increasing evidence suggests that disruption of these homeostatic and plasticity events involves an array of deregulated epigenetic processes [50]. Appreciation of the potential involvement of epigenetic mechanisms in the incidences and outcomes of stroke has begun to motivate studies of these mechanisms in relation to cerebral ischemia and stroke. DNA methylation has been suggested to contribute to delayed ischemic brain injury in mice and has been correlated with stroke risk in humans. Histone modifications have been implicated in LPS-induced cerebral inflammation and oxidative neuronal injury and may be neuroprotective following ischemia in rodent brains [17].

DNA methylation has been suggested to contribute to delayed ischemic brain injury in mice and has been correlated with stroke risk in humans. Histone modifications have been implicated in LPS-induced cerebral inflammation and oxidative neuronal injury and may be neuroprotective following ischemia in rodent brains [17]. 6. ncRNAs and RNA Regulatory Networks The most recently recognized category of epigenetic mechanisms includes the pathways involved in the transcription, processing, and action of a class of short (≈20–25 nucleotides) RNA molecules identified as micro-RNAs (miRNAs) [51]. MicroRNAs are first transcribed as longer primary miRNA transcripts that can have multiple functional miRNAs embedded within a single transcript. These primary miRNAs are processed to form mature molecules of approximately 22 nucleotides that regulate the expression of large numbers of target genes through sequence-specific interactions with messenger RNA (mRNA) molecules. MicroRNAs bind to the 3 regulatory regions and to particular coding regions of their cognate mRNAs, leading to sequestration for storage or degradation and to translational repression. Cerebral ischemia in animal models is associated with highly selective and temporally regulated profiles of miRNAs in the postischemic brain [52]. Differential expression of miRNAs in the postischemic brain correlates with differential expression of their target mRNAs, including many implicated in transcriptional regulation, ionic flux, inflammation, and other stress responses. These results suggest that miRNA networks regulate a spectrum of processes in the postischemic brain. MicroRNA-140 is one of the miRNAs that was rapidly up regulated in the brain 3 hours after middle cerebral artery occlusion and sustained for 72 hours. One of the validated target mRNAs for miRNA-140 encodes stromal cell-derived factor 1, which plays an important role in the CNS by mediating neural progenitor cell proliferation and migration and tissue repair after cerebral ischemia [53]. This observation suggests that miRNA-140 may be responsible, in part, for mitigating the regenerative response in the postischemic brain. Furthermore, some miRNAs that are highly differentially expressed in brain tissue can similarly be detected in peripheral blood [54], suggesting not only that these may serve as novel clinical biomarkers but also that these miRNAs may be involved in mediating systemic responses to cerebral ischemia.

postischemic brain. Furthermore, some miRNAs that are highly differentially expressed in brain tissue can similarly be detected in peripheral blood [54], suggesting not only that these may serve as novel clinical biomarkers but also that these miRNAs may be involved in mediating systemic responses to cerebral ischemia. Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell-cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In another study, using Multipotent mesenchymal stromal cells (MSCs) treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR-133b) level in the ipsilateral hemisphere. In vitro, cultured neurons treated with exosome-enriched fractions from MSCs exposed to MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR-133b to neural cells via exosomes [55]. Further, microRNAs (miRNAs) regulate formation of myelinating oligodendrocytes. Overexpression of miR-219 and miR-338 as oligodendrocyte-specific miRNAs is sufficient to promote oligodendrocyte differentiation. These findings illustrate that miRNAs are important regulators of oligodendrocyte differentiation, providing new targets for myelin repair [56].

e formation of myelinating oligodendrocytes. Overexpression of miR-219 and miR-338 as oligodendrocyte-specific miRNAs is sufficient to promote oligodendrocyte differentiation. These findings illustrate that miRNAs are important regulators of oligodendrocyte differentiation, providing new targets for myelin repair [56]. Long ncRNAs represent another important and emerging subclass of ncRNAs that may also play a role in stroke. Long ncRNAs have roles in local and long-range chromatin remodelling, transcriptional regulation, and alternative splicing and other forms of post transcriptional RNA processing [57]. They are implicated in the development of axonal and dendritic connections and synaptic modulation associated with neural network plasticity. Long ncRNAs may also participate in the generation of the long-term potentiation that underlies learning and memory [58]. An lncRNA can bind to the cyclin D1 gene, a critical mediator of ischemic neuronal cell death [59]. ANRIL (NCBI EntrezGene 100048912) is an lncRNA with an unknown function that is associated with the development of atherosclerosis, diabetes, and aneurysms, possibly through effects on vascular smooth-muscle proliferation and migration. GOMAFU (NCBI Entrez Gene 440823) is another lncRNA that is expressed in the nucleus of developing neural cells. Although the function of GOMAFU is unknown, a case-control association study identified a single nucleotide polymorphism associated with the GOMAFU locus as a susceptibility factor for cardiovascular disease [60]. In addition to miRNAs and lncRNAs, other ncRNA transcripts, such as those resembling the virus-like 30 family of interspersed, repeated, mobile genetic elements (i.e., retrotransposons), are also increased in mouse brain after cerebral ischemia. These virus-like 30 ncRNAs are induced by ischemia and paradoxically bound to polyribosomes, although they are not translated. The distribution of these virus 30-like ncRNAs in ribosomal fractions is distinct from the distribution of mRNAs that are translated or translationally repressed and suggests a novel structural or catalytic role for these ncRNAs. Together, these observations imply that the expression and function of several newly identified subclasses of ncRNAs may be associated with the pathogenesis of stroke [61]. Figure 1 represents biogenesis of microRNAs.

lated or translationally repressed and suggests a novel structural or catalytic role for these ncRNAs. Together, these observations imply that the expression and function of several newly identified subclasses of ncRNAs may be associated with the pathogenesis of stroke [61]. Figure 1 represents biogenesis of microRNAs. 7. RNA Editing and DNA Recoding in Stroke This process is intimately linked with ncRNA expression and is another epigenetic process, RNA editing, a mechanism for altering nucleotides in RNA molecules that allows the generation of significant diversity of transcripts in a highly environmental-responsive manner. In transient global cerebral ischemia models, the death of hippocampal CA1 pyramidal neurons is mediated by selective downregulation of an RNA-editing enzyme leading to defective editing of the ionotropic glutamatergic -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid GluR2 receptor subunit, which influences the vulnerability of hippocampalCA1 pyramidal neurons to ischemia-associated cell death [62]. By alteration of RNA nucleotides, not only does editing have the capacity to change amino acids and modulate splice-site choice in protein-coding transcripts, but it also has roles in ncRNA-related processes such as miRNA localization, target diversification, and function [63]. MicroRNA regulatory network dynamics mediated by RNA editing may be implicated in stroke. For example, miRNA-151 is found in neurons and up-regulated after middle cerebral artery occlusion, and the immature form of miRNA-151 (primary miRNA-151) is subject to RNA editing that influences processing of the primary miRNA into mature miRNA within the CNS [64]. Intriguingly, miRNA-151 is thought to target various cell cycle regulators as well as protein tyrosine kinase 2 (focal adhesion kinase), a nonreceptor tyrosine kinase involved in integrin and growth factor signalling pathways that is differentially regulated after middle cerebral artery occlusion and implicated in modulating neurite outgrowth, neuronal plasticity, and restoration of neural network integrity within the ischemic penumbra [65]. These observations imply that multiple layers of interleaved epigenetic controls that include RNA editing and miRNA regulatory networks are involved in stroke. Another, miR120 is positively correlated with better prognosis in stroke patients and antagonists to miR497, infused prior to stroke, reduce infarct volume.

5]. These observations imply that multiple layers of interleaved epigenetic controls that include RNA editing and miRNA regulatory networks are involved in stroke. Another, miR120 is positively correlated with better prognosis in stroke patients and antagonists to miR497, infused prior to stroke, reduce infarct volume. However, to date, no neuroprotective miRNA mimics or antagomirs have been identified that are effective when delivered poststroke. To identify neuroprotective miRNAs, Selvamani et al. studied a known neuroprotectant, Insulin-like Growth Factor (IGF-) 1, for specific miRNA target sites, with the goal of inhibiting these miRNA to elevate local levels of IGF-1 poststroke. IGF-1 is a critical endogenous neuroprotectant and low normal levels of peptide hormone are associated with increased morbidity and mortality in ischemic heart disease and stroke. Exogenous IGF-1 reduces ischemic injury in many species, stimulates stroke induced neurogenesis and promotes neuronal survival, neuronal myelination, and angiogenesis. Two conserved IGF pathway regulatory microRNAs, Let7f and miR1, can be inhibited to mimic and even extend the neuroprotection afforded by IGF-1. Collectively these data support a novel miRNA-based therapeutic strategy for neuroprotection following stroke in experimental model [66].

uronal myelination, and angiogenesis. Two conserved IGF pathway regulatory microRNAs, Let7f and miR1, can be inhibited to mimic and even extend the neuroprotection afforded by IGF-1. Collectively these data support a novel miRNA-based therapeutic strategy for neuroprotection following stroke in experimental model [66]. In addition, the apolipoprotein-B-(ApoB-) editing catalytic subunit (APOBEC) family of RNA editing and DNA recoding enzymes may also play a role in stroke. These enzymes are cytidine deaminases that edit (deoxy)-cytidine to (deoxy)-uridine and act on RNA and DNA molecules [67]. One of the substrates for these enzymes is APOB (NCBI Entrez Gene 338) mRNA, which encodes an important apolipoprotein found in chylomicrons and low-density lipoproteins. Mutations of the APOB gene and its regulatory region cause dyslipidemias (eg hypobetalipoproteinemia and hypercholesterolemia), and genetic variants of APOBEC1 and APOBEC2 are associated with high levels of serum low-density lipoproteins and increased atherosclerosis [68]. The APOBECs may affect stroke risk through effects on APOB mRNA editing; however, APOBECs may play additional roles within the brain. APOBEC-mediated DNA recoding protects the stability of the genome and also enhances its diversity and plasticity [64]. Although these functions have largely been characterized within the immune system, it is intriguing that the APOBEC3 enzyme subfamily has significantly expanded in primates and that certain members (i.e., APOBEC3G) are expressed in postmitotic neurons [69]. Furthermore, accumulating evidence suggests that RNA editing and DNA recoding may be functionally linked through specific classes of reverse transcriptases within the CNS that can mediate RNA-directed DNA modifications [70]. Also, like the immune system, the CNS exhibits exquisite degrees of functional plasticity by modulating cell identity and connectivity. Because of these observations, we have previously suggested that DNA recoding in the brain might represent a novel mechanism for transmitting productive RNA editing events back into the postmitotic neuronal genome [61].

NS exhibits exquisite degrees of functional plasticity by modulating cell identity and connectivity. Because of these observations, we have previously suggested that DNA recoding in the brain might represent a novel mechanism for transmitting productive RNA editing events back into the postmitotic neuronal genome [61]. This suggests a possible evolutionary mechanism to account for the multigenerational inheritance of complex cognitive and behavioural traits and risk profiles for stroke in response to both productive and adverse environmental events. Apart from the molecular mechanisms responsible for epigenetic regulation, a broad variety of evidence has implicated epigenetic regulation in long-term environmental influences on gene regulation. One of the best-known such examples is the epidemiological work of Pearce, who identified a cohort of Dutch individuals with a uniquely elevated risk of coronary artery disease [17]. The common feature among this cohort was maternal food restriction during the Dutch famine in World War II. These early studies established that foetal nutritional stress could produce life-long changes in the vulnerability to cardiovascular disease, and subsequent work has further established the epigenetic basis of such “vascular programming”. Similarly, other studies have implicated epigenetic mechanisms in long-term responses to hypoxia [71–74] and ischemia [75–77]. Of particular relevance to stroke are findings that miRNA is involved in ischemic preconditioning [79] and may even play a role in ischemic post conditioning. Together, these results emphasize that environmental influences can produce long-term changes in physiological patterns of gene expression through epigenetic mechanisms.

cular relevance to stroke are findings that miRNA is involved in ischemic preconditioning [79] and may even play a role in ischemic post conditioning. Together, these results emphasize that environmental influences can produce long-term changes in physiological patterns of gene expression through epigenetic mechanisms. 8. Epigenetics and Transient Ischemia Transient global cerebral ischemia (TGCI) following systemic hypoperfusion is associated with selective and delayed death of hippocampal CA1 pyramidal neurons through the mediation of a series of parallel epigenetic processes. Within vulnerable neurons, there is selective downregulation of ADAR2 and defective Q/R site editing of the ionotropic glutamatergic AMPA, GluR2 receptor subunit, resulting in the expression of the death-promoting calcium permeable GluR2 isoform and associated impairment in GluR2 mRNA and protein expression, receptor assembly, membrane trafficking, and synaptic targeting. Heterogeneity in ADAR2- mediated GluR2 Q/R site editing enhances the vulnerability of hippocampal CA1 pyramidal neurons to global ischemia-associated neurodegeneration. In parallel, TGCI induces the selective expression of REST within these vulnerable neurons with associated suppression of GluR2 and the CA1- selective m-opioid receptor 1 (MOR1) in inhibitory interneurons through a series of histone modifications, including MOR1 promoter H3/4 deacetylation, H3K9 dimethylation and associated recruitment of the G9a histone methyltransferase. This has been postulated to represent a failed attempt of inhibitory interneurons to dampen the excitotoxicity of CA1 pyramidal neurons by disinhibiting GABA release. Ischemia-induced alterations in the histone code may be the result of early dephosphorylation and inactivation of components of the neuronal ERK1 and CREB1 signal transduction pathways that simultaneously reduce expression of the antiapoptotic, bcl2 gene and activate expression of the proapoptotic, caspase-3 effector pathway [80–83].

d alterations in the histone code may be the result of early dephosphorylation and inactivation of components of the neuronal ERK1 and CREB1 signal transduction pathways that simultaneously reduce expression of the antiapoptotic, bcl2 gene and activate expression of the proapoptotic, caspase-3 effector pathway [80–83]. There is also evidence that the more common type of focal stroke syndrome due to occlusion of the middle cerebral artery is associated with aberrant DNA methylation and histone H3 deacetylation, and that systemic administration of a potent HDAC inhibitor reduces the volume of the ischemic infarction whereas concurrent application of an HDAC inhibitor with a DNA demethylating agent confers neuroprotection against mild but not severe ischemic injury [84]. Increasing evidence suggests that intricate epigenetic processes may also operate to modulate premorbid vascular pathology and responses to agents that attenuate ischemic risk factors. For example, a novel deubiquinating enzyme, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), mutated in a rare inherited form of Parkinson's disease, is normally present in vascular endothelial cells of atherosclerotic lesions of human carotid arteries and attenuates pathological vascular remodeling by inhibiting tumor necrosis factor a-induced NF-kappaB activation [85]. Interestingly, the normal balance of transcriptional activity and associated histone acetylation and methylation that is disrupted in cerebral ischemia depends, in part, on maintenance of the balance of histone H2A and H2B mono-ubiquitylation that is mediated through the actions of UCHL1 [86]. Moreover, statins have recently been shown to act through inhibition of HDAC activity and associated enhancement of histone H3 acetylation [87].

s disrupted in cerebral ischemia depends, in part, on maintenance of the balance of histone H2A and H2B mono-ubiquitylation that is mediated through the actions of UCHL1 [86]. Moreover, statins have recently been shown to act through inhibition of HDAC activity and associated enhancement of histone H3 acetylation [87]. Transient focal ischemia in adult rat brain regulates the expression of microRNAs predicted to target proteins known to mediate inflammation, transcription, neuroprotection, receptor function, and ionic homeostasis in the brain. The mRNA levels for proteins important to microRNA biogenesis pathways, including Drosha, Dicer, the cofactor Pasha, and the precursor microRNA transporter Exportin 5, were not altered after transient ischemia. However, transient ischemia repressed miR-145 expression, which resulted in increased translation of its mRNA target, superoxide dismutase-2, in post-ischemic adult rat brain. It is interesting to note that in silico studies revealed eight microRNAs induced by transient ischemia with complementarity to 877 gene promoters, suggesting that microRNAs also regulate gene expression [88]. There is also specific induction of miR-497 in mouse brain after transient ischemia, and in mouse N2A neuroblastoma (N2A) cells after oxygen-glucose deprivation [86]. Levels of miR-497 correlated with oxygen-glucose deprivation-induced effects on N2A cells: decreased miR-497 suppressed cell death, whereas increased miR-497 increased neuronal loss. As miR-497 directly binds to the 30-UTR of Bcl-2/-w, the knockdown of cerebral miR-497 in mice enhanced Bcl-2/-w protein levels in the ischemic region, attenuated brain infarction, and improved neurological outcome after focal ischemia. These studies show that miR-497 promotes ischemic neuronal death by repressing expression of Bcl-2 and Bcl-w, supporting the role of apoptosis in the pathogenesis of ischemic brain injury [89, 90].

rotein levels in the ischemic region, attenuated brain infarction, and improved neurological outcome after focal ischemia. These studies show that miR-497 promotes ischemic neuronal death by repressing expression of Bcl-2 and Bcl-w, supporting the role of apoptosis in the pathogenesis of ischemic brain injury [89, 90]. 9. Molecular Studies of MicroRNAs in Human Stroke Whole genome expression microarrays can be used to study gene expression in blood, which comes in part from leukocytes, immature platelets, and red blood cells. Since these cells are important in the pathogenesis of stroke, RNA provides an index of these cellular responses to stroke. Human studies show gene expression changes following ischemic stroke. These gene profiles predicted the cause of stroke in 58% of cryptogenic patients. New techniques to measure all coding and noncoding RNAs along with alternatively spliced transcripts will markedly advance molecular studies of human stroke [91]. Platelets are crucial for the maintenance of haemostasis and contribute to thrombosis and vessel occlusion that underlies stroke and acute coronary syndromes. Although platelets are anucleate, they do contain mRNAs and are capable of protein synthesis [89]. Human platelets have been shown to contain microRNAs and Dicer in Ago2 protein complexes, as well as mRNA for the P2Y purinoceptor 12 that is involved in platelet aggregation, suggesting a role for microRNAs in this system [92].

h platelets are anucleate, they do contain mRNAs and are capable of protein synthesis [89]. Human platelets have been shown to contain microRNAs and Dicer in Ago2 protein complexes, as well as mRNA for the P2Y purinoceptor 12 that is involved in platelet aggregation, suggesting a role for microRNAs in this system [92]. Mutations in mitochondrial DNA are responsible for a spectrum of mitochondrial encephalomyopathies, including mitochondrial encephalopathy with lactic acidosis and stroke like episodes. Although the DNA sequences that harbor these mutations generally do not code for proteins, many of them encode transfer (tRNAs) and ribosomal RNAs (rRNAs). The array of clinical symptoms seen in mitochondrial disorders highlights the functional importance of nonprotein-coding RNAs (ncRNAs) such as tRNAs and rRNAs that are transcribed from nonprotein-coding DNA sequences. In fact, the pathogenesis of a spectrum of neurodevelopmental, neurodegenerative, and neuropsychiatric diseases is increasingly being associated with mutations of ncRNAs [61].

the functional importance of nonprotein-coding RNAs (ncRNAs) such as tRNAs and rRNAs that are transcribed from nonprotein-coding DNA sequences. In fact, the pathogenesis of a spectrum of neurodevelopmental, neurodegenerative, and neuropsychiatric diseases is increasingly being associated with mutations of ncRNAs [61]. 10. MicroRNAs as Novel Biomarkers in Brain Ischemia The brain is a conspicuous consumer of energy resources, and a major consequence of cerebral ischemia is the disruption of energy metabolism and exhaustion of adenosine triphosphate. Because RNA can rapidly be activated, modified, transported, and degraded, it serves as a highly flexible, high fidelity, information encoding, and functional molecule. The ability of RNA molecules to dynamically store, transform, and transmit both “digital” and “analogue” information is a key feature of RNA-based systems [61].

NA can rapidly be activated, modified, transported, and degraded, it serves as a highly flexible, high fidelity, information encoding, and functional molecule. The ability of RNA molecules to dynamically store, transform, and transmit both “digital” and “analogue” information is a key feature of RNA-based systems [61]. Studies support the potential for microRNAs as novel biomarkers for vascular injury and diseases. Expression profiling of microRNAs in ischemic rat brains revealed significant changes in several micro-RNAs, and some of the microRNAs highly expressed in ischemic brain were detected in blood samples [93]. Peripheral blood examined in ischemic stroke patients revealed differential expression of microRNAs implicated in endothelial cell and vascular function, erythropoiesis, angiogenesis, neural function, and hypoxia, and altered microRNAs were detectable even several months after the onset of stroke [94]. Rat models of ischemia, brain haemorrhage, and kainate-induced seizures also revealed regulated expression of microRNAs in hippocampus and blood in each treatment group, many of which changed >1.5-fold in both tissues [95].

poxia, and altered microRNAs were detectable even several months after the onset of stroke [94]. Rat models of ischemia, brain haemorrhage, and kainate-induced seizures also revealed regulated expression of microRNAs in hippocampus and blood in each treatment group, many of which changed >1.5-fold in both tissues [95]. Evidence also suggests that microRNAs serve as effectors in neointimal lesion formation, and in angiogenesis in normal and injured brain. The miR-17–92 cluster is highly expressed in human endothelial cells and miR-92a, a component of this cluster, targets several mRNAs for proangiogenic proteins. Overexpression of miR-92a in endothelial cells blocked angiogenesis, and systemic administration of an miR-92a antagomir led to enhanced blood vessel growth and functional recovery of damaged tissue in mouse models of limb ischemia and myocardial infarction [93]. In a similar vein, profiling of microRNAs in vascular walls after balloon injury revealed that miR-21 is overexpressed in injured vascular tissue, and that miR-21 depletion inhibited formation of neointimal lesions. Depletion of miR-21 decreased cell proliferation and increased cell apoptosis, and targets of miR-21 include the phosphatase and tensin homolog protein (PTEN) and Bcl-2 [96].

oon injury revealed that miR-21 is overexpressed in injured vascular tissue, and that miR-21 depletion inhibited formation of neointimal lesions. Depletion of miR-21 decreased cell proliferation and increased cell apoptosis, and targets of miR-21 include the phosphatase and tensin homolog protein (PTEN) and Bcl-2 [96]. 11. The Era of Epigenomic Medicine For treatment of stroke, RNA-based therapies and additional epigenetic strategies are extremely promising. Indeed, approaches for gene silencing that use short regulatory ncRNAs, including miRNAs and related short interfering RNAs (RNA interference) have already been used to identify new molecular targets for treating stroke, such as Bcl-2 and 19-kDa interacting protein 3 [97] and carboxyterminalmodulator protein [98]; however, RNA interference-based gene silencing for treating stroke has yet to advance beyond preliminary studies. Therapeutic approaches using other customized oligonucleotides are also being developed for modulation of endogenous RNA transcripts. For example, novel antisense oligonucleotides have now been constructed with the capacity to repair and reprogram aberrant disease-associated RNAs. The mechanism of action of these agents includes alteration of pre mRNA processing (e.g., alternative splicing) and promotion of trans-splicing, which results in the creation of a composite mRNA from 2 separate pre-mRNAs [99]. Although RNA-based approaches such as these are still in their infancy, they offer the potential for dynamic and highly selective reprogramming of gene expression and function. Because of their unique properties, functional RNA molecules may be ideal candidates for a number of future therapeutic strategies. For example, through sequence-specific digital interactions with DNA, RNA-based therapeutic molecules may serve as guideposts for a certain genomic sequence. Through analog interactions with proteins, RNAs may also act as molecular beacons for recruitment of DNA methylation and histone-modifying enzyme complexes to a given genomic locus. Thus, multifunctional RNA molecules with binding domains for DNA and for these enzyme complexes may be used for targeting epigenetic modifications to a single gene locus or to multiple gene loci that harbour a shared genomic sequence.

t of DNA methylation and histone-modifying enzyme complexes to a given genomic locus. Thus, multifunctional RNA molecules with binding domains for DNA and for these enzyme complexes may be used for targeting epigenetic modifications to a single gene locus or to multiple gene loci that harbour a shared genomic sequence. Furthermore, because RNA molecules can interact with DNA, RNAs, proteins, and small molecules, RNA-based therapeutics may also provide the flexibility and specificity necessary to selectively manipulate intricate profiles of gene transcription, posttranscriptional RNA processing, and translation by targeting epigenetic effectors such as nucleosome- and chromatin-remodeling complexes, multiple ncRNAs (e.g., miRNAs and lncRNAs), and RNA editing and DNA recoding enzymes. Although these approaches have yet to be validated, the evolution of CNS drug delivery methods and rapid advances in RNA-based therapeutics, including the advent of RNA aptamers (RNA molecules engineered to bind with high affinity to specific molecular targets such as small molecules, proteins, and nucleic acids), suggest that such strategies are now possible [100]. Future therapies may also be designed to target factors that serve as key modulators of CNS-specific epigenetic events and thereby promote neural cell- and tissue selective epigenetic reprogramming. For example, these strategies may use novel agents that activate or inhibit special AT-rich sequence-binding protein 2 (SATB2), the repressor element-1 silencing transcription factor/neuron restrictive silencing factor (REST/NRSF), and the corepressor for element-1-silencing transcription factor (CoREST). As an environmentally sensitive regulator of neuronal cell fate decisions during development [101], SATB2 modulates neuronal gene expression by promoting coordinate regulation of multiple genes on different chromosomes involved in functionally integrated gene networks. These molecular processes involve dynamic reorganization of the nuclear architecture to allow a seamless link between transcriptional and posttranscriptional processing events and associated RNA quality control mechanisms. SATB2 is also associated with a regulatory lncRNA that is coexpressed with SATB2 [102]. These observations suggest that therapeutic agents targeting SATB2or its associated lncRNA could lead to dynamic reprogramming of neuronal gene expression and even neural cell identityand patterns of neural connectivity that is essential for neural regeneration.

a regulatory lncRNA that is coexpressed with SATB2 [102]. These observations suggest that therapeutic agents targeting SATB2or its associated lncRNA could lead to dynamic reprogramming of neuronal gene expression and even neural cell identityand patterns of neural connectivity that is essential for neural regeneration. REST and CoREST are critical epigenetic factors that mediate predominantly site-specific gene repression, gene activation, and long-term gene silencing for a large spectrum of genes involved in neural development, homeostasis, and plasticity, including but not limited to those that encode growth factors, axon guidance cues, ion channels, neurotransmitter receptors, synaptic vesicle proteins, components of the cytoskeleton, and elements of the extracellular matrix [103].

r a large spectrum of genes involved in neural development, homeostasis, and plasticity, including but not limited to those that encode growth factors, axon guidance cues, ion channels, neurotransmitter receptors, synaptic vesicle proteins, components of the cytoskeleton, and elements of the extracellular matrix [103]. In addition, REST and CoREST modulate the expression of several classes of ncRNAs, including miRNAs and lncRNAs. These molecules act as dynamic modular platforms for the recruitment of a broad array of epigenetic factors to neural gene loci in which they orchestrate site-specific and genome-wide chromatin remodelling. One of the molecular mechanisms that underlie cell death after transient global ischemia is REST dependent repression of the GluR2 subunit and μ opioid receptor 1 [104, 105]. REST also regulates the expression of a significant number of the miRNAs that are differentially expressed after cerebral ischemia [52]. These observations suggest that therapeutic targeting of REST and CoREST may have significant effects on highly integrated epigenetic regulatory mechanisms that could promote reprogramming of neural cells to enhance neural regeneration in stroke by recapitulating developmental events responsible for establishing and remodelling neural cell identity and neural network connectivity. Additional treatment strategies may also be developed to fine-tune epigenetic mechanisms that mediate RNA modifications and trafficking within cells. Among the more salient molecular targets may be regulatory ncRNAs (e.g., miRNAs and lncRNAs), RNA binding proteins, and cytoskeletal proteins (e.g., dyneins and kinesins) that have prominent roles in a diverse array of processes that are under epigenetic regulation, including alternative splicing; editing; nuclear export; stabilization; temporal, spatial, and activity-dependent localization; and translation of RNAs. For example, rationally designed small molecules that bind to miRNAs and modulate their activity are now under early stage development, and these agents may specifically be designed to target miRNAs that are deregulated in stroke [106]. Furthermore, novel therapies may act by selectively influencing the composition of complexes that carry mRNAs, ncRNAs, proteins, and other functionally related factors. These structures, referred to as RNA operons,play key roles in axodendritic transport and mediate local mRNA translation and synaptic plasticity [107].

Furthermore, novel therapies may act by selectively influencing the composition of complexes that carry mRNAs, ncRNAs, proteins, and other functionally related factors. These structures, referred to as RNA operons,play key roles in axodendritic transport and mediate local mRNA translation and synaptic plasticity [107]. Higher-order regulatory mechanisms coordinate the dynamics of interrelated RNA operons by modulating their individual components, the kinetics of anterograde and retrograde axodendritic transport and activity-dependent deployment and function of neuronal RNAs. These mechanisms are termed RNA regulons. In postischemic neurons, vulnerability to cell death is associated with pathological alterations in RNA operon and regulon dynamics and stress responses that lead to translational [108]. These observations imply that manipulating RNA posttranscriptional processing may be useful in postischemic neurons to promote cellular reprogramming and to selectively activate responses that favorneuronal survival and the maintenance of neural network integrity. Furthermore, RNA operons and regulons are implicated in bidirectional axodendritic transport responsible for relaying RNA editing events from the synapse to the nucleus for DNA recoding within postmitotic neurons. Because these processes are implicated in multigenerational inheritance, therapeutic interventions targeting RNA editing events and associated recoding of the neuronal genome may be implemented to directly alter stroke risk even in future generations. Epigenetic mechanisms are also involved in regulating cell-cell communication, including the active transport of RNAs between adjacent nerve cells through multiple signalling pathways, to more distant sites within the same tissue, to other organ systems through blood-borne routes, and even back to the germline; these processes may represent novel targets for future therapeutic initiatives [109]. Specific transmembrane proteins required for the systemic spread of RNA interference are expressed in the adult brain preferentially in areas associated with learning and memory. Moreover, microvesicles (i.e., exosomes) containing mRNAs and ncRNAs are produced by neural cells and secreted locally and into the peripheralcirculation [110].

ane proteins required for the systemic spread of RNA interference are expressed in the adult brain preferentially in areas associated with learning and memory. Moreover, microvesicles (i.e., exosomes) containing mRNAs and ncRNAs are produced by neural cells and secreted locally and into the peripheralcirculation [110]. These microvesicles may be responsible for cell-cell communication through local and more long distance intercellular RNA transfer because they express cell recognition molecules on their surfaces for selective targeting and uptake into recipient cells, in which mRNAs may be translated and ncRNAs may exert their regulatory effects. Modulation of microvesicle composition and transport pathways may serve as novel targets for regulating anterograde and retrograde signalling across synapses, reinforcing local and long-range neural network connectivity, and signalling to other organ systems (i.e., the immune system) that may play seminal roles in the pathogenesis and evolution of stroke syndromes and associated co-morbidities. As epigenetic processes begin to reveal the many previously hidden layers of functional information embedded within the genome, many future strategies can be envisioned that exploit these processes to develop novel therapies. In fact, the epigenome provides multiple layers of contextual controls that are intricately interlaced and potentially modifiable, and a single epigenetic intervention may even have a cascade of effects on many interrelated processes, including those that may be important for circumventing the pathogenesis and sequelae of stroke. For example, the DNA double helix itself has the potential to form alternative structural conformations with unique epigenetic properties that can be harnessed for the treatment of stroke. Indeed, when the neuroprotective cytokine, colony-stimulating factor 1, is activated by the BRG1 chromatin-remodeling enzyme, a left-handed DNA stereoisomer referred to as a Z-DNA structurecan be found in the region actively being transcribed [111]. The formation of Z-DNA stereoisomers can, in turn, modulate a range of processes responsible for fine-tuning transcriptional events, regulating chromatin architecture, and promoting specific forms of RNA editing [112].

eoisomer referred to as a Z-DNA structurecan be found in the region actively being transcribed [111]. The formation of Z-DNA stereoisomers can, in turn, modulate a range of processes responsible for fine-tuning transcriptional events, regulating chromatin architecture, and promoting specific forms of RNA editing [112]. Thus, understanding complex epigenetic mechanisms and their complementary roles in mediating CNS functions, both in health and in disease, is important for developing next-generation technologies to dynamically reprogram neural cells for treatment of complex neurological disease states, including stroke [61].

eoisomer referred to as a Z-DNA structurecan be found in the region actively being transcribed [111]. The formation of Z-DNA stereoisomers can, in turn, modulate a range of processes responsible for fine-tuning transcriptional events, regulating chromatin architecture, and promoting specific forms of RNA editing [112]. Thus, understanding complex epigenetic mechanisms and their complementary roles in mediating CNS functions, both in health and in disease, is important for developing next-generation technologies to dynamically reprogram neural cells for treatment of complex neurological disease states, including stroke [61]. 12. Final Conclusions Studies of epigenetic mechanisms in stroke are in their infancy but offer great promise for better understanding of stroke pathology and the potential viability of new strategies for its treatment. Correspondingly, inhibitors of histone modification have been suggested to be neuroprotective in animal models of cerebral ischemia and intracranial haemorrhage. In turn, miRNAs have been shown to play diverse roles in neuronal, glial, and endothelia responses to stroke. In addition, miRNAs have been suggested to regulate the effects of ischemia on aquaporin expression and function and in some cases may be neuroprotective. miRNAs may also help explain gender-based differences in responses to cerebral ischemia. RNA editing, a related epigenetic mechanism that is partly responsible for generating the exquisite degrees of environmental responsiveness and molecular diversity. In addition, the development of future therapeutic strategies for locus-specific and genome-wide regulation of genes and functional gene networks through the modulation of RNA transcription, posttranscriptional RNA processing (e.g., RNA modifications, quality control, intracellular trafficking, and local and long distance intercellular transport), and RNA translation. These novel approaches for neural cell- and tissue-selective reprogramming of epigenetic regulatory mechanisms are likely to promote more effective neuroprotective and neural regenerative responses for safeguarding and even restoring central nervous system function. Data accumulated to date strongly suggest that further studies of these mechanisms are well justified and that future publications resulting from these studies are worthy of careful attention.

ffective neuroprotective and neural regenerative responses for safeguarding and even restoring central nervous system function. Data accumulated to date strongly suggest that further studies of these mechanisms are well justified and that future publications resulting from these studies are worthy of careful attention. Figure 1 There are three main epigenetic mechanisms. (1) The first includes the mechanisms mediating DNA methylation, typically at cytosine residues in gene promoter regions. These reactions attenuate gene expression and are catalyzed by multiple different isoforms of DNA methyltransferases. An important requirement for these reactions is a methyl donor, typically folic acid supplied through the diet. (2) The second epigenetic category of mechanisms includes the enzymes that acetylate and deacetylate lysine residues on histone proteins. These enzymes regulate chromatin structure and include histone acetyltransferases and histone deacetylases. In general, histone acetylation promotes dissociation from DNA and facilitates gene expression, whereas deacetylation promotes reassociation and reduced gene expression. (3) The third epigenetic category includes the pathways that transcribe, process, and transport microRNA, endogenous short interfering RNAs (siRNAs), and exogenous siRNAs. Endogenous microRNAs are transcribed from nuclear genes into primary microRNA transcripts, which are cleaved into precursor microRNA transcripts. The nuclear protein, Exportin 5, transports precursor microRNAs onto the cytoplasm, where it is cleaved by Dicer to an imperfect miR-X : miR-X* duplex. One strand of the duplex is degraded and the remaining, mature microRNA binds Dicer and Argonaute (Ago) proteins to form RNA-induced silencing complexes (RISCs). MicroRNAs target sequences within cellular mRNAs. Parallel processes in the cytoplasm produce siRNAs derived from endogenous transposons, or from exogenous siRNAs and target cellular or viral mRNAs.

1. Introduction People with stroke results in several neurological impairments, affecting around 1 million subjects in Europe. Hence, stroke effects are the leading cause of long-term disability in industrialized societies [1, 2]. Rehabilitation's outcomes often conclude in incomplete motor recovery and over 60% of patients cannot use their paretic hands in functional activities. Furthermore, presence of severe paresis after four weeks is considered a negative predictive factor for the motor recovery [2], indicating for these patients serious difficulties in the activities of daily living in their future. To facilitate recovery of upper limb function, many different rehabilitative treatments are still proposing. Among them, researchers are focusing their attention on non-invasive brain stimulations (NIBS), worldwide. Tools of NIBS are the repetitive transcranial magnetic stimulation (rTMS) and the transcranial direct current stimulation (tDCS). Use of tDCS is increasing in patients with stroke for its modulatory effects on cognitive and motor functions [3–5]. In particular for the motor domain, the cortical target of tDCS application has been showed to enhance execution and skills [6], producing interest for the improvement of rehabilitative stroke's course. Moreover, respect than rTMS, it is less expensive, more mobile, and, therefore, more comfortable, making its use easier in clinical settings.

tor domain, the cortical target of tDCS application has been showed to enhance execution and skills [6], producing interest for the improvement of rehabilitative stroke's course. Moreover, respect than rTMS, it is less expensive, more mobile, and, therefore, more comfortable, making its use easier in clinical settings. This technique applies electrical current directly on the scalp and modulates the membrane potential dependently by type of electrode's application. In fact, anode is able to facilitate the depolarization of neurons, while, on the contrary, cathode hyperpolarizes the resting membrane potential, reducing the neuronal firing [7]. Application in motor domain for subjects with stroke has been showed to be effective in enhancing the performance of functional tasks and muscle force [4, 8, 9]. At the same time, a recent meta-analysis has underlined as small sample size, different setups, and a large effect size in studies concerning motor recovery on patients with stroke may reduce the clinical meanings of this preliminary evidence. The aim of this study was to evaluate the effects on manual dexterity and pinch and grip force of a tDCS single stimulation, compared to Sham stimulation, and if this improvement was different among the three possible electrodes' montages (anodal, cathodal, or bipolar). Secondary outcome was to evaluate the satisfaction of patients in using this advanced rehabilitative technology.

terity and pinch and grip force of a tDCS single stimulation, compared to Sham stimulation, and if this improvement was different among the three possible electrodes' montages (anodal, cathodal, or bipolar). Secondary outcome was to evaluate the satisfaction of patients in using this advanced rehabilitative technology. 2. Material and Methods This is a single-blind, crossover, sham-controlled study. Patients were admitted for an inpatient rehabilitation with a diagnosis of stroke to our hospital. The inclusion criteria to participate to this study were selected as follows: first-ever stroke; cortical or cortical-subcortical lesion, confirmed by diagnostic imaging (CT or MRI scans); mild to moderate hemiparesis with presence of minimal hand movement (proved by possibility to perform grip or pinch test). The following exclusion criteria were considered: presence of a history of chronic disabling pathologies of upper limb; spasticity; presence of pacemaker or severe cardiovascular conditions; a history of tumor, prior neurosurgical brain intervention, or severe cardiovascular conditions, including the presence of a pacemaker; a diagnosis of epilepsy or major psychiatric disorders. Demographic and clinical characteristics of the nine patients undergone the experimental procedure are summarized in Table 1. The protocol was approved by the local independent ethics committee, and all participants gave written informed consent.

2. Material and Methods This is a single-blind, crossover, sham-controlled study. Patients were admitted for an inpatient rehabilitation with a diagnosis of stroke to our hospital. The inclusion criteria to participate to this study were selected as follows: first-ever stroke; cortical or cortical-subcortical lesion, confirmed by diagnostic imaging (CT or MRI scans); mild to moderate hemiparesis with presence of minimal hand movement (proved by possibility to perform grip or pinch test). The following exclusion criteria were considered: presence of a history of chronic disabling pathologies of upper limb; spasticity; presence of pacemaker or severe cardiovascular conditions; a history of tumor, prior neurosurgical brain intervention, or severe cardiovascular conditions, including the presence of a pacemaker; a diagnosis of epilepsy or major psychiatric disorders. Demographic and clinical characteristics of the nine patients undergone the experimental procedure are summarized in Table 1. The protocol was approved by the local independent ethics committee, and all participants gave written informed consent. 2.1. Transcranial Direct Current Stimulation Stimulation was delivered for 15 minutes, both in real and sham condition, in two consecutive days, randomized for sham/tDCS and anodal/bipolar/cathodal stimulations. In both sessions, the stimulation was preceded by 60 seconds where the current was gradually increased until intensity of 1.5 mA, eliciting transient sensations that disappeared over seconds, consistently with previous reports [9, 10]. The stimulator (Eldith DC Stimulator, NeuroConn, Germany) provided the direct current using two gel-sponge electrodes with a surface area of 35 cm2 (5 × 7 cm for each electrode) embedded in a saline-soaked solution.

liciting transient sensations that disappeared over seconds, consistently with previous reports [9, 10]. The stimulator (Eldith DC Stimulator, NeuroConn, Germany) provided the direct current using two gel-sponge electrodes with a surface area of 35 cm2 (5 × 7 cm for each electrode) embedded in a saline-soaked solution. Positioning of active electrode varied according to randomized different montage: for anodal stimulation, the active electrode was placed on the projection of the hand knob area of the primary motor cortex of the affected hemisphere; for cathodal stimulation, the electrode was placed on unaffected hemisphere in an analogue position of the anodal stimulation. For these electrodes' setup, referent electrode was positioned on the skin overlying the contralateral supraorbital region. In bilateral montage, cathode and anode were positioned as active electrode in the same way described above. In the context of electrical stimulation, anode indicates the relative positive terminal where current flows into the body, while cathode indicates the relative negative terminal where the current exits from the body [11].

l montage, cathode and anode were positioned as active electrode in the same way described above. In the context of electrical stimulation, anode indicates the relative positive terminal where current flows into the body, while cathode indicates the relative negative terminal where the current exits from the body [11]. 2.2. Test Protocol Patients were asked to perform the 9-hole peg test (9HPT) before- and after-tDCS or Sham. This test consists of a squared board with 3 rows of 3 holes. Participants were asked to fill the 9-holes with pegs as fast as possible. Researchers recorded the time spent to execute the task with a stopwatch starting when the subject touched the first peg and stopping it when the subject filled the last hole or when time was longer than 50 s, as previous researches reported [12, 13]. Velocity of execution was computed in terms of holes filled per second (number of filled holes/time). 9HPT-index, as an index of manual dexterity, was obtained. To perform a data normalization among subjects, the 9HPT-index was computed as follows: 9HPT-index = velocity LS/velocity HS ∗ 100. The percentage improvement between pre and post treatment of 9HPT-index was computed as (9HPT-indexpost – 9HPT-indexpre)/9HPT-indexpre ∗ 100.

as an index of manual dexterity, was obtained. To perform a data normalization among subjects, the 9HPT-index was computed as follows: 9HPT-index = velocity LS/velocity HS ∗ 100. The percentage improvement between pre and post treatment of 9HPT-index was computed as (9HPT-indexpost – 9HPT-indexpre)/9HPT-indexpre ∗ 100. As other outcome's measure, for each participant, the maximum pinch force and the maximum grasp force were measured by means of specific dynamometers. Both hands were evaluated with the patients seated, the elbow at 90° of flexion and a neutral position of the wrist. Grip force was determined according to Jamar method, with the arm as more stretched as possible and handlebars fixed to 5 cm, the most appropriate distance to develop the maximal force [14]. The maximum forces recorded between two trials were analyzed. Each participant performed these tests before- and after-tDCS or Sham. Finally, four questions were asked about the satisfaction with the tool from patients' perspective. Inspired by QUEST (Quebec User Evaluation of Satisfaction with Assistive Technology) questionnaire [15], items were concerned on dimension and utility of the device, modality of application, comfort in the using. Answers were graded on the Likert-type scale, the most widely used approach to scaling responses in survey research, from “not satisfied at all” to “very satisfied.”

with Assistive Technology) questionnaire [15], items were concerned on dimension and utility of the device, modality of application, comfort in the using. Answers were graded on the Likert-type scale, the most widely used approach to scaling responses in survey research, from “not satisfied at all” to “very satisfied.” 2.3. Statistical Analysis All measurements are reported in terms of mean ± standard deviation. A repeated measure analysis of variance was performed on the 9HPT-index using as within-subjects factors pre versus post treatment and tDCS versus Sham, whereas as between-subjects factor the type of setup (A, B, or C). Post hoc analyses had been performed using Tukey correction for the inflation or type I error for multiple comparisons. Similarly, same analyses were performed on the pinch and grasp forces recorded for the affected limb of subjects. For verifying the applicability of analysis of variance, we previously performed the Levene's test of equality of error variances for verifying the homogeneity of data of the 3 recorded variables (9HPT-index, pinch and grasp forces) for both the stimulations (tDCS versus Sham), before and after-stimulation. SPSS 17.0 was used and significant threshold was set at 0.05. 3. Results Table 2 reports the experimental data of all nine selected patients.

2.3. Statistical Analysis All measurements are reported in terms of mean ± standard deviation. A repeated measure analysis of variance was performed on the 9HPT-index using as within-subjects factors pre versus post treatment and tDCS versus Sham, whereas as between-subjects factor the type of setup (A, B, or C). Post hoc analyses had been performed using Tukey correction for the inflation or type I error for multiple comparisons. Similarly, same analyses were performed on the pinch and grasp forces recorded for the affected limb of subjects. For verifying the applicability of analysis of variance, we previously performed the Levene's test of equality of error variances for verifying the homogeneity of data of the 3 recorded variables (9HPT-index, pinch and grasp forces) for both the stimulations (tDCS versus Sham), before and after-stimulation. SPSS 17.0 was used and significant threshold was set at 0.05. 3. Results Table 2 reports the experimental data of all nine selected patients. Before applying analysis of variance, the data homogeneity was verified with Levene's test of equality of error variances, of the 3 recorded variables (9HPT-index, pinch and grasp forces) for both stimulations (tDCS versus Sham), before and after-stimulation. Eleven of 12 datasets resulted homogenous (P > 0.05), with a significant reduction of homogeneity observed just for grasping after sham stimulation (P = 0.039). According to these results, we applied repeated measure analysis of variance.

p forces) for both stimulations (tDCS versus Sham), before and after-stimulation. Eleven of 12 datasets resulted homogenous (P > 0.05), with a significant reduction of homogeneity observed just for grasping after sham stimulation (P = 0.039). According to these results, we applied repeated measure analysis of variance. The improvements recorded after tDCS treatment were significantly higher with respect to the changes observed after Sham treatment, as shown in Figure 1 and Table 3 (P = 0.022 of interaction Pre versus Post ∗ tDCS versus Sham). Despite the high data variability, anodal and cathodal showed the higher improvements, but the differences between setups were significant only as main factor (P = 0.008), but not for the interaction Pre versus Post ∗ tDCS versus Sham ∗ ABC (P = 0.212). Post hoc analyses revealed that the A group had a lower 9HPT-index already before treatment (P = 0.017, analysis of variance, factor group).

mprovements, but the differences between setups were significant only as main factor (P = 0.008), but not for the interaction Pre versus Post ∗ tDCS versus Sham ∗ ABC (P = 0.212). Post hoc analyses revealed that the A group had a lower 9HPT-index already before treatment (P = 0.017, analysis of variance, factor group). In terms of manual force, the interaction among factors (Pre versus Post ∗ tDCS versus Sham ∗ ABC) significantly affected the pinch force of the affected limb (F(2,6) = 5.60, P = 0.042). Main factor ABC did not affect significantly the pinch force (F(2,6) = 1.22, P = 0.360). We found a significant improvement of +13.1 ± 7.5% after cathodal stimulation, a reduction of force of −6.5 ± 19.8% after bipolar stimulation and no changes (0% in mean) after anodic stimulation or Sham simulation. Grasping forces were not altered, with just a slight but not significant effect of tDCS versus Sham ∗ ABC interaction (F(2,6) = 4.00, P = 0.079), again with higher improvement after cathodal stimulation. Finally, regarding the user evaluation, overall satisfaction with the device was very good. Results of the short survey was reported in Table 4.

In terms of manual force, the interaction among factors (Pre versus Post ∗ tDCS versus Sham ∗ ABC) significantly affected the pinch force of the affected limb (F(2,6) = 5.60, P = 0.042). Main factor ABC did not affect significantly the pinch force (F(2,6) = 1.22, P = 0.360). We found a significant improvement of +13.1 ± 7.5% after cathodal stimulation, a reduction of force of −6.5 ± 19.8% after bipolar stimulation and no changes (0% in mean) after anodic stimulation or Sham simulation. Grasping forces were not altered, with just a slight but not significant effect of tDCS versus Sham ∗ ABC interaction (F(2,6) = 4.00, P = 0.079), again with higher improvement after cathodal stimulation. Finally, regarding the user evaluation, overall satisfaction with the device was very good. Results of the short survey was reported in Table 4. No statistically significant changes were found among anodal, bilateral, and cathodal montages in terms of patients' judgment assessed by means of user satisfaction scale in terms of dimension (P = 0.848, Kruskal-Wallis analysis), perceived utility (P = 0.846), perceived easiness to use (P = 0.230), and comfort during treatment (P = 0.656) (Figure 2). Just a little surprising trend was observed indicating that bipolar montage was perceived as less invasive, despite the presence of two electrodes on the head and being more comfortable.

analysis), perceived utility (P = 0.846), perceived easiness to use (P = 0.230), and comfort during treatment (P = 0.656) (Figure 2). Just a little surprising trend was observed indicating that bipolar montage was perceived as less invasive, despite the presence of two electrodes on the head and being more comfortable. 4. Discussion The purpose of this study was to determine the effects of a single transcranial direct current stimulation (real versus sham) on dexterity and manual force in patients with stroke, performing the stimulation through three different electrodes' setups, and determining if the montage was perceived by the patients as satisfactory. Our results suggest that tDCS treatment was more effective than Sham treatment on manual dexterity, while no significant differences were recorded in terms of manual force, even if a slight improvement was noted after the cathodal stimulation. Furthermore, no difficulties in performing treatment were complained by the patients.

ts suggest that tDCS treatment was more effective than Sham treatment on manual dexterity, while no significant differences were recorded in terms of manual force, even if a slight improvement was noted after the cathodal stimulation. Furthermore, no difficulties in performing treatment were complained by the patients. Recently, many studies have been focused on devices to facilitate the motor recovery. The tDCS is emerging as one of the most interesting device to apply in stroke rehabilitation, both for the cognitive and motor impairments. Treatments with tDCS can be supplied up to 30 minutes, similarly to the timing of rehabilitative session, before or in synchrony with it, enhancing the rehabilitation outcomes [9, 10]. Moreover, compared to other forms of NIBS, tDCS is more comfortable, more mobile, and cheaper, and no major adverse effects have been reported. Common side effects include mild headache, itching, and erythema at the electrode site [16].

ion, before or in synchrony with it, enhancing the rehabilitation outcomes [9, 10]. Moreover, compared to other forms of NIBS, tDCS is more comfortable, more mobile, and cheaper, and no major adverse effects have been reported. Common side effects include mild headache, itching, and erythema at the electrode site [16]. In spite of these advantages, use of this technique in rehabilitation is counteracted due to still too preliminary evidence. In fact, studies vary widely in terms of phase of stroke, functional impairments, targeting of the outcomes, stimulation set-ups, and rehabilitative integration. Hence, in a recent meta-analysis, Bastani and Jaberzadeh concluded that tDCS (in that case, as anodal stimulation) seems to produce significant effects in subjects with stroke but any conclusion should be considered cautiously [3]. At the same, they also noted its potential role as add-on technique to improve motor function and corticomotor excitability. In our study, we have focused our attention on different electrodes' montages, being an increasing interest on type of stimulation. Our results show as anodal stimulation provided the higher improvement in terms of manual dexterity. These findings are consistent with previous reports [8, 17–19]. In such cases, effects can last up to 2 weeks after the treatment [17]. Most of these studies are concerning a chronic phase of the stroke, while only Kim and colleagues have showed a stimulation effect on patients in a subacute phase. Noteworthy, a recent report has observed that tDCS does not seem to be effective in an acute phase [20].

ts can last up to 2 weeks after the treatment [17]. Most of these studies are concerning a chronic phase of the stroke, while only Kim and colleagues have showed a stimulation effect on patients in a subacute phase. Noteworthy, a recent report has observed that tDCS does not seem to be effective in an acute phase [20]. The tasks utilized to measure the manual dexterity, including the Jebsen-Taylor test, the Box and Block test, and the 9HPT, need a complex sensory information and sensorimotor integration for accurate performance. Moreover, the successful performance requires a complex pattern of activation of muscles and joints as well as the use of targets and tools [9, 21]; hence, the role of enhancer of motor rehabilitation should be more appropriate for the anodal stimulation of tDCS. In fact, also the stimulation with cathode of the unaffected hemisphere seems to be effective in motor function improvement, but reports are not always concordant [17, 19]. On the contrary, our results showed that cathodal tDCS seemed to be a little effect in terms of force, differently by other setups. In our study, bipolar stimulation seemed to be the less effective. In a previous study, it was reported as simultaneous application of anodal tDCS over the motor cortex and cathodal tDCS over the contralateral motor cortex induced an increase in cortical excitability [22]. Our study supports these findings in terms of dexterity, suggesting a global effect of treatments based on electrical stimulation respect than sham conditions, also for the bipolar montage of electrodes.

otor cortex and cathodal tDCS over the contralateral motor cortex induced an increase in cortical excitability [22]. Our study supports these findings in terms of dexterity, suggesting a global effect of treatments based on electrical stimulation respect than sham conditions, also for the bipolar montage of electrodes. Finally, the overall satisfaction by the patient was maintained during a brief protocol treatment, confirming the facility of using this device [10]. The main limitation of our study was the reduced sample size. Although the number of subjects involved in this study was in line with other studies on tDCS [4, 8–10, 17–19], it suggests cautions in data interpretation. On the other hand, from a statistical point of view, the significant effects found in our study (P = 0.022 for interaction Pre versus Post ∗ tDCS versus Sham for 9HPT-index and P = 0.042 for interaction Pre versus Post ∗ tDCS versus Sham ∗ ABC for pinch force) obtained on a small sample were potentially larger than equivalent results obtained with larger samples, supporting the importance of our results. Anyway, further researches on wider samples are needed. Moreover, the group of anodal stimulation had a generally lower manual dexterity (but not force) that could limit the interpretation of our results. Further research on larger samples is hence needed.

with larger samples, supporting the importance of our results. Anyway, further researches on wider samples are needed. Moreover, the group of anodal stimulation had a generally lower manual dexterity (but not force) that could limit the interpretation of our results. Further research on larger samples is hence needed. In conclusion, the present study contributes to the panel of evidence that strengthen the role of tDCS inside the rehabilitative stroke's course, in particular for the more complex activities of daily living as add-on technique. More studies are needed to define the better montage set-ups, targeting more specific outcome measures. Figure 1 Percentual of improvement in the manual dexterity, measured as 9HPT index, for the real and sham stimulation in the three different electrodes' montages. Abbreviations for the stimulation: A: anodal; B: bilateral; C: cathodal. Figure 2 4-item user satisfaction questions, based on a Likert scoring, for the three electrodes' setups (A: anodic, B: bilateral, C: cathodic montage). Box (thin lines: first and third quartiles, wide line: median) and whiskers (minimum and maximum values) plot for patients' judgments about dimension, utility, easiness of use, and comfort. Table 1 Demographic and clinical characteristics of participants.

Figure 2 4-item user satisfaction questions, based on a Likert scoring, for the three electrodes' setups (A: anodic, B: bilateral, C: cathodic montage). Box (thin lines: first and third quartiles, wide line: median) and whiskers (minimum and maximum values) plot for patients' judgments about dimension, utility, easiness of use, and comfort. Table 1 Demographic and clinical characteristics of participants. Patient (number-initial of surname) 1-A. 2-R. 3-A. 4-S. 5-B. 6-D.M. 7-O. 8-R. 9-V. Mean & S.D. Age 36 27 57 67 82 33 65 37 78 53.5 ± 20.7 Gender (male/female) F F M M M F M F M Handedness (right/left) R R R R R R R R R Type of lesion (hemorrhagic/ischaemic) I I I I I I I I H Time after stroke (days) 29 36 47 21 32 22 32 10 26 28.3 ± 10.4 Site of hemiparesis (right/left) R R L R L L R L R Type of tDCS (anodal/bipolar/cathodal) B A A A B C B C C Sequence of stimulation (tDCS/Sham) T-S T-S T-S S-T S-T S-T T-S T-S S-T Mean ± standard deviation of demographic characteristics and clinical features are reported. Abbreviations in the table above: M: male; F: female; R: right; L: left; H: hemorrhagic stroke; I: ischaemic stroke; A: Anodal; B: Bipolar; C: Cathodal; T: tDCS stimulation; S: sham stimulation; S.D.: standard deviation. Table 2 Data of 9HPT-index and manual force recorded for each patient.

Mean ± standard deviation of demographic characteristics and clinical features are reported. Abbreviations in the table above: M: male; F: female; R: right; L: left; H: hemorrhagic stroke; I: ischaemic stroke; A: Anodal; B: Bipolar; C: Cathodal; T: tDCS stimulation; S: sham stimulation; S.D.: standard deviation. Table 2 Data of 9HPT-index and manual force recorded for each patient. Type of stimulation Type of setup stimulation Prestimulation Poststimulation 9HPT-index (%) Pinch (kg) Grasp (kg) 9HPT-index (%) Pinch (kg) Grasp (kg) tDCS Anodal 26.7 3.5 14 28.0 3.5 12 tDCS Anodal 25.6 3.5 10 29.6 3.5 9 tDCS Anodal 19.6 6 18 24.0 6 18 tDCS Cathodal 44.7 4.5 14 48.3 6 12 tDCS Cathodal 88.9 5.5 16 75.0 6 22 tDCS Cathodal 81.1 2.5 14 100.0 4.5 14 tDCS Bilateral 88.9 6 24 75.0 5 15 tDCS Bilateral 77.3 9.5 34 88.9 11 36 tDCS Bilateral 57.1 5 18 75.0 2.5 16 Sham Anodal 35.1 4 10 21.3 4 10 Sham Anodal 20.4 2 8 26.7 2 10 Sham Anodal 22.2 5 14 25.3 5 16 Sham Cathodal 30.2 3 15 32.0 3 14 Sham Cathodal 70.6 6 22 61.1 6 20 Sham Cathodal 106.3 4 18 96.6 4 20 Sham Bilateral 125.0 6 16 94.4 6 18 Sham Bilateral 93.3 9 36 77.8 10 38 Sham Bilateral 133.3 4.5 20 125.0 3.5 20 Table 3 Repeated measure ANOVA results. Factors and interactions df F P Pre versus Post 1 0.475 0.516 Pre versus Post ∗ ABC 2 0.404 0.685 tDCS versus Sham 1 1.457 0.273 tDCS versus Sham ∗ ABC 2 3.167 0.115 Pre versus Post ∗ tDCS versus Sham 1 9.507 0.022 Pre versus Post ∗ tDCS versus Sham ∗ ABC 2 2.030 0.212 ABC 2 11.808 0.008 df: degrees of freedom (df of error = 6), F and P values (in bold if statistically significant).

Pre versus Post 1 0.475 0.516 Pre versus Post ∗ ABC 2 0.404 0.685 tDCS versus Sham 1 1.457 0.273 tDCS versus Sham ∗ ABC 2 3.167 0.115 Pre versus Post ∗ tDCS versus Sham 1 9.507 0.022 Pre versus Post ∗ tDCS versus Sham ∗ ABC 2 2.030 0.212 ABC 2 11.808 0.008 df: degrees of freedom (df of error = 6), F and P values (in bold if statistically significant). Table 4 4-item satisfaction of the user and the 5-point scale used to rate each item. Patient (number-initial of surname) 1-A. 2-R. 3-A. 4-S. 5-B. 6-D.M. 7-O. 8-R. 9-V. Mean & S.D. Dimension 5 5 4 4 3 5 5 4 3 4.2 ± 0.8 Utility 4 5 4 4 4 5 5 4 3 4.2 ± 0.6 Application 4 5 3 4 3 3 5 3 3 3.6 ± 0.9 Comfort 4 5 3 3 3 4 5 3 3 3.6 ± 0.9 1: not satisfied at all, 2: not very satisfied, 3: more or less satisfied, 4: quite satisfied, and 5: very satisfied. Abbreviations in the table above: S.D.: standard deviation.

“Real progress happens only when advantages of a new technology become available to everybody” said Henry Ford; we would add “or to the most disadvantaged people.” Stroke is the leading cause of disability in all industrialized countries. Common rehabilitation usually allows about 50% of patients with stroke to recover walking, leaving the others not independent in walking and other activities of daily living [1]. For these reasons, an increasing number of researches are pursuing the use of new technologies to improve the efficacy of rehabilitation. For example, over the last decade, many devices for robotic-assisted training have been developed to allow patients to perform early, intensive, and task-oriented exercises [2, 3], and wearable devices for an objective assessment of human movements have been developed for substituting in clinical settings the expensive and difficulty to use stereophotogrammetric systems of research gait laboratories [4]. Moreover, invasive and noninvasive techniques allowing the manipulation of brain excitability and plasticity appeared in the last decades. If their promises will be confirmed in the next future, these techniques associated to rehabilitation may boost the recovery of stroke patients.

s of research gait laboratories [4]. Moreover, invasive and noninvasive techniques allowing the manipulation of brain excitability and plasticity appeared in the last decades. If their promises will be confirmed in the next future, these techniques associated to rehabilitation may boost the recovery of stroke patients. Differently from other fields of engineering, studies about the effectiveness of these technologies often occur after their development and their insertion in the rehabilitation settings. A number of studies showed the efficacy of these new technological approaches, whereas some others did not show any improvement in respect of conventional therapies. This uncertainty about efficacy, together with high purchase cost for some of these devices, some difficulties in their use by untrained staff, the absence of clear guidelines about better dosage and parameter values to select, and a somewhat diffuse scepticism by some members of the rehabilitation teams may limit the transfer of these new technologies from research laboratories to clinical settings, where patients are waiting to benefit from them.

ined staff, the absence of clear guidelines about better dosage and parameter values to select, and a somewhat diffuse scepticism by some members of the rehabilitation teams may limit the transfer of these new technologies from research laboratories to clinical settings, where patients are waiting to benefit from them. This special issue aimed to provide an overview on the use of new technologies for the rehabilitation of people with stroke. It contains two reviews and seven original researches. There is a generic review of M. Iosa and colleagues about seven promising technologies for stroke rehabilitation: robots, brain computer interfaces, noninvasive brain stimulators, neuroprostheses, virtual reality, wearable devices, and tablet-pcs. A more specific review is that of E. Q. van Delden and colleagues about twenty different devices for bilateral upper limb training. Upper limb rehabilitation was also the object of the study of G. Thielman and P. Bonsall, reporting the combined use of different technologies such as a unilateral robotic device used for interacting with a virtual task on a monitor, together with an auditory biofeedback for improving the trunk control. Also the study of P. Sale et al. was focused on the use of robotic rehabilitation of upper limb, in their case using a unilateral device designed for hand rehabilitation. Positive outcomes were found in all these studies, suggesting that robotic devices may provide a useful extension of currently available forms of therapy, as also stated in the paper by E. Q. van Delden and colleagues. But this special issue was not only focused on robotic devices. A. R. Bowers and colleagues investigated the on-road detection performance by drivers with hemianopia using oblique peripheral prisms. The study of G. Morone and colleagues showed the potentiality of using a neuroprosthesis not only for correcting the foot drop in chronic phase, but also for improving the sensorimotor relearning during rehabilitation in subacute phase of stroke, facilitating the gait recovery. P. J. Manns and R. G. Haennel investigated the use of a wearable device for assessing walking capacity in people after stroke in terms of energy expenditure and step measurement, concluding that this device is not enough accurate as step counter. So not all technologies can be used also in stroke population, and not in a nonspecific manner. The study of A.

ated the use of a wearable device for assessing walking capacity in people after stroke in terms of energy expenditure and step measurement, concluding that this device is not enough accurate as step counter. So not all technologies can be used also in stroke population, and not in a nonspecific manner. The study of A. Fusco and colleagues, in fact, analysed three different montages of the electrodes of transcranial direct current stimulation, suggesting different effects among anodal montage acting on manual dexterity, cathodal montage improving manual force, and bilateral montage, the less effective. Also, G. Thielman and P. Bonsall suggested that the degree of changes varied per protocol and may be due to the appropriateness of the technique chosen, as well as based on patients impairments. These observations result in line with the proposal to change the research question from “is this specific technology effective?” into “how may I use this technology in an effective way?” as suggested by M. Iosa et al. [5] and “which patients is this technology effective for?” as suggested by G. Morone et al. [6]. Robotic devices, for example, were shown to be more effective for severely affected patients, whereas rehabilitative outcomes after robotic training resulted similar to those of conventional manual therapy for moderately affected patients [6, 7].

is this technology effective for?” as suggested by G. Morone et al. [6]. Robotic devices, for example, were shown to be more effective for severely affected patients, whereas rehabilitative outcomes after robotic training resulted similar to those of conventional manual therapy for moderately affected patients [6, 7]. This special issue would contribute to provide clear results on the potential benefits of the use of new technologies for patients with stroke, avoiding overstatements of results, and, at the same time, reducing the scepticism of those who say “With machines they would perform miracles. What sort of miracles?” as asked by the Inquisitor in the drama of Bertolt Brecht Life of Galileo. Marco Iosa Stefan Hesse Antonio Oliviero Stefano Paolucci

1. Background Regaining independent ambulation is important to those with stroke [1, 2] and is the most frequently reported rehabilitation goal [3, 4]. Therefore, walking should be an integral part of in-patient rehabilitation. However, accelerometer-based monitoring of walking activity has revealed that the amount of daily walking completed by individuals with stroke during in-patient rehabilitation is low [5, 6]. Importantly, the majority of walking bouts are of short duration (<1 minute) [5–7] and typically involve walking to essential activities (e.g., washroom, dining area, or therapy) [5]. While activity monitors provide insight into total daily activity [5–10], they do not inform the possible determinants or consequences of this activity. Aerobic capacity is reduced in the early months following stroke [11–13]. Furthermore, poststroke gait is inefficient, and there are increased aerobic demands on those with stroke when walking compared to healthy controls, even when walking at the same speed [14]. Therefore, individuals with stroke are closer to their maximal aerobic threshold when walking than healthy controls. This potentially limits the intensity (i.e., speed) and total duration of walking activity during daily life.

h stroke when walking compared to healthy controls, even when walking at the same speed [14]. Therefore, individuals with stroke are closer to their maximal aerobic threshold when walking than healthy controls. This potentially limits the intensity (i.e., speed) and total duration of walking activity during daily life. Walking can be a valuable means to improve aerobic capacity [15]. Aerobic exercise can help to improve aerobic capacity following stroke and improve recovery from stroke [16, 17]. Furthermore, there is evidence that increased amount of rehabilitation early after stroke improves recovery [18, 19]. However, limited resources within rehabilitation hospitals may impede the ability to provide formalized or structured aerobic training. Unstructured and unsupervised activities, such as daily walking, provide an opportunity to benefit aerobic fitness afterstroke. Presently, it is not known if in-patients engage in episodes of walking that have aerobic benefit outside of therapy.

y impede the ability to provide formalized or structured aerobic training. Unstructured and unsupervised activities, such as daily walking, provide an opportunity to benefit aerobic fitness afterstroke. Presently, it is not known if in-patients engage in episodes of walking that have aerobic benefit outside of therapy. This study aims to answer two questions: (1) does unsupervised, unstructured daily walking activity provide aerobic benefit to individuals after stroke and (2) is daily walking activity limited after stroke due to increased energy demands of walking? This initial study was specifically focused on a sample of stroke patients who were able to walk independently and who resided within a rehabilitation hospital. We view this subacute phase to be particularly important for enhancing aerobic training after stroke. To address the first objective, we determined if individuals with stroke engaged in walking bouts that were at least 10 minutes long at an intensity of 40%–80% of heart rate reserve (HRR), totaling at least 20 minutes per day [15]. To address the second question, we determined the duration of walking activity that reached or exceeded the aerobic threshold of 80% of HRR [20]. The latter, if it occurred, would reflect that the challenge of everyday walking may pose a potential barrier to being more active.

totaling at least 20 minutes per day [15]. To address the second question, we determined the duration of walking activity that reached or exceeded the aerobic threshold of 80% of HRR [20]. The latter, if it occurred, would reflect that the challenge of everyday walking may pose a potential barrier to being more active. 2. Methods 2.1. Patients We included individuals who were attending in-patient rehabilitation following stroke and who were able to walk independently without supervision (with or without use of a walking aid). We excluded patients who used heart-rate (HR) altering medication (e.g., beta-blockers) as HR response would be variable depending on when medication was taken. Eight individuals volunteered to participate and provided informed consent. The study was approved by the institution's research ethics board, and study procedures were in accordance with institutional guidelines. Characteristics of the eight volunteers are presented in Table 1. Participants underwent clinical assessment of gait, functional balance, and motor impairment. Spatiotemporal characteristics of gait were collected using a pressure-sensitive mat (GAITRite, CIR Systems Inc., Havertown, PA, USA). Participants walked across the mat three times at their preferred speed and the location and timing of each footstep were sampled at 30 Hz. We then calculated walking speed, cadence, and temporal symmetry [21]. Motor impairment was assessed using the Chedoke-McMaster Stroke Assessment (CMSA) [22]. Functional balance was assessed using the Berg Balance Scale (BBS) [23].

heir preferred speed and the location and timing of each footstep were sampled at 30 Hz. We then calculated walking speed, cadence, and temporal symmetry [21]. Motor impairment was assessed using the Chedoke-McMaster Stroke Assessment (CMSA) [22]. Functional balance was assessed using the Berg Balance Scale (BBS) [23]. 2.2. Ambulatory and Heart Rate Data Acquisition The ABLE system [5] (Figure 1) was used to collect ambulatory data. The ABLE system is comprised of two triaxial accelerometers (SparkFun Electronics, Boulder, CO, USA) worn bilaterally around the ankles, which transmit data wirelessly to a personal digital assistant (PDA) (Hewlett Packard, Palo Alto, CA, USA) worn around the waist. The accelerometers were placed just proximal to the lateral malleoli using custom ankle sleeves, and the PDA was secured to the participant's waist using a polyester belt and pouch. Data from each accelerometer unit were recorded on the PDA at 50 Hz. HR data were acquired using a commercially available HR monitoring system (Polar Electro, Kempele, Finland). Participants wore a chest strap and a wristwatch. Heart beats were recorded by the chest strap and transmitted wirelessly to the wristwatch, which logged HR data at 0.2 Hz. The two collection systems were synchronized by initiating data collection in tandem.

lable HR monitoring system (Polar Electro, Kempele, Finland). Participants wore a chest strap and a wristwatch. Heart beats were recorded by the chest strap and transmitted wirelessly to the wristwatch, which logged HR data at 0.2 Hz. The two collection systems were synchronized by initiating data collection in tandem. Patients were fitted with the ABLE and HR monitoring systems in the morning after routine activities were completed (e.g., bathing). The investigator checked every one to two hours to ensure that there was no discomfort and that all devices remained operational. Data collection continued for approximately eight hours, between approximately 9 am and 5 pm. Periods of walking activity were identified and delimited to “bouts” of walking in our analysis. A bout of walking consisted of at least 10 consecutive steps; shorter bouts would not likely have yielded a measurable HR response. Individual bouts of walking were differentiated by a pause of at least 5 seconds prior to the next bout of walking [5].

were identified and delimited to “bouts” of walking in our analysis. A bout of walking consisted of at least 10 consecutive steps; shorter bouts would not likely have yielded a measurable HR response. Individual bouts of walking were differentiated by a pause of at least 5 seconds prior to the next bout of walking [5]. 2.3. Physiological Change Detection The Karvonen formula [20] was used to determine the cardiovascular intensity (i.e., % of HRR) during bouts of walking. Using HR collected from the HR monitor (HRobserved), the % of HRR was determined for each bout of walking by using the following modified Karvonen formula: (1) %HRR  =(HRobserved−HRrest)∗100(HRmax⁡−HRrest). HRmax⁡ was the estimated maximum HR and was determined by subtracting the participant's age from 220 [15]. While the patient remained seated, resting HR (HRrest) was determined by recording the lowest HR measured within the initial 10-minute period of collection. For each identified bout of walking, HR response (HRobserved) for that specific bout was determined by averaging the three highest consecutive HR measures. This approach was taken in order to acquire a sustained HR response over 15 s (e.g., three HR measurement points), as opposed to using a single-point HR, which has the potential to be influenced by transient mutant HR responses.

r that specific bout was determined by averaging the three highest consecutive HR measures. This approach was taken in order to acquire a sustained HR response over 15 s (e.g., three HR measurement points), as opposed to using a single-point HR, which has the potential to be influenced by transient mutant HR responses. 3. Results Mean data collection duration was 8.4 hours (standard deviation: 0.8 hours). Six of the eight participants required a walking aid during both daily walking and clinical data collection (Table 1). At the time of collection, participant B had begun independently ambulating only recently with a walking aid after having been limited to a wheelchair since the onset of her stroke. 3.1. Characteristics of Daily Walking Walking characteristics are outlined in Table 2. The mean number and duration of bouts throughout the collection period were 62.6 bouts (standard deviation: 21.4 bouts) and 57.1 s (standard deviation: 31.6 s), respectively. Overall, 80.8% of all walking bouts were less than 1 min in duration, only 1.8% of all bouts were greater than 5 minutes, and only two walking bouts were greater than 10 minutes. Average step count was 3,708 steps (standard deviation: 1,452). Participant B demonstrated the lowest number of walking bouts with 33 (1,774 steps), while participant E demonstrated the greatest number of walking bouts with 91 (4,778 steps). The single longest bout duration was 13 minutes by participant F, which was performed during structured therapy.

(standard deviation: 1,452). Participant B demonstrated the lowest number of walking bouts with 33 (1,774 steps), while participant E demonstrated the greatest number of walking bouts with 91 (4,778 steps). The single longest bout duration was 13 minutes by participant F, which was performed during structured therapy. 3.2. Did Patients Meet Recommended Physiological Intensities and Durations for Aerobic Exercise during Daily Walking? Overall, none of the participants fulfilled both requirements of duration (bouts ≥10 minutes long for a total of 20 minutes per day) and intensity (% of HRR ≥ 40%) for aerobic benefit (Figure 2). This was most profoundly limited by the duration of the bouts of walking as detailed in the preceding section. With respect to amplitude only 3.1% of all bouts detected, occurring in only two participants, were found to be above 40% HRR. The overall average intensity for bouts of walking was 19.4% HRR. Of the 8 participants tested, only participants B and G exhibited bouts of walking that exceeded the minimum 40% HRR. Participant B had the greatest number of bouts over 40% HRR, which occurred in 31 bouts (93% of their total bouts); participant G exhibited 3 bouts (3.3% of their total bouts) during which HRR exceeded 40%. The remaining participants did not present walking intensities above 40% HRR in any bout.

ed the minimum 40% HRR. Participant B had the greatest number of bouts over 40% HRR, which occurred in 31 bouts (93% of their total bouts); participant G exhibited 3 bouts (3.3% of their total bouts) during which HRR exceeded 40%. The remaining participants did not present walking intensities above 40% HRR in any bout. 3.3. Did Patients Exceed Recommended Intensities during Daily Walking? Participant B was the only participant found to exceed the 80% HRR threshold for any bout of walking (Figure 2). This participant presented 3 bouts (9% of her total bouts) above 80% HRR which ranged from 85.8% to 97.5% HRR. These bouts were not long in duration (<1 minute) and were not performed at high cadences (70–74 steps/minute). These bouts were performed while the participant walked with a rollator on a pedestrian pathway outside the hospital under the supervision of a physiotherapist. 4. Discussion The present investigation sought to determine the extent to which individuals with stroke met or exceeded the recommended cardiovascular intensities of everyday walking activity during in-patient rehabilitation. Together, the quantity and intensity of everyday walking have the potential to positively or negatively influence poststroke recovery. Such information provides insight into the potential value of everyday walking and contributes to the understanding of adaptations to current rehabilitative practices that can be made to maximize time spent in therapeutically beneficial activities.

he potential to positively or negatively influence poststroke recovery. Such information provides insight into the potential value of everyday walking and contributes to the understanding of adaptations to current rehabilitative practices that can be made to maximize time spent in therapeutically beneficial activities. 4.1. Participants Did Not Meet Recommended Duration and Intensity of Walking Activity In agreement with previous work [5, 6, 24] the total amount of spontaneous walking activity was low. As our prior study has also indicated [5], durations of walking throughout an in-patient's day primarily consist of short bouts (e.g., less than one minute). Although there were no data available on healthy individuals for comparison, it is possible that short durations of walking activity observed are not just specific to patients with stroke, but more broadly reflect common walking patterns for inside environments. However, among all eight participants, only one spontaneous walking bout was longer than 10 minutes in duration; one other walking bout was longer than 10 minutes, but this occurred during scheduled physiotherapy. In terms of step counts, it is recommended that individuals with disability take at least 6,000 steps per day, of which 3,000 should be engaged in moderate-to-vigorous physical activity [25]. No participant in the current study attained 6,000 steps in one day.

ted finger movement coordination and combined structured piano lessons to home practice. The specific objective was to estimate the short-term and retention effects of a 3-week piano training program on manual dexterity, finger movement coordination, and functional use of upper extremity in persons with chronic stroke. 2. Methods Three male participants with a mild to moderate deficits of upper extremity motor function due to a first supratentorial chronic stroke (6 to 24 months duration) in the middle cerebral artery territory were recruited after being discharged from rehabilitation (Table 1). Participants had (1) some capacity of dissociation of upper extremity movements as reflected by scores of 3 to 6 on the arm and hand components of the Chedoke-McMaster Stroke Assessment and (2) the ability to follow simple instructions. They had corrected to normal vision and were free of visual field defects (Goldman perimetry), hemineglect (<6 omissions, Bell's test), and cognitive deficits (scores > 23, Montreal Cognitive Assessment). None had musical experience. The study was approved by the Ethics Committee of the Centre for Interdisciplinary Research in Rehabilitation (CRIR), and informed consent was obtained from each participant.

minutes, but this occurred during scheduled physiotherapy. In terms of step counts, it is recommended that individuals with disability take at least 6,000 steps per day, of which 3,000 should be engaged in moderate-to-vigorous physical activity [25]. No participant in the current study attained 6,000 steps in one day. The majority of walking was at <40% HRR, and no participant completed 3,000 steps at a moderate-vigorous level. The overall mean of 19.4% HRR for everyday walking (including periods of structured therapy) was similar to the 24.2% (SD 21.2) HRR of walking found in patients with stroke performing standing and walking tasks exclusively during physical therapy [26]. HRR provides a more informative measure of intensity than cadence or walking speed as it is linked to the patients' aerobic capacity. While the present and previous studies were based on single day “snapshot” of activity, the low duration and intensity of activity for in-patients residing in a rehabilitation facility are striking. The absence of therapeutically beneficial walking activity may be attributed to factors such as short durations, low walking speeds, and the purpose for which patients walked. It is likely that low walking duration and low intensity levels may well be associated with more conventional walking activities, such as activities of everyday living (e.g., going to meals). Subsequent measurement will need to be extended to longer periods of time. However, the modest HR responses confirmed other work that monitored HR through the day [27] leading to the view that such moderate within-day HR responses may be “typical” of patients' experiences.

ies of everyday living (e.g., going to meals). Subsequent measurement will need to be extended to longer periods of time. However, the modest HR responses confirmed other work that monitored HR through the day [27] leading to the view that such moderate within-day HR responses may be “typical” of patients' experiences. 4.2. Excessive Walking-Related Cardiovascular Load Does Not Limit Walking Duration We sought to determine if HR response might be a limiter to walking duration and a potential barrier to activity. As noted the occurrence of HR response greater than 80% HRR was rare and occurred in only one participant (B). This participant had only begun to start walking independently one day prior to data collection after having been mostly wheelchair bound for one month following her stroke. Therefore, this individual was likely extremely deconditioned as a result of limited mobility and, consequently, reached the threshold of her aerobic capacity easily during daily walking. Limited aerobic capacity potentially limited total walking time for this participant; she had the lowest total walking duration and fewest total steps among all participants. However, reduced balance control and increased motor impairment may also have limited total daily walking. Participant B also had the lowest BBS score, CMSA scores, and self-selected walking speed. Increased motor impairment may have increased the energy demands of walking and caused participant B to reach the threshold of aerobic capacity more easily than other participants [14, 28]. Alternatively, reduced walking duration may have been a strategy to prevent a fall given impaired balance control [24].

ing speed. Increased motor impairment may have increased the energy demands of walking and caused participant B to reach the threshold of aerobic capacity more easily than other participants [14, 28]. Alternatively, reduced walking duration may have been a strategy to prevent a fall given impaired balance control [24]. Among the remaining seven participants, there was no evidence that reduced aerobic capacity limited total daily walking activity. No other participant attained >80% HRR while walking during the day. Furthermore, long-duration walking bouts (e.g., 5 minutes or longer) were not associated with increased HR response. Therefore, aerobic capacity did not limit frequency of long-duration walking bouts. The barriers to increasing total spontaneous walking activity following stroke remain to be determined. While HR was typically low, it is possible that patients' perceived fatigue caused them to limit walking speed and time. We did not record perceived exertion or fatigue in the current study; this should be considered in future work. In hospital, patients are likely to be reluctant to walk outside, particularly if balance control is impaired or if the weather is poor. Reduced balance control may play a role, and patients may limit physical activity due to fear of falling [29, 30]. Long-duration walking bouts may be influenced by the size of the unit or the length of the corridors within the hospital, although the current unit features an approximately 50 m long hallway where patients can walk unencumbered. Such distances would provide a much greater opportunity for someone to walk indoors than would be possible in many indoor living settings in the community. However, patients who were transferred from in-patient care to the community were found to increase bout durations in the community, amounting to an extra 30 minutes of activity per day [6]. Such an increase may be partially attributed to opportunity and willingness to walk outside and participate in community activities [6] or may reflect the improved functional capacity at the time after discharge from in-patient rehabilitation. Finally, patients may not be aware of their limited walking activity, and interventions may be required to increase spontaneous walking activity during in-patient rehabilitation. Additional work is required to determine the factors contributing to reduced daily walking activity during in-patient stroke rehabilitation.

Finally, patients may not be aware of their limited walking activity, and interventions may be required to increase spontaneous walking activity during in-patient rehabilitation. Additional work is required to determine the factors contributing to reduced daily walking activity during in-patient stroke rehabilitation. 4.3. Clinical Significance Profiling the relationship between ambulatory activity and HR response can have important health-related implications to poststroke rehabilitation. The absence of activity that may benefit cardiorespiratory health and fitness during supervised and unsupervised periods of the day highlights a larger problem associated with conventional in-patient care. Feasibility studies have demonstrated that structured equipment-based exercise programs can be implemented safely and without negative effects on conventional therapy [17]. Patients receiving aerobic training in addition to standard care have significant improvements in indices of neuromuscular control and functional ambulation [17]. However, these exercise programs require therapist supervision and other resources such as space and equipment. These results can be viewed as a missed or lost opportunity for supplementary rehabilitation practice. The benefit of measuring both walking and HR, as in the present study, is to help clinicians consider periods of unstructured activity. Emphasizing additional therapeutically relevant activities throughout the day may be one method to better address “down” time frequently experienced by patients [31, 32]. However, it can be argued that simply requesting patients to engage in additional walking activities outside of therapy may not occur or may occur at inadequate intensities or durations to provide meaningful improvements in cardiovascular health. By using heart rate and activity monitors to identify the absence of walking bouts required for therapeutic benefits, clinicians would be able to better guide treatment decisions regarding walking exercise programs. Future studies will need to examine intensity and duration of patient activity across multiple days to develop a better understanding of patient activity levels and barriers to increased activity. In addition, the potential use of bout duration and its associated physiological response as an outcome measure for clinical practice requires further study [6].

tensity and duration of patient activity across multiple days to develop a better understanding of patient activity levels and barriers to increased activity. In addition, the potential use of bout duration and its associated physiological response as an outcome measure for clinical practice requires further study [6]. 5. Summary/Conclusions This preliminary investigation between walking activity and task-related HR responses provides insight into the physiological demands associated with daily walking on patients residing in a rehabilitation hospital. These results indicate that daily walking (performed indoors) likely does not provide cardiorespiratory benefit within this group. Consequently, to achieve aerobic benefits from daily walking patients should be encouraged to increase the quantity of walking, and additional emphasis needs to be placed on increasing the intensity of walking if possible. Among those patients for whom the recommended walking intensity is not yet possible aerobic training should be formally included into structured therapy or performed using equipment that poses no risk of falling (e.g., recumbent stepper). Ideally, encouraging adaptations to daily walking activity, to increase both duration and intensity, may help promote and facilitate recovery after stroke and reduce the risk of subsequent vascular events.

uded into structured therapy or performed using equipment that poses no risk of falling (e.g., recumbent stepper). Ideally, encouraging adaptations to daily walking activity, to increase both duration and intensity, may help promote and facilitate recovery after stroke and reduce the risk of subsequent vascular events. Acknowledgments This study was supported by contributions made by the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Institutes of Health Research and Collaborative Health Research and Development Program (no.323492-06), and the Heart & Stroke Foundation Centre for Stroke Recovery. The authors also acknowledge support of the Toronto Rehabilitation Institute, which receives funding under the Provincial Rehabilitation Research Program from the Ontario Ministry of Health and Long-Term Care. D. brooks holds a Canada Research Chair in Rehabilitation (tier 2). Figure 1 Placement of the ABLE system on a patient. Highlighted in the figure is the personal digital assistant (PDA) data logger worn around the waist of the patient, bilateral placement of accelerometer straps worn superior to the lateral malleolus, and heart rate wrist watch data logger. Figure 2 Mean heart rate response, expressed as a percentage of heart rate reserve (HRR), versus duration of walking bout throughout the collection period for all participants. Each point represents a single bout of walking performed during the collection period. The shaded areas indicate the recommended intensity (40%–80% maximum heart rate) and duration (10 minutes of continuous walking) of walking.

HRR), versus duration of walking bout throughout the collection period for all participants. Each point represents a single bout of walking performed during the collection period. The shaded areas indicate the recommended intensity (40%–80% maximum heart rate) and duration (10 minutes of continuous walking) of walking. Table 1 Demographic and clinical characteristics of patients. Participant Gender Age (years) Time after stroke (days) CMSA BBS (score) Resting HR (beats/min) Gait speed (m/s) Cadence (steps/min) Temporal symmetry (ratio) Leg Foot A* M 59 13 4 4 51 64 0.54 72.8 1.26 B* F 31 28 3 4 32 72 0.28 67.2 1.15 C* M 76 14 5 6 51 59 1.11 103.5 1.08 D M 57 25 4 4 38 51 0.83 93.9 1.10 E* M 38 68 5 5 41 64 1.10 100.6 0.98 F M 54 46 5 5 49 55 0.86 98 1.03 G* F 63 30 4 3 44 82 0.53 81.5 1.29 H* F 47 32 3 4 33 51 0.31 60.0 1.20 Mean   53.1 32 4.1 4.4 42.3 62.3 0.70 88.2 1.15 Standard deviation   14.2 17.9 0.8 0.9 7.7 10.7 0.33 14.4 0.96 *Denotes use of an assistive device (e.g., single-point cane/rollator) throughout data collection. BBS: Berg balance scale; CMSA: Chedoke-McMaster stroke assessment; F: female; HR: heart rate; M: male. Table 2 Summary of walking measures for each patient collected throughout the day.

Mean   53.1 32 4.1 4.4 42.3 62.3 0.70 88.2 1.15 Standard deviation   14.2 17.9 0.8 0.9 7.7 10.7 0.33 14.4 0.96 *Denotes use of an assistive device (e.g., single-point cane/rollator) throughout data collection. BBS: Berg balance scale; CMSA: Chedoke-McMaster stroke assessment; F: female; HR: heart rate; M: male. Table 2 Summary of walking measures for each patient collected throughout the day. Participant Total collection time (hours) Total walking time (minutes) Number of walking bouts Mean bout duration (s) Total step count Mean cadence (steps/minute) A 7.06 33.3 46 43.4 2643 73.2 B 8.85 24.6 33 57.6 1774 79.2 C 7.86 31.6 55 34.5 3743 85.8 D 8.65 31.7 79 31.8 2201 78.5 E 8.91 59.3 91 45.8 4778 87.2 F 7.87 58.4 60 131.3 5621 103.6 G 8.41 78.3 89 52.4 5377 78 H 9.69 74.7 48 60.2 3532 61.6 Mean 8.4 49 62.6 57.1 3708 80.8 Standard deviation 0.8 21.2 21.3 31.6 1452 12.1

1. Introduction Persistent contralateral motor impairments are common following a stroke. It is estimated that 80% to 95% of patients experience sensorimotor upper extremity impairments as well as activity and participation limitations, which persist beyond 6 months after stroke onset [1]. This is a major concern as in order to manage daily activities, chronic stroke survivors often use nonoptimal compensation strategies that can lead to a pattern of learned disuse of the paretic arm and further exacerbate the level of disability. Existing therapies that aim at improving upper extremity function show modest to moderate improvements [2], possibly due to insufficient training intensity [3] and lack of adherence. It was also shown that well beyond the optimal recovery window that occurs within the first 6 months after a stroke, rehabilitation still has the potential to induce neurological and functional changes [4, 5]. There is a need to develop and implement interventions that will meet the patient's interests to actively engage them during and beyond the supervised rehabilitation period so that long-term improvements in upper extremity function can be achieved.

has the potential to induce neurological and functional changes [4, 5]. There is a need to develop and implement interventions that will meet the patient's interests to actively engage them during and beyond the supervised rehabilitation period so that long-term improvements in upper extremity function can be achieved. Music-supported therapy (MST) is an innovative approach that has been shown to yield larger improvements in fine and gross motor dexterity compared to conventional rehabilitation and constraint-induced movement therapy in acute stroke survivors [6]. MST was also shown to yield enhanced motor skills and neuroplastic changes of auditory-motor network in chronic stroke participants [7]. In addition to integrating key principles of motor learning and providing instantaneous auditory feedback on performance, the rapid establishment of auditory-motor coupling during music playing would underlie the efficacy of MST [7, 8]. Such coupling can be observed within 20 minutes of musical training and is largely enhanced after 5 weeks of training in nonmusicians [9]. Existing MST programs, however, involve 5 days/week of training and may be difficult to implement in an outpatient and community rehabilitation settings. Furthermore, no previous MST program has focused on finger movement accuracy, timing, and speed, which are important determinants of finger coordination. We have developed, using a user-friendly computerized piano program, a piano training paradigm that provides feedback on note accuracy, timing and speed while allowing participants to progress through finger sequences of increasing complexity. The purpose of this study was to investigate the feasibility of an individually tailored piano training intervention that targeted finger movement coordination and combined structured piano lessons to home practice. The specific objective was to estimate the short-term and retention effects of a 3-week piano training program on manual dexterity, finger movement coordination, and functional use of upper extremity in persons with chronic stroke.

etry), hemineglect (<6 omissions, Bell's test), and cognitive deficits (scores > 23, Montreal Cognitive Assessment). None had musical experience. The study was approved by the Ethics Committee of the Centre for Interdisciplinary Research in Rehabilitation (CRIR), and informed consent was obtained from each participant. Subjects participated in a step-by-step musical training consisting of three individual 1-hour sessions per week for 3 consecutive weeks, for a total of 9 sessions. The individual sessions were complemented with a home program consisting of biweekly piano exercises of 30 min duration. Synthesia, an MIDI piano program, was used to program and display the musical pieces played by the participants on the electronic piano keyboard (Yamaha P155) during the training sessions. The musical pieces involved all 5 fingers of the paretic hand, and participants were cued to press the piano key(s) indicated by the visual stimuli (illuminated, blue dot) presented on the computer screen. Nine musical pieces were created and were introduced to the participants in an increasing order of difficulty: (1) simple, or “following notes” involving movements of consecutive fingers; (2) intermediate, or third, fourth, and fifth intervals involving movements of nonconsecutive fingers; and (3) complex, which involves chords, that is 2 fingers played at the same time. Within each musical piece, the participants started at a tempo of 30 bpm. When reaching a note accuracy and timing score of 80%, as measured in Synthesia, the tempo increased by steps of 10% until reaching a tempo of 60 bpm. Home piano exercises were executed on a roll up flexible piano (Hand Roll Piano 61 K).

me time. Within each musical piece, the participants started at a tempo of 30 bpm. When reaching a note accuracy and timing score of 80%, as measured in Synthesia, the tempo increased by steps of 10% until reaching a tempo of 60 bpm. Home piano exercises were executed on a roll up flexible piano (Hand Roll Piano 61 K). Changes in fine motor (nine hole peg test (NHPT)) and gross motor dexterity (box and block test (BBT)) were measured at multiple baseline time points (week0, week3), immediately prior to (week6) and after the intervention (week9), and at a 3-week follow-up (week12). The Jebsen hand function Test (JHFT), which reflects the functional use of the hand, is time-consuming and was administered only at pre- and post-intervention, as well as follow-up. Piano performance measures, including timing and note accuracy, were collected with Synthesia throughout the training sessions. Participants recorded their home practice duration and frequency in a logbook.

use of the hand, is time-consuming and was administered only at pre- and post-intervention, as well as follow-up. Piano performance measures, including timing and note accuracy, were collected with Synthesia throughout the training sessions. Participants recorded their home practice duration and frequency in a logbook. 3. Results All participants showed improvements in note accuracy and timing accuracy within and across the training sessions. Participant 3 completed 3 musical pieces and the two others completed 5 pieces during the 3-week intervention. They progressed through finger sequences of increasing complexity, involving movements of consecutive fingers followed by movements of nonconsecutive fingers (intervals). Each musical piece started at 30 bpm and were practiced on an average of 25 times over 2 to 3 training sessions before reaching a note accuracy > 80% at 60 bpm. When considering a tempo of 60 bpm, the duration of musical pieces also increased from 17 s to 48 s (participants no. 1 and no. 2) and from 17 s to 32.5 s (participant no. 3) between the first and last training session.

n average of 25 times over 2 to 3 training sessions before reaching a note accuracy > 80% at 60 bpm. When considering a tempo of 60 bpm, the duration of musical pieces also increased from 17 s to 48 s (participants no. 1 and no. 2) and from 17 s to 32.5 s (participant no. 3) between the first and last training session. A mean increase of 6 blocks (range: 4–10 blocks) and a mean reduction of 24.8 s (16–31 s) were observed on the BBT and NHPT, respectively, between pre- and post-intervention (Figure 1). At variance, little variations were observed between baseline measurements at week0 (BBT = 23.3; NHPT = 139.2 s) and week3 (BBT = 24.7; NHPT = 134.8 s) and pre-intervention (BBT = 23.7; NHPT = 138.1 s). None of the participants were able to complete the writing subtest of the JHFT. The 3 participants, however, showed larger scores on all other subtests of the JHFT at post-intervention compared to pre-intervention, with mean increments ranging from 36.2% to 44.2% (Figure 1). Post-intervention scores for the BBT, NHPT, and JHFT were maintained at the 3-week follow-up. Average home practice duration was 50 minutes per session, which exceeded the 30 minutes practice time required. All participants reported enjoying the piano training program, and they all expressed the desire to continue piano lessons after their participation in the study. No adverse reactions to the intervention were reported, with the exception of one participant (no. 2) displaying occasional “hand stiffness” typical of spasticity during the intervention, as well as a fatigue described as a “general fatigue” after the training sessions. The stiffness was going away with frequent breaks, and the fatigue resolved within a few hours after the sessions.

exception of one participant (no. 2) displaying occasional “hand stiffness” typical of spasticity during the intervention, as well as a fatigue described as a “general fatigue” after the training sessions. The stiffness was going away with frequent breaks, and the fatigue resolved within a few hours after the sessions. 4. Discussion This case study is, to our knowledge, the first to report the immediate and retention effects of a structured piano training program combined to home practice in chronic stroke survivors. Improvements in fine and gross manual dexterity, as well as in the functional use of the hand, were observed in all three participants immediately after, but also at the 3-week follow-up. These changes were accompanied by improvements in the speed of execution, as well as in the timing accuracy and note accuracy for each musical piece. Such positive training effects are especially remarkable, considering that participants involved in this study were suffering from a chronic stroke and were exposed to an intervention of short duration. Similar improvements in manual dexterity were observed in a 3-week MST program involving piano and drum pad playing in acute stroke survivors [6], although changes in fine dexterity in the present study (NHPT: +24.8 s) do appear to exceed those reported in the combined drum-piano MST paradigm (NHPT: +13 s). Present results contrast, however, with findings from a case report involving the combined drum pad and piano playing intervention in chronic stroke survivors, where no changes on the NHPT and BBT were observed following the intervention [10]. We hypothesize that the impact on manual dexterity observed in the present study is attributed to the intensity and specificity of the piano exercises that were specifically designed to target dissociated and coordinated finger movements, with an emphasis on note and timing accuracy, as well as speed of execution. Rich feedback was provided to the participants throughout the supervised training session using a computerized program that provided knowledge of performance (note accuracy and timing) and knowledge of result (final speed and error score).

ts, with an emphasis on note and timing accuracy, as well as speed of execution. Rich feedback was provided to the participants throughout the supervised training session using a computerized program that provided knowledge of performance (note accuracy and timing) and knowledge of result (final speed and error score). Comparison of present training effects with other existing upper extremity interventions in chronic stroke such as constraint-induced movement therapy is difficult, due to the use of different outcome measures and inclusion of participants with different characteristics. Present findings, however, can be interpreted in the light of the smallest real differences, or true changes, for the BBT (6 blocks) and the NHPT (32.8 s) [11]. Despite of their chronic stage, our participants either approached (nos. 1 and 3) or exceeded (no. 2) the smallest real difference on the BBT. Similarly, one participant (no. 1) almost reached the smallest real difference for the NHPT while the two others made more modest gains. These observations suggest that MST has the potential to yield real improvements in upper extremity function in chronic stroke participants with different levels of hand and arm motor recovery. The presence of an enhanced functional use of the upper extremity post-intervention also suggests that a better coordination of finger movements can impact on the upper extremity as a whole, and may be a prime target for rehabilitation. Finally, the persistence of positive effects at follow-up further indicates that improvements can be maintained even after the cessation of the training. Compared to other therapies such as constraint-induced movement therapy MST may be less time consuming and labor intensive. It has the potential to be safely self-managed and pursued well beyond the rehabilitation period, such that gains can be maintained or further enhanced.

d even after the cessation of the training. Compared to other therapies such as constraint-induced movement therapy MST may be less time consuming and labor intensive. It has the potential to be safely self-managed and pursued well beyond the rehabilitation period, such that gains can be maintained or further enhanced. MST relies on key principles of motor learning, including repeated and task-specific practice as well as the involvement of multisensory feedback, which gives instantaneous knowledge of result and performance. It would further take advantage of a rapid establishment of auditorimotor coactivation induced by the musical training [8], while engaging the participants in an individually tailored and rewarding program. Participant's remarkable adherence to the home practice sessions, which led to practice times beyond expectations, indicates high levels of motivation. This motivation, as well as a perception of being engaged in an enjoyable, socially valued leisure activity, are factors that may help patients pursuing musical lessons beyond the usual rehabilitation time frame. The small sample size in this pilot study limits the generalization of results. Present results, however, support the feasibility of MST in chronic stroke and provide useful information that can be used to generate further hypotheses and design larger intervention studies. Longer-term benefits of the training on upper extremity function and quality of life should also be investigated.

of results. Present results, however, support the feasibility of MST in chronic stroke and provide useful information that can be used to generate further hypotheses and design larger intervention studies. Longer-term benefits of the training on upper extremity function and quality of life should also be investigated. 5. Conclusion This study provides preliminary evidence indicating that a piano training program combined to home practice is feasible and can lead to meaningful improvements in manual dexterity, finger movement coordination, and functional use of upper extremity in chronic stroke survivors. For the first time, it was also demonstrated that MST training effects are maintained at a 3-week follow-up. This unique intervention, which targeted finger movement coordination, engaged the participants in an individually tailored and highly motivating program. It has the potential to be self-managed and pursued on the long term, outside the rehabilitation setting, and lead to further and sustainable improvements in upper extremity function. Acknowledgments The authors want to thank all the participants who took part in this study. This project was supported by the Foundation of the Jewish Rehabilitation Hospital.

5. Conclusion This study provides preliminary evidence indicating that a piano training program combined to home practice is feasible and can lead to meaningful improvements in manual dexterity, finger movement coordination, and functional use of upper extremity in chronic stroke survivors. For the first time, it was also demonstrated that MST training effects are maintained at a 3-week follow-up. This unique intervention, which targeted finger movement coordination, engaged the participants in an individually tailored and highly motivating program. It has the potential to be self-managed and pursued on the long term, outside the rehabilitation setting, and lead to further and sustainable improvements in upper extremity function. Acknowledgments The authors want to thank all the participants who took part in this study. This project was supported by the Foundation of the Jewish Rehabilitation Hospital. Figure 1 Scores of the three participants at different time points on the box and block test (a), nine hole peg test (b), and the Jebsen hand function test (c). For the box and block test, the scores represent the maximum number of blocks transported from one box to the other in 60 seconds. For the nine hole peg test, the time required to place and remove nine dowels into a nine holes is represented. For the Jebsen hand function test, the time required to complete the following subtests is shown: (1) simulating page turning; (2) lifting small common objects; (3) simulated feeding; (4) stacking checkers; (5) lifting large, light objects; (6) lifting large, heavy objects.

nine dowels into a nine holes is represented. For the Jebsen hand function test, the time required to complete the following subtests is shown: (1) simulating page turning; (2) lifting small common objects; (3) simulated feeding; (4) stacking checkers; (5) lifting large, light objects; (6) lifting large, heavy objects. Table 1 Initial participant characteristics. Participant 1 Participant 2 Participant 3 Age (years) 60 67 58 Gender (male/female) Male Male Male Time since stroke (months) 9 10 16 Side of stroke (left/right) Right Left Right Type of stroke (ischemic/hemorrhage) Ischemic Ischemic Hemorrhage CMSA arm/hand score (max = 7) 3/3 3/3 4/5 Piano experience (years) 0 0 0 Handedness Right Right* Left* *Affected hand is the dominant hand; CMSA: Chedoke-McMaster Stroke Assessment.

1. Introduction Hypoxic-ischemic brain damage caused by intrapartum disastrous events is still an important problem in modern obstetrics even in developed countries. It accounts for 10% to 20% of infants with cerebral palsy [1, 2]. Since 1997, we have been performing a regional population-based study on intrauterine fetal deaths, neonatal deaths, and severely handicapped infants [1]. From a total of 140,000 deliveries in the last 13 years, we found a perinatal mortality rate of 4 per 1,000. This is the lowest rate in the world (perinatal mortality includes stillbirths ≥22 weeks of gestation and neonatal deaths ≤7 days of life). However, even where the most advanced perinatal services are available, the incidence of brain damage is 2/1,000, similar to rates around the world [2]. Among infants with brain damage, the most frequent cause is congenital abnormality (1/3), and hypoxic-ischemic encephalopathy constitutes 15%. Thus, it is important for us to study (1) how to predict fetal hypoxic-ischemic events early enough to prevent brain damage, (2) how to treat severely damaged neonates immediately after birth to prevent brain damage, and (3) how to individualize fetuses at high-risk of brain damage?

Since 1997, we have been performing a regional population-based study on intrauterine fetal deaths, neonatal deaths, and severely handicapped infants [1]. From a total of 140,000 deliveries in the last 13 years, we found a perinatal mortality rate of 4 per 1,000. This is the lowest rate in the world (perinatal mortality includes stillbirths ≥22 weeks of gestation and neonatal deaths ≤7 days of life). However, even where the most advanced perinatal services are available, the incidence of brain damage is 2/1,000, similar to rates around the world [2]. Among infants with brain damage, the most frequent cause is congenital abnormality (1/3), and hypoxic-ischemic encephalopathy constitutes 15%. Thus, it is important for us to study (1) how to predict fetal hypoxic-ischemic events early enough to prevent brain damage, (2) how to treat severely damaged neonates immediately after birth to prevent brain damage, and (3) how to individualize fetuses at high-risk of brain damage? We have been performing clinical and basic animal studies to elucidate the pathogenesis of hypoxic-ischemic brain damage of neonates. In this context, we have also performed animal studies to seek neuroprotective therapies against hypoxia-ischemia. In this paper, we would like to show some of the results of our recent studies on neuroprotective therapies in animal experiments, as well as some literature reviews on neuroprotective therapies.

neonates. In this context, we have also performed animal studies to seek neuroprotective therapies against hypoxia-ischemia. In this paper, we would like to show some of the results of our recent studies on neuroprotective therapies in animal experiments, as well as some literature reviews on neuroprotective therapies. 2. The Levine-Rice Model We have been using the Levine-Rice model to study neonatal hypoxic-ischemic brain damage. This model has been widely used for 3 decades for histological analysis as well as behavioral tests. 2.1. Preparation of the Model The hypoxic-ischemic encephalopathy model in adult can be done in a variety of ways. One method involves introducing a ligation the unilateral carotid artery and exposing it to whole-body hypoxia [3]. This Levine preparation was modified for neonatal rats, for example, in order to examine birth asphyxia [4]. In employing the Levine-Rice model to study perinatal hypoxic-ischemic encephalopathy, we used 7-day-old Wistar rats because the developmental maturity of their brains roughly corresponds to that of near-term or term fetal brain in human beings [4].

or neonatal rats, for example, in order to examine birth asphyxia [4]. In employing the Levine-Rice model to study perinatal hypoxic-ischemic encephalopathy, we used 7-day-old Wistar rats because the developmental maturity of their brains roughly corresponds to that of near-term or term fetal brain in human beings [4]. The Levine-Rice model was made as follows [5] (Figure 1). The 7-day-old Wistar rat was lightly anesthetized by ether inhalation, and the left carotid artery was sectioned between a double ligature with 4–0 surgical silk. The rat was allowed to recover for 2 hours or more and then exposed to 8% hypoxia, by being placed in a hypoxic chamber at 32 degrees Celsius, which is the usual ambient temperature of the neonatal rat. After hypoxia, the rats were removed from the chamber and returned to their dams. 2.2. Histological Grading of Severity of Brain Damage In this model, the ligated side of the brain hemisphere is exposed to hypoxia and hypoperfusion (hypoxia-ischemia) and the nonligated side is exposed to hypoxia only. From a histological standpoint, the nonligated side has long been used as the control and the ligated side is used as the experimental side. Severity of brain damage was graded into 4 categories: normal (no damage), mild (<25%), moderate (25–50%), and severe (>50%) of the surface area on a single section with neuronal loss [5] (Figure 2).

standpoint, the nonligated side has long been used as the control and the ligated side is used as the experimental side. Severity of brain damage was graded into 4 categories: normal (no damage), mild (<25%), moderate (25–50%), and severe (>50%) of the surface area on a single section with neuronal loss [5] (Figure 2). 2.3. Blood Flow Distribution to the Brain Hemispheres As mentioned, this model causes hypoperfusion in the ligated side of the brain, while the nonligated side is exposed to hypoxia alone. Wistar rats have anatomical arterial connections between the right and left side of the brain hemispheres. With a radioactive tracer technique, regional cerebral blood flow was decreased in the ligated side following carotid artery ligation and hypoxia [6]. We used a colored microsphere technique and investigated cerebral blood flow distribution and the resulting grade of hypoxic-ischemic brain damage [7]. Colored microspheres 15 micrometers in diameter were administered directly into the left cardiac ventricle percutaneously at the end of hypoxia. The rats were killed 24 hours after insult, and brain damage was classified into mild and severe damage groups. In control animals, the blood flow was equally distributed in both hemispheres (Figure 3). Cerebral blood flow distribution of the ligated side decreased significantly, 45% in the mild damage group and 66% in the severe damage group. Thus, in the Levine-Rice model, the more severe the developing brain damage, the greater the percentage difference of blood flow distribution.

). Intracerebral injection of 2 or 4 micrograms of GDNF significantly decreased the incidence and severity of brain damage (controls 76–93% versus GDNF 34–64% in 2 micrograms and 7–29% in 4 micrograms, in incidence). This study suggests that GDNF may be protective against perinatal hypoxic-ischemic encephalopathy [19]. We studied the spatial and temporal patterns of GDNF after hypoxia-ischemia in neonatal rat brain and found that significant upregulation of the GDNF protein occurred in a bimodal fashion in the damaged brain hemisphere. The early rise is during the first 3 hours and is probably related to enhanced neuronal release. The second rise is during 72 hours to 1 week and is probably related to progressive astrogliosis after injury [20]. Following the above-mentioned studies, we administered GDNF for hypoxic-ischemic encephalopathy to prevent brain damage in neonatal rats. GDNF is a rather large protein that is impermeable to the blood-brain barrier. For this purpose, we used encapsulated GDNF-secreting cells by using baby hamster kidney cells transfected with human GDNF [21]. The capsule was implanted in the brain at 12 days of life, and hypoxia-ischemia was loaded 2 days after implantation. Compared with the control group, serum GDNF concentrations were significantly elevated and neuronal damage was significantly less in the experimental group [22].

ed in both hemispheres (Figure 3). Cerebral blood flow distribution of the ligated side decreased significantly, 45% in the mild damage group and 66% in the severe damage group. Thus, in the Levine-Rice model, the more severe the developing brain damage, the greater the percentage difference of blood flow distribution. 2.4. Behavioral Tests We used 3 different learning and memory tasks. Details of these have been described elsewhere [8]. 2.4.1. Choice Reaction Time Task (Figure 4(a)) The choice reaction time task represents the first step of cognition and memory and is related to attention and immediate memory retention ability. Rats were trained for 1 to 2 weeks before the test, in which the rats should press either of 2 levels by varying the correct lever; a cue lamp was randomly lighted above the correct lever. A pellet dispenser released a pellet only when the rat pressed the correct lever. The time between pellet presentation with the cue-lamp on and the correct lever being pressed was defined as choice-reaction time. Parameters were correct responses, incorrect lever pressings, and so forth. 2.4.2. Water Maze Task (Figure 4(b)) The water maze task tests permanent spatial learning ability and reference memory. The pool was divided into 4 quadrants where one quadrant had a hidden platform in the middle. The test rat was placed in 1 of 3 quadrants (excluding the platform containing one), facing the wall of the pool. Parameters were the time taken to reach the platform (or 120 seconds elapsed), total swimming distance, and swimming speed.

s divided into 4 quadrants where one quadrant had a hidden platform in the middle. The test rat was placed in 1 of 3 quadrants (excluding the platform containing one), facing the wall of the pool. Parameters were the time taken to reach the platform (or 120 seconds elapsed), total swimming distance, and swimming speed. 2.4.3. 8-Arm Radial Maze Task (Figure 4(c)) The 8-arm radial maze task is a test for spatial learning ability, which indicates long-term reference memory as well as short-term working memory. The test rat was placed in a circular plastic ring on the platform at the center of the 8-arm maze. After 1 minute, the ring was lifted, and the rat was allowed to move freely in the maze. The task continued until the rat entered all 8 arms to eat a pellet or until 10 minutes had elapsed. The test was performed every day for 21 days. Test performance was assessed by 3 parameters: correct choices in the initial 8 chosen arms, errors of entry into an already entered arm, and total time. 3. Neuroprotective Therapies Plenty of studies seeking effective neuroprotective therapies have investigated perinatal hypoxic-ischemic encephalopathy. In the last 2 decades, we have also contributed to this field. Some of the findings from our research terms are provided here.

The test rat was placed in a circular plastic ring on the platform at the center of the 8-arm maze. After 1 minute, the ring was lifted, and the rat was allowed to move freely in the maze. The task continued until the rat entered all 8 arms to eat a pellet or until 10 minutes had elapsed. The test was performed every day for 21 days. Test performance was assessed by 3 parameters: correct choices in the initial 8 chosen arms, errors of entry into an already entered arm, and total time. 3. Neuroprotective Therapies Plenty of studies seeking effective neuroprotective therapies have investigated perinatal hypoxic-ischemic encephalopathy. In the last 2 decades, we have also contributed to this field. Some of the findings from our research terms are provided here. 3.1. Hypothermia A protective effect of mild to moderate hypothermia against hypoxic-ischemic brain damage has been shown by a number of studies in adult and neonatal rats. A 3°C reduction in the systemic temperature during 3 h hypoxia provides partial benefit, whereas a 6°C reduction completely protects the brain [9]. A detrimental effect of mild hyperthermia on hypoxicischemic brain damage, in which a 2°C increase exacerbates postischemic brain damage and functional neurologic outcome, has been reported.

the systemic temperature during 3 h hypoxia provides partial benefit, whereas a 6°C reduction completely protects the brain [9]. A detrimental effect of mild hyperthermia on hypoxicischemic brain damage, in which a 2°C increase exacerbates postischemic brain damage and functional neurologic outcome, has been reported. We also performed the hypoxic-ischemic experiments on 7-day-old Wistar rats to see the histological and functional changes in brain development under three different temperature conditions: hypothermia (27°C), normothermia (33°C), and hyperthermia (37°C) [10]. Histologically, hyperthermia during hypoxia-ischemia significantly worsened brain damage, while hypothermia protected against brain damage, compared with normothermic conditions (Figure 5). We also evaluated the influence of temperature conditions on long-lasting neurologic deficits after hypoxia-ischemia in the same animal model. Hypothermia significantly decreased attention deficits in the choice reaction time task and spatial learning deficits in the water maze task. Hyperthermia, however, aggravated those behavioral and memory deficits. Thus, temperature regulation during hypoxia-ischemia is important, such that hypothermia reduces histological and behavioral deficits after hypoxia-ischemia, but hyperthermia worsens them.

We also performed the hypoxic-ischemic experiments on 7-day-old Wistar rats to see the histological and functional changes in brain development under three different temperature conditions: hypothermia (27°C), normothermia (33°C), and hyperthermia (37°C) [10]. Histologically, hyperthermia during hypoxia-ischemia significantly worsened brain damage, while hypothermia protected against brain damage, compared with normothermic conditions (Figure 5). We also evaluated the influence of temperature conditions on long-lasting neurologic deficits after hypoxia-ischemia in the same animal model. Hypothermia significantly decreased attention deficits in the choice reaction time task and spatial learning deficits in the water maze task. Hyperthermia, however, aggravated those behavioral and memory deficits. Thus, temperature regulation during hypoxia-ischemia is important, such that hypothermia reduces histological and behavioral deficits after hypoxia-ischemia, but hyperthermia worsens them. 3.2. Rehabilitative Training Rehabilitative manipulations have been demonstrated to improve learning and behavioral disability caused by hypoxia-ischemia in humans as well as in rats [11]. We also tested whether rehabilitative training improves spatial learning impairment in the water maze, after hypoxia-ischemia, in rats. We demonstrated a late-onset, slowly progressive brain damage 5 weeks after hypoxia ischemia [12]. We hypothesized that these progressive histological defects and their related functional impairments could be improved by rehabilitation, since rehabilitative training would increase neurotropic factors in some experimental models. We used 7-day-old Wistar rat models to make hypoxic-ischemic brain damage. Six weeks later, the rats were divided into training and no training groups. We used the water maze task to evaluate spatial learning ability in both groups and then euthanized the rats to evaluate histological changes. Interestingly, the training tasks did not change the hemispheric area of brain damage between the training and no training groups, but swimming distance and speed were significantly improved in the training group. These results suggested that rehabilitative training prevented long-lasting hypoxic-ischemic functional deficits such as learning and memory disability.

ge the hemispheric area of brain damage between the training and no training groups, but swimming distance and speed were significantly improved in the training group. These results suggested that rehabilitative training prevented long-lasting hypoxic-ischemic functional deficits such as learning and memory disability. 3.3. Edaravone Free radicals are reactive chemicals which are important mediators of cell death and tissue injury after hypoxia-ischemia. Hypoxia-ischemia causes free radical reactions, leading to tissue toxicity, including oxidation of lipid, protein, and polysaccharides. Newborns are at higher risk of oxidative stress and more susceptible to free radical oxidative damage than more mature infants. Thus, we investigated the effect of the free radical scavenger, edaravone, 3-methyl-1-phenyl-2-pyrazolin-5-one, on the development of hypoxic-ischemic brain damage in newborn rats [13]. A Levine-Rice model of 7-day-old rat was made and edaravone was given intraperitoneally. A control group was given saline. Edaravone significantly reduced the brain-damaged area in a dose-response fashion (3, 6, or 9 mg/kg) (Figure 6).

lin-5-one, on the development of hypoxic-ischemic brain damage in newborn rats [13]. A Levine-Rice model of 7-day-old rat was made and edaravone was given intraperitoneally. A control group was given saline. Edaravone significantly reduced the brain-damaged area in a dose-response fashion (3, 6, or 9 mg/kg) (Figure 6). Since edaravone has been approved in Japan for use in patients with cerebral infarction, this is a promising candidate for the treatment of neonatal hypoxic-ischemic encephalopathy. We then performed an experiment to find out whether long-term edaravone treatment is more effective than short-term treatment [14]. With the same Levine-Rice models, edaravone was given after hypoxic-ischemic insult every 24 hours for 2, 5, or 10 consecutive days, and behavioral and histological deficits were evaluated. The 2-day treatment improved learning and memory performance, as well as histological recovery, compared with controls. The 5-day treatment showed histological improvement but no behavioral improvement. However, the 10-day treatment resulted in no improvement in histological or behavioral changes, compared with the controls. These 3 different treatments of edaravone had different impacts on brain histology and behavioral parameters, suggesting that its use is most beneficial for the acute phase after hypoxia-ischemia.

However, the 10-day treatment resulted in no improvement in histological or behavioral changes, compared with the controls. These 3 different treatments of edaravone had different impacts on brain histology and behavioral parameters, suggesting that its use is most beneficial for the acute phase after hypoxia-ischemia. Possible mechanisms by which the free radical scavenger is protective against hypoxic-ischemic brain impairment have also been studied. Hypoxia-ischemia produces free radicals, which initiate lipid peroxidation and maintain generation in a chain reaction, ultimately damaging the cell membrane and causing cell death. So, we studied whether edaravone inhibits lipid peroxidation in hypoxic-ischemic newborn rats [15]. Edaravone significantly decreased lipid peroxidation (thiobarbituric acid reactive substance levels) of the damaged brain hemisphere, compared with saline controls. Furthermore, edaravone significantly decreased the level of nitric oxide metabolites in cerebrospinal fluid at 5 hours after hypoxia. Thus, edaravone improves hypoxic-ischemic brain damage in the developing rat, probably through mechanisms such as transient inhibition of lipid peroxidation and nitric oxide production.

Furthermore, edaravone significantly decreased the level of nitric oxide metabolites in cerebrospinal fluid at 5 hours after hypoxia. Thus, edaravone improves hypoxic-ischemic brain damage in the developing rat, probably through mechanisms such as transient inhibition of lipid peroxidation and nitric oxide production. Protective effects of edaravone against hypoxic-ischemic damage were investigated with the aid of an in vivo microdialysis technique. We placed a microdialysis probe into the hippocampus and induced hypoxic-ischemic stress in the Levine-Rice rat model. Edaravone or saline was perfused with a spin trap agent and then analyzed by electron paramagnetic resonance spectroscopy. We found that edaravone directly and dose-dependently inhibited lipid free radical formation during the hypoxic-ischemic insult in the neonatal rat brain [16]. Following these experiments with edaravone, we then looked at changes in gene expression caused by hypoxia-ischemia to elucidate molecular events occurring in the brain, as well as the impact of edaravone on gene expression. We performed comprehensive gene expression and gene network analyses using a DNA microarray system. After hypoxia-ischemia alone, there are many upregulated genes, relating to cell death signaling and immune responses, and many downregulated genes reflecting progressive damage, in the contralateral cerebral hemisphere. Comparing these changes, edaravone caused much less gene expression, probably reflecting the protective effect of edaravone against hypoxic-ischemic brain damage [17, 18].

to cell death signaling and immune responses, and many downregulated genes reflecting progressive damage, in the contralateral cerebral hemisphere. Comparing these changes, edaravone caused much less gene expression, probably reflecting the protective effect of edaravone against hypoxic-ischemic brain damage [17, 18]. 3.4. Neurotrophic Factors One of the new approaches toward the prevention and treatment of hypoxic-ischemic brain damage is neurotrophic factor, which includes nerve growth factor, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor (GDNF), basic fibroblast growth factor, a transforming growth factor group, and a neurotrophin group. GDNF is a potent neurotrophic peptide and is present in neuronal and nonneuronal cells throughout all regions in the developing brain, suggesting its protective role against hypoxic-ischemic damage. We first investigated the effects of GDNF in hypoxic-ischemic brain injury in developing rats (Levine-Rice model). Intracerebral injection of 2 or 4 micrograms of GDNF significantly decreased the incidence and severity of brain damage (controls 76–93% versus GDNF 34–64% in 2 micrograms and 7–29% in 4 micrograms, in incidence). This study suggests that GDNF may be protective against perinatal hypoxic-ischemic encephalopathy [19].

cells transfected with human GDNF [21]. The capsule was implanted in the brain at 12 days of life, and hypoxia-ischemia was loaded 2 days after implantation. Compared with the control group, serum GDNF concentrations were significantly elevated and neuronal damage was significantly less in the experimental group [22]. We also investigated the effects of GDNF on long-lasting learning and behavioral changes in the rat model. The encapsulated GDNF was implanted in the 7-day-old Wistar rats, and, 2 days after implantation, a hypoxic-ischemic insult was given. Then several learning tasks were examined, such as the 8-arm radial maze task, the choice-reaction time task, and the water maze task. Improved performance was observed in all three tasks for the GDNF group compared with the control group [23]. Thus, GDNF treatment is effective not only in reducing brain injury, but also in improving learning and memory performances after hypoxic-ischemic insults in the developing rats. GDNF is also effective in the reduction of the peripheral nerve injury. We produced the Erb's palsy model by transecting the anterior and posterior roots of the left C5–C7 nerves of 7-day-old Wistar rats [24]. The transected edges were kept in contact by each other and nestled by Gelform soaked with 10 microgram GDNF, or saline as control. The behavioral evaluation by foot-fault test was significantly improved by GDNF. As well, the number of anterior horn cells was preserved by GDNF but significantly reduced in saline controls.

transected edges were kept in contact by each other and nestled by Gelform soaked with 10 microgram GDNF, or saline as control. The behavioral evaluation by foot-fault test was significantly improved by GDNF. As well, the number of anterior horn cells was preserved by GDNF but significantly reduced in saline controls. 3.5. Dexamethasone Corticosteroid therapy has been widely used antenatally to prevent neonatal respiratory distress syndrome, intraventricular hemorrhage, and intestinal perforation, as well as chronic lung disease postnatally. Furthermore, antenatal corticosteroids reduced the risk of periventricular leukomalacia, the most popular cause of neurological complication of the premature infants [25]. Therefore, we performed several studies on the neuroprotective effects of corticosteroid. Dexamethasone (0.4 mg/kg, intraperitoneally) was injected 4 h before hypoxic-ischemic insult at the postnatal day 7 of the Wistar rat, and learning and memory impairment as well as histological deficits were studied. Dexamethasone treatment completely prevented histological brain damage and significantly improved behavioral and learning abilities (Figure 7). Dexamethasone without hypoxic-ischemic insult caused no adverse effects on learning and memory tests [26].

and memory impairment as well as histological deficits were studied. Dexamethasone treatment completely prevented histological brain damage and significantly improved behavioral and learning abilities (Figure 7). Dexamethasone without hypoxic-ischemic insult caused no adverse effects on learning and memory tests [26]. Similarly, dexamethasone also prevents behavioral and histological damage caused by a combination of lipopolysaccharide and hypoxia-ischemia in neonatal rats [27]. Lipopolysaccharide worsens the hypoxic-ischemic brain damage in a dose-response fashion and in a synergetic manner [28]. Thus, dexamethasone treatment can be a promising candidate for the prevention of inflammation and hypoxia-associated brain damage in clinical settings. 3.6. Magnesium Magnesium is a nonspecific competitive blocker of calcium channel and plays many important roles in maintaining homeostasis of the body. One of its roles is a gating function against calcium influx through the NMDA (N-methyl-D-asparate) receptor-associated ion channels in the brain. Hypoxia-ischemia causes intracellular energy failure that initiates a series of additional mechanisms, such as membrane depolarization, accumulation of excitatory amino acids, and accumulation of cytosolic calcium, which lead to a variety of cascading deleterious effects. We hypothesized that magnesium ion possibly blocks calcium ion influx through the calcium channels and prevents hypoxic-ischemic brain damage.

anisms, such as membrane depolarization, accumulation of excitatory amino acids, and accumulation of cytosolic calcium, which lead to a variety of cascading deleterious effects. We hypothesized that magnesium ion possibly blocks calcium ion influx through the calcium channels and prevents hypoxic-ischemic brain damage. Using the Levine-Rice neonatal rat model, we first found that prehypoxic treatment of magnesium sulfate ameliorates the severity of brain damage, but posthypoxic treatment deteriorates it. This deleterious effect may be attributable to hypotension caused by high-dose magnesium sulfate, which further worsens cerebral perfusion [29]. From a clinical standpoint, prehypoxic treatment is not practical. So, we studied possible rescue treatment modalities of magnesium sulfate to decrease brain deficits after hypoxia-ischemia. In adult animals, brain magnesium ion concentrations are significantly decreased for several hours or days after ischemia or trauma, and restoration of magnesium ion concentration of the brain improved brain damage. Therefore, we evaluated the effects of long-term (3 days), low-dose magnesium administration on hypoxic-ischemic brain injury in neonatal rats [30, 31]. The serum concentrations of magnesium ion were significantly decreased by hypoxia-ischemia for 3 days in controls. Compared with the controls, magnesium infusion with an osmotic pump restored its concentrations. Brain damage was significantly improved by long-term magnesium administration in a dose-dependent manner, compared with the controls (Figure 8).

m ion were significantly decreased by hypoxia-ischemia for 3 days in controls. Compared with the controls, magnesium infusion with an osmotic pump restored its concentrations. Brain damage was significantly improved by long-term magnesium administration in a dose-dependent manner, compared with the controls (Figure 8). Magnesium may indirectly affect brain damage by, for example, increasing blood flow distribution to the brain. To elucidate this possibility, we used a chronically instrumented fetal goat model (Figure 9) and a colored microsphere technique [32]. Magnesium sulfate was directly infused to the fetal cervical vein in a bolus dose of 270 mg/kg followed by 80 mg/kg/h, which is equivalent to the clinical dosage. Hypoxia was induced by adding nitrogen gas to the maternal inhaling air. Fetal PO2 significantly decreased from 30 mmHg to 14 mmHg. Hypoxia significantly increased cerebral blood flow, and hypoxia combined with magnesium administration further increased cerebral blood flow (P < 0.05) in the cerebral cortex (Figure 10). 3.7. Vagal Stimulation In fetal life, parasympathetic responses are relatively more dominant than sympathetic ones in resting and in hypoxemic conditions, implying their beneficial effects on the fetus. In adults, neuroprotective effects of parasympathetic activation on brain damage have been reported, including inhibition of glutamate release, activation of cholinergic anti-inflammatory pathways to inhibit cytokine release, increase in cerebral blood flow via nitric oxide induction, and enhancement of neurogenesis [33].

adults, neuroprotective effects of parasympathetic activation on brain damage have been reported, including inhibition of glutamate release, activation of cholinergic anti-inflammatory pathways to inhibit cytokine release, increase in cerebral blood flow via nitric oxide induction, and enhancement of neurogenesis [33]. We hypothesized that acetylcholine receptor agonists reduce hypoxic-ischemic brain damage in the Levine-Rice model. We injected subcutaneously 0.1 mg/kg of parasympathetic agonist, carbachol (carbamylcholine chloride), or saline as control, just before 2-hour 8% hypoxia-ischemia. The severity of the brain damage was compared between the carbachol group and the saline control. In the cerebral cortex, 25% of the carbachol group showed mild neural damage, and the remaining 75% showed no damage (Figure 11). In contrast, more than 80% of the saline group had severe damage (P < 0.05). Thus, vagal stimulation through acetylcholine receptor agonist has a beneficial effect against perinatal hypoxic-ischemic brain damage [34]. This neuroprotective effect is likely related to the effect on microglial activation during hypoxia-ischemia [35]. We also confirmed that, contrary to the neuroprotective effects of acetylcholine receptor agonists, its antagonists worsen hypoxia-ischemia brain damage in neonatal rats [35]. These observations imply that vagal stimulation during hypoxic-ischemic insult is a promising treatment of choice against hypoxic-ischemic neonatal encephalopathy.

hat, contrary to the neuroprotective effects of acetylcholine receptor agonists, its antagonists worsen hypoxia-ischemia brain damage in neonatal rats [35]. These observations imply that vagal stimulation during hypoxic-ischemic insult is a promising treatment of choice against hypoxic-ischemic neonatal encephalopathy. 3.8. Osteopontin Osteopontin is a glycosylated phosphoprotein and is involved in multiple biological functions such as antiapoptotic processes. Its neuroprotective effect is investigated in the neonatal rat brain after hypoxia-ischemia [36]. First, endogenous expression of osteopontin in the rat brain was significantly decreased during development after birth. Second, osteopontin expression in the brain was significantly increased after hypoxic-ischemia with a peak at 48 hours. Third, osteopontin treatment (both 0.03 and 0.1 microgram) significantly reduced infarct volume compared with the vehicle control. And finally, osteopontin treatment significantly improved some behavioral tests for memory and learning functions. Osteopontin is thought to function through interactions with proteins preferable to apoptosis. 3.9. Isoflurane Some minor insults before the major injurious events may act as preconditioning or tolerance so as to reduce the brain damage. For example, mild degrees of hypoxia, heat stress, and inflammation by lipopolysaccharide are well known for preconditioning activities [37].

3.8. Osteopontin Osteopontin is a glycosylated phosphoprotein and is involved in multiple biological functions such as antiapoptotic processes. Its neuroprotective effect is investigated in the neonatal rat brain after hypoxia-ischemia [36]. First, endogenous expression of osteopontin in the rat brain was significantly decreased during development after birth. Second, osteopontin expression in the brain was significantly increased after hypoxic-ischemia with a peak at 48 hours. Third, osteopontin treatment (both 0.03 and 0.1 microgram) significantly reduced infarct volume compared with the vehicle control. And finally, osteopontin treatment significantly improved some behavioral tests for memory and learning functions. Osteopontin is thought to function through interactions with proteins preferable to apoptosis. 3.9. Isoflurane Some minor insults before the major injurious events may act as preconditioning or tolerance so as to reduce the brain damage. For example, mild degrees of hypoxia, heat stress, and inflammation by lipopolysaccharide are well known for preconditioning activities [37]. Anesthetics may also play a unique role as preconditioning. One of them is isoflurane, which improved neuronal injury induced by oxygen-glucose deprivation in vitro [38] and hypoxic-ischemic brain injury in the 7-day-old Levine-Rice model [39]. On the other hand, other reported in the same animal model that isoflurane exerted only a short-term, but not a long-term neuroprotective effect [40]. The differences between these studies may be attributed to varying levels of preconditioning such as duration of isoflurane exposure and recovery time from the prior minor insult to the hypoxia-ischemia.

in the same animal model that isoflurane exerted only a short-term, but not a long-term neuroprotective effect [40]. The differences between these studies may be attributed to varying levels of preconditioning such as duration of isoflurane exposure and recovery time from the prior minor insult to the hypoxia-ischemia. 3.10. Granulocyte-Colony Stimulating Factor (G-CSF) and Erythropoietin (EPO) Similar to the neurotrophic factors, it has long been studied whether blood cell growth factors such as G-CSF and EPO act as neuroprotective in animals as well as in humans. G-CSF mainly stimulates the development of progenitor cells to neutrophils, but it also has trophic effects on the different cells including neuronal cells. G-CSF also has an anti-inflammatory effect on central nervous system, an antiapoptotic effect on neurons, and a stimulatory effect on neurogenesis [41]. Although the antioxidants data suggest that G-CSF plays a role as a neuroprotectant [41]. In the developing brain, G-CSF also improves hypoxic-ischemic brain damage in the Levine-Rice model. When injected 1 hour before the insult and once per day for 5 days or 10 days thereafter, G-CSF prevented brain atrophy and heart underdevelopment, improved motor and behavioral functions, and improved tests for short-term memory [42].

g brain, G-CSF also improves hypoxic-ischemic brain damage in the Levine-Rice model. When injected 1 hour before the insult and once per day for 5 days or 10 days thereafter, G-CSF prevented brain atrophy and heart underdevelopment, improved motor and behavioral functions, and improved tests for short-term memory [42]. EPO is an endogenous cytokine that enhances red blood cell production to increase oxygen delivery as a hypoxic physiological response and promotes cell survival via mechanisms of antiapoptotic functions [43]. EPO is neuroprotective in neonatal rat models [44, 45] as well as in clinical settings [46]. 3.11. Antioxidants Antioxidants such as dipyridamole, apotransferrin, vitamin E, and N-acetylcysteine are known to have some neuroprotective potentials against oxidative stress including reactive oxygen species and reactive nitrogen species, which are increased during hypoxia and postischemic reperfusion stages. In the developing brain of neonatal rat model, administration of these antioxidants attenuates white matter damage and induces remyelination processes [47, 48]. 3.12. Stem Cells Cell therapy containing stem cells has been found to protect neurons from hypoxic-ischemic damage and some degenerative disorders. One of the targets is hypoxic-ischemic brain damage that occurs during labor and delivery and neonatal period.

3.11. Antioxidants Antioxidants such as dipyridamole, apotransferrin, vitamin E, and N-acetylcysteine are known to have some neuroprotective potentials against oxidative stress including reactive oxygen species and reactive nitrogen species, which are increased during hypoxia and postischemic reperfusion stages. In the developing brain of neonatal rat model, administration of these antioxidants attenuates white matter damage and induces remyelination processes [47, 48]. 3.12. Stem Cells Cell therapy containing stem cells has been found to protect neurons from hypoxic-ischemic damage and some degenerative disorders. One of the targets is hypoxic-ischemic brain damage that occurs during labor and delivery and neonatal period. Mesenchymal stromal cells, bone marrow mesenchymal stem cells, and umbilical cord stem cells have been used in the treatment of neonatal hypoxic-ischemic encephalopathy and show reduction in sensorimotor and cognitive impairments [49–51]. Review articles show that these stem cell therapies are one of the promising options for the treatment of neonatal neurological diseases in the future. 3.13. Modulators of K(+)-ATP Channels K(+)-ATP channels exist on the cell surface and on the inner membrane of the mitochondria [52]. Some controversies exist concerning their role in neuroprotection.

Mesenchymal stromal cells, bone marrow mesenchymal stem cells, and umbilical cord stem cells have been used in the treatment of neonatal hypoxic-ischemic encephalopathy and show reduction in sensorimotor and cognitive impairments [49–51]. Review articles show that these stem cell therapies are one of the promising options for the treatment of neonatal neurological diseases in the future. 3.13. Modulators of K(+)-ATP Channels K(+)-ATP channels exist on the cell surface and on the inner membrane of the mitochondria [52]. Some controversies exist concerning their role in neuroprotection. Activation of certain ion channels that are able to attenuate neuronal depolarization may produce neuronal protective effects. One of the candidates is K(+)-ATP channels. Hypoxia reduces intracellular ATP levels that activate K(+)-ATP channels to prevent membrane potential depolarization, leading to neuroprotection [51]. Neuronal hyperpolarization inactivates calcium channels and in turn inhibits calcium-dependent glutamate release, thereby protecting against excitatory neurotoxicity [52]. On the other hand, recent studies also proposed that inhibition of these channels might have protective effects on neuronal survival. For example, in in vitro hippocampal slice preparation, K(+)-ATP channel blockers such as tolbutamide and glibenclamide produce neuroprotective effects [53].

Activation of certain ion channels that are able to attenuate neuronal depolarization may produce neuronal protective effects. One of the candidates is K(+)-ATP channels. Hypoxia reduces intracellular ATP levels that activate K(+)-ATP channels to prevent membrane potential depolarization, leading to neuroprotection [51]. Neuronal hyperpolarization inactivates calcium channels and in turn inhibits calcium-dependent glutamate release, thereby protecting against excitatory neurotoxicity [52]. On the other hand, recent studies also proposed that inhibition of these channels might have protective effects on neuronal survival. For example, in in vitro hippocampal slice preparation, K(+)-ATP channel blockers such as tolbutamide and glibenclamide produce neuroprotective effects [53]. 4. Summary From the basic animal studies on perinatal hypoxic-ischemic brain damage, we have now obtained some possible candidates for the therapeutic measures against it. They are hypothermia, rehabilitation, free radical scavenger, trophic factors, steroid, calcium channel blocker, vagal stimulation, some antiapoptotic agents, before and after conditioning, antioxidants, cell therapy with stem cells, and modulators of K(+)-ATP channels. Some of them have already been introduced to clinical practice, for example, hypothermia, magnesium, G-CSF, and EPO. Whether combination of these therapies may be more beneficial than any single therapy needs to be clarified.

conditioning, antioxidants, cell therapy with stem cells, and modulators of K(+)-ATP channels. Some of them have already been introduced to clinical practice, for example, hypothermia, magnesium, G-CSF, and EPO. Whether combination of these therapies may be more beneficial than any single therapy needs to be clarified. Hypoxia-ischemia is a complicated condition, in which the cause, severity, magnitude, and deteriorating speed are different in each case. Likewise, each fetus has its own inherent potentials against an hypoxic-ischemic insult, for example, adaptation, preconditioning-tolerance, and intolerance. Therefore, our final goal is an individualized strategy for neuroprotection against perinatal hypoxic-ischemic insult. Further extensive studies are required. Acknowledgments This study was partly supported by a Grant (79-258, 2009–2013) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and a Grant from the Ogyaa Donation, Japan, and a Grant (no. 24592476) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The authors are grateful to all our colleagues for actively contributing to basic and clinical researches and to Mr. Rick White for English editing of this manuscript. Figure 1 The Levine-Rice model of 7-day-old Wistar rat (from left to right). Under ether anesthesia, skin was incised, and unilateral common carotid artery was doubly ligated. After recovery, they were transferred to a chamber containing humidified 8% hypoxic gas. Brain was removed for histological study. The ligated side of the hemisphere was atrophic.

-day-old Wistar rat (from left to right). Under ether anesthesia, skin was incised, and unilateral common carotid artery was doubly ligated. After recovery, they were transferred to a chamber containing humidified 8% hypoxic gas. Brain was removed for histological study. The ligated side of the hemisphere was atrophic. Figure 2 Severity of brain damage was classified into normal, mild, moderate, or severe according to the damaged area. Figure 3 Percent difference of cerebral blood flow distribution between the ligated and nonligated side was expressed. The more severely damaged, the less blood flow distributed to the damaged brain hemisphere. Bars represent mean ± SD. Figure 4 Behavioral tests: (a) choice reaction time task, (b) water maze task, and (c) 8-arm radial maze task. Figure 5 Effect of hypothermia and hyperthermia on histological changes in brain. White bars represent the nonligated side (NL) and black bars represent the ligated side (L) of the brain. Brain hemisphere area was significantly decreased by hyperthermia, whereas it was preserved by hypothermia. Figure 6 Effect of edaravone on the infracted area. White bars represent controls and black bars represent the edaravone group, where there is a dose-response relationship. Figure 7 Effect of dexamethasone on brain hemisphere area. White bars represent the nonligated side (NL) and black bars represent the ligated side (L) of the brain. Dexamethasone reversed hypoxic-ischemic brain damage, while dexamethasone under normoxemic condition had no deleterious effects.

Figure 6 Effect of edaravone on the infracted area. White bars represent controls and black bars represent the edaravone group, where there is a dose-response relationship. Figure 7 Effect of dexamethasone on brain hemisphere area. White bars represent the nonligated side (NL) and black bars represent the ligated side (L) of the brain. Dexamethasone reversed hypoxic-ischemic brain damage, while dexamethasone under normoxemic condition had no deleterious effects. Figure 8 Effect of magnesium sulfate on mortality and brain cyst formation. A positive dose-response relationship existed between magnesium dosage and brain protection. Figure 9 The chronic preparation model using goat fetuses at 0.9 gestation. Under general anesthesia, (a) fetal head and neck was exteriorized, (b) catheters and electrodes were placed, (c) fetus was returned to the uterine cavity and recovered from surgical stresses for 4 days, and (d) hypoxic experiments were performed by adding nitrogen gas through the maternal endotracheal catheter. Figure 10 Blood flow changes by hypoxemia alone and hypoxemia under magnesium administration. Hypoxemia significantly increased the cerebral blood flow, and hypoxemia with magnesium further increased cerebral blood flow. Mean ± SE. Figure 11 Effect of a parasympathetic agonist, carbachol, on the hypoxic-ischemic brain damage in the neonatal rat. HI is hypoxia-ischemia. Carbachol significantly decreased neuronal damage in the cortex.

1. Introduction Of all stroke survivors, more than half experience impairments of the upper limb in the chronic phase, including loss of strength and dexterity, spasticity, muscle contracture, pain, and edema [1–3]. Patients with a more severe paresis have a higher risk of developing spasticity [4] and muscle contractures of the wrist and finger flexor muscles [5–7]. Without appropriate spasticity treatment or contracture prevention, patients are at risk of developing a clenched fist, a hand which is deformed into a fist by shortening of flexor muscles of the fingers and soft tissue [8]. The abnormal position of the hemiplegic hand and wrist due to spasticity and muscle contractures may interfere with daily activities and hygiene maintenance, both negatively influencing the quality of life [9–11].

, a hand which is deformed into a fist by shortening of flexor muscles of the fingers and soft tissue [8]. The abnormal position of the hemiplegic hand and wrist due to spasticity and muscle contractures may interfere with daily activities and hygiene maintenance, both negatively influencing the quality of life [9–11]. Different approaches are used to inhibit spasticity, prevent contractures, reduce pain and edema, or improve hygiene maintenance of the hand in stroke patients with a nonfunctional spastic upper limb. However, there is no consensus about the most effective treatment [12]. A commonly used and widely accepted intervention is prolonged splinting using static orthoses [12–17]. Two reviews on the effect of upper limb splinting after stroke have been published [18, 19]. Both reviews showed no effect of static orthoses on upper limb function, range of motion, and pain after an intervention period less than 13 weeks. However, conclusions should be interpreted with caution because of the lack of high quality randomised controlled trials. There is a considerable heterogeneity of included study designs, clinical aims, and orthosis wearing protocols, materials, and regimes. In addition, all published studies focused on the short-term effect of splinting with splinting periods no longer than 13 weeks. Despite controversies concerning splinting of the hemiplegic upper limb, static orthoses continue to be advised in clinical practice.

, and orthosis wearing protocols, materials, and regimes. In addition, all published studies focused on the short-term effect of splinting with splinting periods no longer than 13 weeks. Despite controversies concerning splinting of the hemiplegic upper limb, static orthoses continue to be advised in clinical practice. When used in clinical practice, a considerable amount of stroke patients complain about increased pain and spasticity since the use of the static orthosis [20, 21]. Due to discomfort, the orthosis cannot be worn for the advised 8 hours per day which leads to nonuse in chronic stroke patients and with that increases the risk of developing clenched fists with which patients may experience problems with daily activities and hygiene maintenance. Given our experiences in clinical practice, the purpose of this pilot study is to describe the long-term use of static hand-wrist orthoses and the experienced comfort of wearing the orthosis in chronic stroke patients in order to acquire preliminary data to further study the treatment of this specific patient population. We hypothesize that, in a number of the chronic stroke patients with upper limb impairments, discomfort—increased pain, and spasticity—is the reason for not wearing a static hand-wrist orthosis for the advised 8 hours per day. The secondary aim is to describe the self-reported complaints before and since the use of the static orthosis to evaluate the effect of the use of the orthosis in chronic stroke patients. Additionally, the use of cointerventions for the impaired upper limb is investigated.

rist orthosis for the advised 8 hours per day. The secondary aim is to describe the self-reported complaints before and since the use of the static orthosis to evaluate the effect of the use of the orthosis in chronic stroke patients. Additionally, the use of cointerventions for the impaired upper limb is investigated. 2. Methods In this pilot study, semistructured interviews were used to explore the long-term use (i.e., more than one year) of the static orthosis in chronic stroke patients, and the experienced comfort with the static orthosis in chronic stroke patients (Figure 1). A selection of stroke patients, who received a static orthosis from the Orthopaedic Centre OIM Brabant Breda, The Netherlands, was taken from the database. All stroke patients who were advised to use a static orthosis at least one year ago and were independently living in the community were included. Patients were excluded when correct contact details were missing or when patients died in the study period. If patients were unable to communicate by telephone, information was obtained from the primary caregiver. Informed consent was obtained prior to each interview.

ndependently living in the community were included. Patients were excluded when correct contact details were missing or when patients died in the study period. If patients were unable to communicate by telephone, information was obtained from the primary caregiver. Informed consent was obtained prior to each interview. Patients were asked about current use, comfort of the orthosis, reasons for wearing the orthosis, self-reported complaints in the hemiplegic upper limb, including spasticity, hygiene maintenance, pain, and edema, and applied cointerventions. Answers to all twelve questions were scored categorically except the complaints scores. Complaints scores were graded from 0 (no complaints) to 10 (major complaints). The telephone interviews were carried out by a physical therapist who was not directly involved in the patients' treatment. Descriptive statistics were used to analyze the results of the semistructured telephone interviews. Nonparametric analyses were applied to evaluate self-reported effect of the orthosis by comparing the data of complaints before and since the use of the static orthosis using a Wilcoxon's signed rank test. Statistical analysis was performed using SPSS 18.0. Statistical significance was set at the 5% level.

interviews. Nonparametric analyses were applied to evaluate self-reported effect of the orthosis by comparing the data of complaints before and since the use of the static orthosis using a Wilcoxon's signed rank test. Statistical analysis was performed using SPSS 18.0. Statistical significance was set at the 5% level. 3. Results 3.1. Study Population A total of 38 patients, diagnosed with stroke, received a static hand-wrist orthosis at the Orthopaedic Centre OIM Brabant Breda, The Netherlands, between January 1, 2008 to October 1, 2009. Participants were retrospectively recruited from the database at October 1, 2010. Nineteen stroke patients matched our inclusion criteria and were invited for the study. Eight patients could not be interviewed, since three died between receiving the static orthosis and data collection, and five could not be reached by telephone. Data of eleven patients (7 female, 4 male, median age 54 years, range 23–80 years) was collected. One interview was conducted with a caregiver. All patients were in chronic stage after stroke (median 86 months poststroke, range 27–163 months) and were advised to use the static orthosis for at least one year ago.

phone. Data of eleven patients (7 female, 4 male, median age 54 years, range 23–80 years) was collected. One interview was conducted with a caregiver. All patients were in chronic stage after stroke (median 86 months poststroke, range 27–163 months) and were advised to use the static orthosis for at least one year ago. 3.2. Long-Term Use of the Orthosis As shown in Table 1, after at least one year from receiving the static orthosis, three patients still wear the orthosis during night time, with different experienced comfort. Four patients wore the orthosis for at least 8 hours per day, all with good experienced comfort. Two patients were unable to wear the orthosis prescribed 8 hours per day, due to poor comfort. Two patients stopped using the orthosis, one because of an increase of spasticity and the other because of an increase in pain. 3.3. Self-Reported Complaints in the Hemiplegic Upper Limb The main reasons reported for wearing the orthosis were reducing spasticity (10/11), improving opening of the hand (7/11), and improving hygiene maintenance of the hand (5/11) (see Table 2). None of the patients wore the orthosis to reduce edema. The complaints score since the use of the orthosis showed a decreasing trend; however differences between complaints in the hemiplegic upper limb before and since the use of the orthosis were not significant (P < 0.05).

aintenance of the hand (5/11) (see Table 2). None of the patients wore the orthosis to reduce edema. The complaints score since the use of the orthosis showed a decreasing trend; however differences between complaints in the hemiplegic upper limb before and since the use of the orthosis were not significant (P < 0.05). 3.4. Cointerventions Ten patients reported cointerventions for their upper limb impairments of whom eight were still using the static orthosis (Table 1). Six patients received regular physical therapy sessions, six patients performed daily home exercises, and two patients used spasticity medication (Botulinum toxin or Baclofen). Only one patient did not use any other form of intervention for the impaired upper limb.

eight were still using the static orthosis (Table 1). Six patients received regular physical therapy sessions, six patients performed daily home exercises, and two patients used spasticity medication (Botulinum toxin or Baclofen). Only one patient did not use any other form of intervention for the impaired upper limb. 4. Discussion In this pilot study, we investigated the long-term use of a static hand-wrist orthosis in chronic stroke patients. Of the 11 interviewed stroke patients, two stopped wearing the orthosis because of discomfort and two could not endure the orthosis for the prescribed wearing time of at least 8 hours per day. These findings support our hypothesis that a substantial number of patients who are at risk of developing contractures in the upper limb, that is, four of the eleven, are not able to endure a static orthosis for the prescribed 8 hours because of discomfort. Concluding that these chronic stroke patients do not receive the appropriate intervention to prevent contractures. Of the seven patients who used the orthosis as prescribed, that is, at least 8 hours, two still complained of discomfort. Without appropriate contracture prevention, these patients will develop contractures in the upper limb which can lead to problems during daily activities and hygiene maintenance, both negatively influencing the quality of life.

d the orthosis as prescribed, that is, at least 8 hours, two still complained of discomfort. Without appropriate contracture prevention, these patients will develop contractures in the upper limb which can lead to problems during daily activities and hygiene maintenance, both negatively influencing the quality of life. The experienced discomfort can be a result of the static characteristics of the orthosis. The position of the orthosis sets the wrist in a fixed position. However, the level of spasticity varies during daytime resulting in different positions of the wrist. With a higher level of spasticity of the muscles, the wrist tends to flex. In contrast, a lower muscle tone can lead to less flexion, or even extension, of the wrist. The chosen position of the static orthosis is seldom adequate to manage these varying levels of spasticity and changing ranges of wrist mobility. When spasticity increases, the hand and fingers will try to flex in the rigid orthosis which causes pain and discomfort. For stroke patients with varying levels of spasticity in the upper limb, a static orthosis with a fixed position of the wrist can lead to problems tolerating the orthosis. Taking this into account, an orthosis for the prevention of contractures in the spastic upper limb needs to allow higher levels of spasticity and flexion of the wrist. A dynamic orthosis using the low-load and prolonged stretch principle, with a hinge which allows the wrist to flex during higher levels of spasticity, might be more appropriate for these stroke patients.

ntion of contractures in the spastic upper limb needs to allow higher levels of spasticity and flexion of the wrist. A dynamic orthosis using the low-load and prolonged stretch principle, with a hinge which allows the wrist to flex during higher levels of spasticity, might be more appropriate for these stroke patients. In our study, most patients use the orthosis because of spasticity in the upper limb, to prevent contractures or to preserve the ability to open the hand for hygiene maintenance. Patients in our study did not report a significant difference of the complaints concerning spasticity, contracture, or pain before and since the use of the static orthosis, although the complaints tended to decrease in this small sample size group. Previous studies on the short-term effect of the static orthosis indicate that stretch does not have clinically important immediate or short-term effect on joint mobility [22]. There is evidence indicating that static orthoses show no effect on upper limb function, range of motion at the wrist, fingers, or thumb, nor pain [19, 23]. Despite the lack of studies of long-term effect, physicians and patients still believe that splinting is an appropriate intervention for contracture prevention. Because contractures develop slowly, studies about the effect of splinting need to focus on long-term use of at least six months. All previous studies focused on an increased joint mobility of the wrist as an effect of the static orthosis. In our opinion maintaining the range of motion of the wrist is already a positive effect of an intervention aiming to prevent contractures.

ct of splinting need to focus on long-term use of at least six months. All previous studies focused on an increased joint mobility of the wrist as an effect of the static orthosis. In our opinion maintaining the range of motion of the wrist is already a positive effect of an intervention aiming to prevent contractures. In conclusion, a static orthosis can be a useful prevention of contractures for a selection of the stroke patients who can tolerate this low-cost orthosis. However, there is a group of chronic stroke patients which is not able to endure a static orthosis and which needs another intervention for the prevention of contractures in the upper limb. In this group, stepped care can be used; when static orthoses are not endured, another intervention has to be applied, for example, dynamic orthosis. 4.1. Limitations of the Study Although explorative, this study offers insight into the long-term use of a static orthosis in chronic stroke patients and the patient's experiences with it. Despite the preliminary character of this study, the presented data are the first about long-term use of the static orthosis and experienced comfort. Patients had to recall scores of complaints before the use of the static orthosis which could have been influenced by recall bias. Taking this into account, in combination with the small sample size, the results about the self-reported complaints of static splinting should be handled with care and should be confirmed in larger studies.

ecall scores of complaints before the use of the static orthosis which could have been influenced by recall bias. Taking this into account, in combination with the small sample size, the results about the self-reported complaints of static splinting should be handled with care and should be confirmed in larger studies. 4.2. Further Research Further studies are important to identify the stroke patients who are able to tolerate the static orthosis and patients who will need other interventions to prevent the development of contractures. For these specific stroke patients who are not able to tolerate the commonly used static orthosis, it will be relevant to study the effect of alternative interventions, for example, dynamic orthoses. 5. Conclusion These pilot data show that a number of chronic stroke patients cannot tolerate a static orthosis for at least 8 hours per day during a long-term period of at least one year. Without appropriate treatment opportunities, these patients will remain at risk of developing a clenched fist and will experience problems with daily activities and hygiene maintenance. It is, therefore, worthwhile to find other interventions which can be endured by these stroke patients. Conflict of Interests The authors declare that they have no conflict of interests. Acknowledgments The authors offer a special thanks to Daisy van Grinsven and Ineke Koolhaas for their valuable participation in this study. The pilot study was financially supported by the “Revant Innovatiefonds.” Figure 1 Example of a prefabricated static hand-wrist orthosis.

Conflict of Interests The authors declare that they have no conflict of interests. Acknowledgments The authors offer a special thanks to Daisy van Grinsven and Ineke Koolhaas for their valuable participation in this study. The pilot study was financially supported by the “Revant Innovatiefonds.” Figure 1 Example of a prefabricated static hand-wrist orthosis. Table 1 Orthosis wearing time, experienced comfort, and the reported cointerventions in addition to the use of the orthosis. Patient Wearing time per 24 hours Day/night use Reported comfort Cointervention 1 4–6 hours Day Poor Handmaster 2 >8 hours Night Poor None 3 0 hours (nonuse)   Very poor Medication, home exercises 4 >8 hours Night Poor Physical therapy 5 6–8 hours Day Very poor Physical therapy, home exercises 6 >8 hours Day Good Physical therapy, home exercises 7 >8 hours Day Good Home exercises 8 0 hours (nonuse)   Poor Home exercises 9 >8 hours Day Good Physical therapy 10 >8 hours Day Good Medication, physical therapy, home exercises 11 >8 hours Night Good Physical therapy Table 2 Self-reported complaints in the hemiplegic upper limb. Reasons for wearing a static orthosis Complaints before orthosis use Complaints since use orthosis Median (range) (0–10) Median (range) (0–10) Spasticity 10/11 8 (5–10) 7.5 (5–10) Hygiene maintenance 5/11 7 (5–10) 6 (1–7) Pain 3/11 8 (7–10) 7 (0–9) Edema 0/11 — — Opening hand 7/11 8.5 (7–10) 7.5 (5–10)

1. Introduction In Western countries, approximately 5.5% of all strokes occur in patients who are younger than 45 years of age [1], and the occurrence of ischemic stroke in young adults is 10.8 per 100,000 per year [2]. Despite the lower mortality rate of cerebral ischemia in the young, there is a high social impact of this disease [3, 4]. Recent years have seen remarkable progress in basic and clinical stroke research, and these advances have resulted in improved clinical care and diagnostic workups, as well as a better understanding of stroke pathogenesis [5]. The most important developments in this respect are represented by the increased attention that is placed on detecting cardiac and coagulation defects, screening for novel risk factors [6], improving advanced noninvasive brain imaging strategies, and using intravenous thrombolysis during acute ischemic stroke [5]. Although there are many published reports concerning stroke in young adults [2–4, 7], only a small number of these studies considers the evolution of the clinical management of young patients with ischemic stroke [2, 8]. The present work is a multicentric retrospective study that included all consecutive patients aged 16 to 44 years with ischemic stroke who were admitted to 10 Northern Italian hospitals. The objectives of the study were (i) to analyze the clinical features of this population and (ii) to examine the changes in patient clinical care over the study period, including changes in the diagnostic process and patient treatment that occurred over the study period.

were admitted to 10 Northern Italian hospitals. The objectives of the study were (i) to analyze the clinical features of this population and (ii) to examine the changes in patient clinical care over the study period, including changes in the diagnostic process and patient treatment that occurred over the study period. 2. Patients and Methods All patients aged between 16 and 44 years with acute ischemic stroke who were admitted to 10 hospitals in the Lombardy region, the most populated of Italy, between January 2000 and December 2005 were included in this study. The clinical centers were categorized as follows: seven were equipped with a stroke unit [10], and the remaining three had a dedicated stroke team but no stroke unit. Furthermore, all of the centers were homogeneous with regard to the availability of intensive care, neurosurgery, vascular surgery, neuroradiology, cardiology, and early rehabilitation. Acute ischemic stroke was defined in accordance with the Cooperative Young Stroke Study Criteria [4]. The exclusion criteria were as follows: ischemic stroke due to complications of other intracranial diseases, such as subarachnoid hemorrhage, sinus venous thrombosis, severe head trauma, or transient ischemic attack (TIA).

e ischemic stroke was defined in accordance with the Cooperative Young Stroke Study Criteria [4]. The exclusion criteria were as follows: ischemic stroke due to complications of other intracranial diseases, such as subarachnoid hemorrhage, sinus venous thrombosis, severe head trauma, or transient ischemic attack (TIA). We constructed our patient dataset by searching each hospital's database. The medical records of all consecutive patients aged between 16 to 44 years with ICD 9 cm codes that were between 433.00 and 434.91 were retrospectively reviewed by a team of stroke neurologists to determine if the cases met the definition of ischemic stroke. All of the clinical departments of each hospital, except pediatrics (which admit patients who are younger than 16 years of age), were involved. The data were collected according to a predefined protocol.

pectively reviewed by a team of stroke neurologists to determine if the cases met the definition of ischemic stroke. All of the clinical departments of each hospital, except pediatrics (which admit patients who are younger than 16 years of age), were involved. The data were collected according to a predefined protocol. All of the following data were registered: demographic characteristics, family history, risk factors [11], neurological examination findings, diagnostic data, and the type of treatment. The evaluated stroke risk factors and applied definitions are shown in the supplemental Appendix (see Appendix: Details of registered cerebrovascular risk factors and their definitions in Supplementary Material available online at http://dx.doi.org/10.1155/2013/715380). We retrospectively applied consistent definitions for risk factors over the study period. The stroke subtypes were classified according to the following Bamford criteria [12]: lacunar infarct (LACI), partial anterior circulation infarct (PACI), total anterior circulation infarct (TACI), and posterior circulation infarct (POCI). The stroke etiology was classified according to the following TOAST criteria [13]: large-vessel disease (LVD), small-vessel disease (SVD), cardioembolic stroke (CE), other determined cause (OTH), and undetermined cause (UND). The time of delay from the stroke onset to the hospital arrival, mortality, and clinical complications during hospitalization were also registered.

All of the following data were registered: demographic characteristics, family history, risk factors [11], neurological examination findings, diagnostic data, and the type of treatment. The evaluated stroke risk factors and applied definitions are shown in the supplemental Appendix (see Appendix: Details of registered cerebrovascular risk factors and their definitions in Supplementary Material available online at http://dx.doi.org/10.1155/2013/715380). We retrospectively applied consistent definitions for risk factors over the study period. The stroke subtypes were classified according to the following Bamford criteria [12]: lacunar infarct (LACI), partial anterior circulation infarct (PACI), total anterior circulation infarct (TACI), and posterior circulation infarct (POCI). The stroke etiology was classified according to the following TOAST criteria [13]: large-vessel disease (LVD), small-vessel disease (SVD), cardioembolic stroke (CE), other determined cause (OTH), and undetermined cause (UND). The time of delay from the stroke onset to the hospital arrival, mortality, and clinical complications during hospitalization were also registered. The study's patient sample was arbitrarily divided into the following three age groups for further comparisons with a previous report of young stroke in Italian population [7]: 16–29 years of age (Group 1), 30–39 years of age (Group 2), and 40–44 years of age (Group 3).

The time of delay from the stroke onset to the hospital arrival, mortality, and clinical complications during hospitalization were also registered. The study's patient sample was arbitrarily divided into the following three age groups for further comparisons with a previous report of young stroke in Italian population [7]: 16–29 years of age (Group 1), 30–39 years of age (Group 2), and 40–44 years of age (Group 3). The statistical analyses were performed using SPSS statistical software (version 10.1). The chi-squared analysis, Student's t-tests, and one-way analysis of variance were used to compare the categorical and continuous variables, respectively. The relationships between the variables were assessed using correlation and regression analyses. The univariate and multivariate logistic regression analyses, which were adjusted for hospital and gender, were performed to test the impact of age at onset of ischemic stroke on the risk of exhibiting the following common risk factors (present in >5% of the population): obesity, hypertension, hypercholesterolemia, and migraine. Additional multivariate models, which were adjusted for hospital and gender, tested the impact of age at onset of stroke on the classification (Bamford) and etiology of the stroke (TOAST). Lastly, a multivariate approach, which was adjusted for hospital and gender, tested the role of stroke subtypes and age at onset of ischemic stroke on the likelihood of returning home following the clinical event.

d the impact of age at onset of stroke on the classification (Bamford) and etiology of the stroke (TOAST). Lastly, a multivariate approach, which was adjusted for hospital and gender, tested the role of stroke subtypes and age at onset of ischemic stroke on the likelihood of returning home following the clinical event. The strength of the association between the predictors and the dependent variable was assessed using the means of the odds ratios (OR) and relative 95% confidence intervals (95% CI). 3. Results Three hundred and twenty-four subjects who were aged between 16 and 44 years and who were affected with ischemic stroke between January 2000 and December 2005 were included in the study. The mean age at onset was 36.7 years, with 162 women and 162 men. This sample represents 2.7% of the total number of ischemic strokes in patients aged 16 to 80 years who were registered in the study period in the Lombardy region (324/12108) [14]. In total, 303 patients (93.5%) were admitted to the neurological ward, and the remaining patients were admitted to other wards (cardiology, internal medicine, neurosurgery, or intensive care units). No significant differences were observed in the demographic data between the centers with respect to gender or the age of onset distributions.

In total, 303 patients (93.5%) were admitted to the neurological ward, and the remaining patients were admitted to other wards (cardiology, internal medicine, neurosurgery, or intensive care units). No significant differences were observed in the demographic data between the centers with respect to gender or the age of onset distributions. The baseline characteristics and the risk factor frequencies, stratified by the age groups, are reported in Table 1. Among the risk factors with frequencies that were greater than 5% (Table 1), one or more were present in 71.5% of the patients (33.3% of the patients had one risk factor, 22.8% of the patients had two risk factors, and 15.4% of the patients had three or more risk factors). No risk factor was present in 28.5% of the patients. Multivariate analyses, which were adjusted for hospital and gender, indicated that hypercholesterolemia is more frequent in older patients (Group 1 versus Group 3: OR 0.97, 95% CI 0.53–1.80, P = 0.04), obesity is more frequent in older patients (Group 2 versus Group 3: OR 0.22, 95% CI 0.07–0.75, P = 0.01), and migraine is more frequent in younger patients (Group 2 versus Group 3: OR 2.30, 95% CI 1.13–4.70, P = 0.02).

mia is more frequent in older patients (Group 1 versus Group 3: OR 0.97, 95% CI 0.53–1.80, P = 0.04), obesity is more frequent in older patients (Group 2 versus Group 3: OR 0.22, 95% CI 0.07–0.75, P = 0.01), and migraine is more frequent in younger patients (Group 2 versus Group 3: OR 2.30, 95% CI 1.13–4.70, P = 0.02). Hypertension was present in 50% of the patients with LVD, in 56.9% of those with SVD, in 38.7% of those with CE, in 36.2% of those with OTH, and in 32.2% of those with UND. Smoking was present in 33.3% of the patients with LVD, in 52.9% of those with SVD, in 8.1% of those with CE, in 18.1% of those with OTH, and in 10.3% of those with UND. Hypercholesterolemia was present in 16.7% of the patients with LVD, in 31.4% of those with SVD, in 21% of those with CE, in 14.9% of those with OTH, and in 19.5% of those with UND. The frequency of hypertension was higher in the LVD than in CE (P = 0.02), OTH (P < 0.001) and UND (P = 0.003) categories. Hypertension was also more prevalent in patients with SVD than in those with CE (P < 0.001) or OTH (P < 0.001). The prevalence of smoking was higher in patients with LVD or SVD; however, the difference was significant only for SVD when compared to OTH (P = 0.02) and UND (P = 0.004). Hypercholesterolemia was present in all of the stroke subtypes, without any significant differences between them.

CE (P < 0.001) or OTH (P < 0.001). The prevalence of smoking was higher in patients with LVD or SVD; however, the difference was significant only for SVD when compared to OTH (P = 0.02) and UND (P = 0.004). Hypercholesterolemia was present in all of the stroke subtypes, without any significant differences between them. The time delay from stroke onset to hospital arrival was <3 hours for 107 patients (33.0%), between 3 and 6 hours for 67 patients (20.7%), between 6 and 24 hours for 68 patients (21.0%), and greater than 24 hours for 63 patients (19.4%). The time delay was unknown for 19 patients (5.9%). No significant changes of time delay over 5-year period of study were observed.

ival was <3 hours for 107 patients (33.0%), between 3 and 6 hours for 67 patients (20.7%), between 6 and 24 hours for 68 patients (21.0%), and greater than 24 hours for 63 patients (19.4%). The time delay was unknown for 19 patients (5.9%). No significant changes of time delay over 5-year period of study were observed. For the diagnostic workup, EKGs and brain CT scans were acquired for all of the patients. Brain MRIs were acquired in 237 patients (73.1% of the total). All of the patients underwent a minimum of one extracranial circulation evaluation: an extracranial duplex ultrasonography was performed in 238 patients (73.4%), an extracranial MR angiography was performed in 67 patients (20.7%), and an extracranial CT angiography was performed in 27 patients (8.3%). Intracranial MR angiography was performed in 118 patients (36.4%), intracranial CT angiography was performed in 19 patients (5.9%), and digital subtraction angiography was performed in 102 patients (31.5%). Cardiologic assessments included transthoracic echocardiography (192 patients, 59.3%), transesophageal echocardiography (146 patients, 45.1%), transthoracic echocardiography with contrast injection (76 patients, 23.5%), and transcranial Doppler ultrasonography (32 patients, 9.9%).

was performed in 102 patients (31.5%). Cardiologic assessments included transthoracic echocardiography (192 patients, 59.3%), transesophageal echocardiography (146 patients, 45.1%), transthoracic echocardiography with contrast injection (76 patients, 23.5%), and transcranial Doppler ultrasonography (32 patients, 9.9%). The type of diagnostic workup that was performed changed over the study period. When we performed the analysis over the 5-year period, the proportion of the patients who underwent a brain MRI progressively increased from 60% (2000) to 80.6% (2005), P = 0.1. The frequency of the patients who underwent a minimum of one noninvasive angiographic study of the cerebral circulation (extracranial and intracranial MR angiography, extracranial and intracranial CT angiography) increased: 26.7% (2000), 37.2% (2001), 50% (2002), 39.2% (2003), 50.7% (2004), 69.4% (2005), P < 0.001. A reduction in the use of digital angiography was observed: 53.3% (2000), 27.9% (2001), 42% (2002), 21.6% (2003), 26.7% (2004), 30.6% (2005), P = 0.03, Figure 1. The proportion of the patients who underwent a minimum of one study to detect a PFO increased from 56.7% (2000) to 66.7% (2005), P = 0.08.

(2005), P < 0.001. A reduction in the use of digital angiography was observed: 53.3% (2000), 27.9% (2001), 42% (2002), 21.6% (2003), 26.7% (2004), 30.6% (2005), P = 0.03, Figure 1. The proportion of the patients who underwent a minimum of one study to detect a PFO increased from 56.7% (2000) to 66.7% (2005), P = 0.08. Coagulation testing (i.e., testing for the presence of antiphospholipid antibodies, protein C, protein S, antithrombin III, factor V Leiden, and prothrombin gene analyses and homocysteine plasma levels) was performed in 271 patients (83.6% of the patients). The findings of these tests were abnormal in at least one test in 84 patients (30.9%). In 19/324 patients (5.9%), thrombophilia was recognized as the underlying cause of stroke: antiphospholipid syndrome was present in ten patients, mutations in the factor V Leiden or prothrombin genes were detected in four patients, a deficiency of protein S was observed in one patient, and hyperhomocysteinemia (persistently elevated plasma levels >20 μmol/L measured at admission and after three months) was observed in four patients. With respect to the stroke type classification, LACI was diagnosed in 44 patients (13.6%), TACI was diagnosed in 35 patients (10.8%), PACI was diagnosed in 130 patients (40.1%), and POCI was diagnosed in 115 (35.5%). No significant changes over the study period were observed in the relative percentages of these diagnoses (P = 0.08).

o the stroke type classification, LACI was diagnosed in 44 patients (13.6%), TACI was diagnosed in 35 patients (10.8%), PACI was diagnosed in 130 patients (40.1%), and POCI was diagnosed in 115 (35.5%). No significant changes over the study period were observed in the relative percentages of these diagnoses (P = 0.08). Multivariate analyses, which were adjusted for age and center, indicated that the patients who were aged between 16 and 29 years (Group 1) exhibited a lower frequency of LACI classification than did older patients (40–44 years of age: OR = 0.42 (95% CI = 0.19–0.91), P = 0.03). The stroke etiology was classified according to the TOAST criteria: LVD in 30 patients (9.3%), SVD in 51 patients (15.7%), CE in 62 patients (19.1%), UND in 87 (26.9%), and OTH in the remaining 94 patients (29%). In the cardioembolic group, high-risk factors were identified in 17 patients (valvular heart disease in 5 patients, dilated cardiomyopathy in 5 patients, atrial fibrillation in 4 patients, myocardial infarction in 2 patients, and congenital cardiac malformation in 1 patient). Low- or uncertain-risk sources were identified in 45 patients (PFO in 32 patients and PFO with atrial septal aneurysm in 13 patients). The subtypes of uncertain diagnoses included an incomplete evaluation in 23 patients, multiple possible etiologies in 13 patients, and a negative evaluation in 51 patients.

In the cardioembolic group, high-risk factors were identified in 17 patients (valvular heart disease in 5 patients, dilated cardiomyopathy in 5 patients, atrial fibrillation in 4 patients, myocardial infarction in 2 patients, and congenital cardiac malformation in 1 patient). Low- or uncertain-risk sources were identified in 45 patients (PFO in 32 patients and PFO with atrial septal aneurysm in 13 patients). The subtypes of uncertain diagnoses included an incomplete evaluation in 23 patients, multiple possible etiologies in 13 patients, and a negative evaluation in 51 patients. The category of ischemic stroke as a result of other causes included the following items: arterial dissection (47), thrombophilia (19), systemic lupus erythematosus (7), inflammatory bowel disease (1), drug abuse (5), migrainous infarct (5), vascular malformations (5), hematological disease (2), Sneddon's syndrome (2), and CADASIL (1). The stroke etiology was stratified by age, as illustrated in Figure 2. According to the multivariate model, the risk of exhibiting a LVD or SVD etiology versus all of the other causes (i.e., CE, UND and OTH) was significantly lower when the patient was under 30 years of age at onset (P = 0.02; OR = 0.21 (95% CI = 0.07–0.56)) when compared to older patients.

ge, as illustrated in Figure 2. According to the multivariate model, the risk of exhibiting a LVD or SVD etiology versus all of the other causes (i.e., CE, UND and OTH) was significantly lower when the patient was under 30 years of age at onset (P = 0.02; OR = 0.21 (95% CI = 0.07–0.56)) when compared to older patients. Only UND and CE showed changes over time. The proportion of undetermined cases decreased as follows: 60% (2000), 30.2% (2001), 22% (2002), 31.4% (2003), 18.7% (2004), 20.8% (2005), P < 0.001. Cardioembolic strokes increased as follows: 3.3% (2000), 18.6% (2001), 12% (2002), 25.5% (2003), 21.3% (2004), 23.6% (2005), P = 0.11, Figure 3. The medical treatments and surgical procedures that were used are listed in Table 2. Percutaneous PFO closure was reserved for cases of PFO that were associated with an atrial septal aneurysm, severe right-left shunting, and/or deep vein thrombosis. The exclusion of other possible causes of ischemia was also required for this procedure to be performed. No significant changes over the study period were observed.

ous PFO closure was reserved for cases of PFO that were associated with an atrial septal aneurysm, severe right-left shunting, and/or deep vein thrombosis. The exclusion of other possible causes of ischemia was also required for this procedure to be performed. No significant changes over the study period were observed. The mortality rate during hospitalization was 2/324 (0.6%). The intrahospital complications included the following: epileptic seizures (6), urinary tract infections (18), respiratory infections (6), fever (12), and depressive symptoms (19). Among the surviving patients, 76.1% returned home, and 23.9% were transferred to a neurorehabilitation department. Multivariate analyses, which were adjusted for age and center, indicated that the probability of returning home was significantly higher in LACI (95.3%) than in non-LACI (73.1%) patients (P = 0.02; OR = 11.03 (95% CI = 2.48–49.08)). A higher probability of returning home was also observed in patients who were aged between 16 and 29 years (80.4%) and for those who were between 30 and 40 years of age (78.4%) when compared to the older group (71.9%). These differences, however, did not reach statistical significance (P = 0.13 and P = 0.08, resp.). 4. Discussion Our study describes the clinical characteristics of 324 Italian patients who were affected with ischemic stroke and who were between 16 and 44 years of age. Here, we evaluate the changes over the study period in the type of clinical care that was given to these patients.

The mortality rate during hospitalization was 2/324 (0.6%). The intrahospital complications included the following: epileptic seizures (6), urinary tract infections (18), respiratory infections (6), fever (12), and depressive symptoms (19). Among the surviving patients, 76.1% returned home, and 23.9% were transferred to a neurorehabilitation department. Multivariate analyses, which were adjusted for age and center, indicated that the probability of returning home was significantly higher in LACI (95.3%) than in non-LACI (73.1%) patients (P = 0.02; OR = 11.03 (95% CI = 2.48–49.08)). A higher probability of returning home was also observed in patients who were aged between 16 and 29 years (80.4%) and for those who were between 30 and 40 years of age (78.4%) when compared to the older group (71.9%). These differences, however, did not reach statistical significance (P = 0.13 and P = 0.08, resp.). 4. Discussion Our study describes the clinical characteristics of 324 Italian patients who were affected with ischemic stroke and who were between 16 and 44 years of age. Here, we evaluate the changes over the study period in the type of clinical care that was given to these patients. The study sample represents 12.4% of all of the ischemic stroke cases in young adults in the Lombardy region during the study period [14]. Furthermore, in agreement with previous studies [15], the collected sample represents 2.7% of all stroke cases that were admitted to the ten participating hospitals. Most of the patients (93.5%) were admitted to a neurological ward. The standard practice in Italy is to treat stroke patients, particularly younger patients, in the neurology department.

revious studies [15], the collected sample represents 2.7% of all stroke cases that were admitted to the ten participating hospitals. Most of the patients (93.5%) were admitted to a neurological ward. The standard practice in Italy is to treat stroke patients, particularly younger patients, in the neurology department. The sample was homogeneous across the participating centers, with no major differences in age or sex. As expected, there was a general increase in the risk of the disease with age [2, 7]. We did not observe a significant prevalence of female patients who were younger than 29 years of age, which is similar to what has been reported in other studies [2, 7]. As is shown in Table 1, modifiable risk factors (particularly smoking and hypertension) are strongly represented. This relationship was observed to be stronger than was reported in earlier studies [16] but was similar to more recent findings [2, 7]. A higher frequency of hypertension, hypercholesterolemia, and obesity was observed in the older age group (40 to 44 years of age). Other observations included (i) the higher prevalence of hypertension in the LVD and SVD category and (ii) the higher prevalence of smoking in the SVD category. Furthermore, 14.8% of the patient sample had experienced previous vascular events, including ischemic stroke and TIA.

older age group (40 to 44 years of age). Other observations included (i) the higher prevalence of hypertension in the LVD and SVD category and (ii) the higher prevalence of smoking in the SVD category. Furthermore, 14.8% of the patient sample had experienced previous vascular events, including ischemic stroke and TIA. Moreover, a high proportion of the patients had previously suffered from symptomatic vascular disease. In conclusion, conventional vascular risk factors are not only important in older patients with ischemic stroke, but also in this younger population under the age of 44. Although a history of migraine was present in approximately 20% of the patients who were younger than 39 years of age, only 5 patients (1.5%) fulfilled the criteria for a probable migraine-induced stroke [9]. The role of migraine in stroke is controversial. Our data contrast with those of most previous reports, which state a 3%–20% prevalence of migrainous stroke [17] but are similar to more recent findings (<1%) [2].

39 years of age, only 5 patients (1.5%) fulfilled the criteria for a probable migraine-induced stroke [9]. The role of migraine in stroke is controversial. Our data contrast with those of most previous reports, which state a 3%–20% prevalence of migrainous stroke [17] but are similar to more recent findings (<1%) [2]. The topography of cerebral infarction appears to confirm the recently described prevalence of posterior circulation involvement in young patients [2, 7]. The proportion of infarcts in the posterior territory varies from 25% to 46% in patients who are under 45 years of age which is significantly higher than what is observed for older patients (11.9%) [18]. The high proportion of vertebrobasilar stroke that was observed in the present study (35.5% of all of the patients) may be attributable to the frequent use of MRI, which is more useful for the analysis of posterior circulation. Arterial dissections involving posterior circulation are also frequent in young adults. Furthermore the young might have distinctive underlying pathophysiological mechanisms compared with the elderly. The relationship between the TOAST classification and the age groups has rarely been detailed [7]. LVD and SVD are underrepresented causes of stroke in patients who are younger than 39 years of age. The prevalence of a vascular cause of stroke increases with age, primarily due to the presence of atherosclerosis in older patients [7, 19]. Additionally, the proportion of cases that are classified as resulting from other causes was similar to that reported in other studies [3].

The relationship between the TOAST classification and the age groups has rarely been detailed [7]. LVD and SVD are underrepresented causes of stroke in patients who are younger than 39 years of age. The prevalence of a vascular cause of stroke increases with age, primarily due to the presence of atherosclerosis in older patients [7, 19]. Additionally, the proportion of cases that are classified as resulting from other causes was similar to that reported in other studies [3]. The second aim of the study was to describe the modification of both the clinical care and the diagnostic and therapeutic workup for the studied patients throughout the study period (2000–2005).

Additionally, the proportion of cases that are classified as resulting from other causes was similar to that reported in other studies [3]. The second aim of the study was to describe the modification of both the clinical care and the diagnostic and therapeutic workup for the studied patients throughout the study period (2000–2005). Only a small number of reports have analyzed changes in the etiological diagnosis of ischemic stroke in young adults over time [2, 8]. In the five-year span of our study, there was a significant reduction in the proportion of undetermined causes based on the TOAST classification. This finding was paralleled by an increase in size of the cardioembolic group. This could be attributable to the major impact that mild cardiac defects, such as PFO and atrial septal aneurysms, can have in stroke pathogenesis in the young compared with the elderly. However, it is necessary to be cautious in interpreting and handling a diagnosis of PFO [20]. First, it is occasionally difficult to detect paradoxical emboli, in situ thrombosis, and transient atrial arrhythmias. Second, other causes of stroke must be carefully excluded. Third, PFO detection can be an incidental finding. Additionally, the systematic application of a neuroradiological diagnostic workup (including brain MRIs and the analysis of extra- and intracranial circulation) could aid in the identification of other causes. A similar evolution in etiological diagnoses was recently reported by Varona et al. [8].

n incidental finding. Additionally, the systematic application of a neuroradiological diagnostic workup (including brain MRIs and the analysis of extra- and intracranial circulation) could aid in the identification of other causes. A similar evolution in etiological diagnoses was recently reported by Varona et al. [8]. Another change of the diagnostic approach was the increased prevalence of noninvasive angiographic studies. It was counterbalanced by a reduction in the use of digital angiography. Although the use of traditional digital angiography is decreasing, it is still used rather frequently (in 30.6% of the cases in this study), principally to identify otherwise unknown causes of stroke. Despite the observed changes in the types of diagnoses that were given over the study period, undetermined cases remain frequent. In our opinion, the prevalence of certain diagnoses (e.g., neurocutaneous syndromes, Fabry's disease and other monogenic disease, arterial vasospasm, and illicit drug use) may have been underestimated given that such causes were not overtly considered by local protocols during the study period. Long-term followup of young patients with ischemic stroke could help to identify rare causes of disease. As previously reported [2, 7], coagulation studies can detect a possible cause of stroke in only a minority of patients.

given that such causes were not overtly considered by local protocols during the study period. Long-term followup of young patients with ischemic stroke could help to identify rare causes of disease. As previously reported [2, 7], coagulation studies can detect a possible cause of stroke in only a minority of patients. With respect to the therapeutic approaches that were adopted, 4% of the patients underwent intravenous thrombolysis, and 3.7% underwent intra-arterial thrombolysis. Therefore, as was observed in older patients [21], only a minority (7.7%) of the patients who arrived within the 6-hour therapeutic window (53.7%) underwent thrombolytic treatment. This proportion did not change over the study period despite the recently reported increase in awareness of the efficacy of thrombolytic treatment in young adults [22–24]. Wagner and Lutsep [21] suggest that poor disease awareness is responsible for the limited administration of thrombolysis in young people. The most frequent surgical intervention was percutaneous PFO closure (8%). This practice reflects the emerging role of septal abnormalities in young stroke patients over the study period. Carotid surgery is restricted to a minority of young patients.

With respect to the therapeutic approaches that were adopted, 4% of the patients underwent intravenous thrombolysis, and 3.7% underwent intra-arterial thrombolysis. Therefore, as was observed in older patients [21], only a minority (7.7%) of the patients who arrived within the 6-hour therapeutic window (53.7%) underwent thrombolytic treatment. This proportion did not change over the study period despite the recently reported increase in awareness of the efficacy of thrombolytic treatment in young adults [22–24]. Wagner and Lutsep [21] suggest that poor disease awareness is responsible for the limited administration of thrombolysis in young people. The most frequent surgical intervention was percutaneous PFO closure (8%). This practice reflects the emerging role of septal abnormalities in young stroke patients over the study period. Carotid surgery is restricted to a minority of young patients. Intrahospital mortality and complications were rare in this study. The short period of observation that corresponded to the acute phase and the young age of the patient sample may explain the low mortality rate that was observed. Short-term outcomes were generally good, particularly for lacunar infarcts. However, the prognostic implications of cerebral small vessel disease are still unclear, as is reported in older patients [25].

ded to the acute phase and the young age of the patient sample may explain the low mortality rate that was observed. Short-term outcomes were generally good, particularly for lacunar infarcts. However, the prognostic implications of cerebral small vessel disease are still unclear, as is reported in older patients [25]. The primary limitation of this study is the retrospective design. The advantages include the exclusion of nonischemic stroke and TIA patients, the large number of the patients in the sample, and the relatively short study period. The multicentric setting of the study does not appear to have influenced our results, as no major differences were identified between the centers. In conclusion, the present study describes the characteristics, the diagnostic flow charts, and the treatment strategies for ischemic stroke in young patients that are currently used in clinical practice. Our results highlight the importance of vascular risk factors in young adults and suggest the requirement for stroke prevention strategy optimization in this population. The improvement in diagnostic workup over the study period reduced the number of undetermined strokes. Certain limitations remain in the management of these patients, especially with regard to the low impact of thrombolysis. The relatively favorable short-term prognosis that was observed in the study sample should be reassessed in a longer follow-up study. Supplementary Material Details of registered cerebrovascular risk factors and their definitions. Click here for additional data file.

In conclusion, the present study describes the characteristics, the diagnostic flow charts, and the treatment strategies for ischemic stroke in young patients that are currently used in clinical practice. Our results highlight the importance of vascular risk factors in young adults and suggest the requirement for stroke prevention strategy optimization in this population. The improvement in diagnostic workup over the study period reduced the number of undetermined strokes. Certain limitations remain in the management of these patients, especially with regard to the low impact of thrombolysis. The relatively favorable short-term prognosis that was observed in the study sample should be reassessed in a longer follow-up study. Supplementary Material Details of registered cerebrovascular risk factors and their definitions. Click here for additional data file. Conflict of Interests The authors state that there is no conflict of interests. Authors' Contribution L. Tancredi and F. Martinelli Boneschi contributed equally to this work. Figure 1 Changes over time (2000–2005) of the frequency of angiographic studies. Figure 2 TOAST criteria stratified by age classes. Figure 3 : TOAST classification: changes over time (2000–2005) of undetermined and cardioembolic strokes. Table 1 Baseline characteristics and risk factors stratified by age classes. Age classes 16–29 years 30–39 years 40–44 years Total Patient number (%) 52 (16.0) 141 (43.5) 131 (40.4) 324 (100) Male/female 27/25 71/70 64/67 162/162 Risk factors (%)

Figure 3 : TOAST classification: changes over time (2000–2005) of undetermined and cardioembolic strokes. Table 1 Baseline characteristics and risk factors stratified by age classes. Age classes 16–29 years 30–39 years 40–44 years Total Patient number (%) 52 (16.0) 141 (43.5) 131 (40.4) 324 (100) Male/female 27/25 71/70 64/67 162/162 Risk factors (%) Smoking 17 (32.7) 51 (36.2) 62 (47.3) 130 (40.1) Hypertension 2 (3.8*) 25 (17.7*) 41 (31.3) 68 (21) Oral contraceptives 12 (23.1) 29 (20.6) 27 (20.6) 68 (21°) Hypercholesterolemia 5 (9.6†) 29 (20.6) 31 (23.7) 65 (20.1) Migraine 11 (21.2) 29 (20.6†) 15 (11.5) 55 (17) Hyperhomocysteinemia 10 (19.2) 23 (16.3) 18 (13.7) 51 (15.7) Previous vascular events 7 (13.5) 20 (14.2) 21 (16) 48 (14.8) Family history of stroke 2 (3.8) 10 (7.1) 13 (9.9) 25 (7.7) Obesity 2 (3.8) 4 (2.8†) 13 (9.9) 19 (5.9) Others°° †P < 0.05 *P < 0.01. °When tested in women is 42% of the sample. °°Other risk factors with frequency below 5% were trauma 12 (3.7%); diabetes mellitus 11 (3.4%); heavy drinking 10 (3.1%); illicit drug use 5 (1.5%); ischemic cardiopathy 5 (1.5%); atrial fibrillation 4 (1.2%); pregnancy/puerperium 6 (1.9%). Table 2 Therapeutic approach in young patients with ischemic stroke. Treatment n  (%) Antiplatelet therapy 238 (73.5) Anticoagulant therapy 70 (21.6) Hypertension treatment 60 (18.5) Vitamin therapy 46 (14.2) Patent foramen ovale closure 27 (8.0) Intravenous thrombolysis 13 (4.0) Intra-arterial thrombolysis 12 (3.7) Carotid stenting 2 (0.6) Thromboendarterectomy 1 (0.3) Basilar stenting 1 (0.3)

1. Introduction Aneurysmal subarachnoid hemorrhage (SAH) is associated with significant morbidity and mortality, accounting for up to ~5% of all stroke cases [1, 2]. The mortality from SAH is estimated at 40–45% by 30 days after hemorrhagic onset and up to 15% mortality before hospital admission [3]. After years of research and extensive pathophysiological investigations of SAH, much is known in animal models about pathways that are activated after SAH and that may contribute to brain injury. However, few have proven to be effective therapeutic targets in humans [4, 5].

15% mortality before hospital admission [3]. After years of research and extensive pathophysiological investigations of SAH, much is known in animal models about pathways that are activated after SAH and that may contribute to brain injury. However, few have proven to be effective therapeutic targets in humans [4, 5]. SAH has been suggested in multiple reports to be complex, multisystem, and multifaceted pathogenesis that likely has multiple ongoing processes activated contributing to its final pathogenesis and highly morbid manifestations [4–8]. There are some common effects, however, such as vasoconstriction of both large and small cerebral arteries. As a result, it is difficult to research one pathway, one protein, and one target for potential therapeutic benefits. There has been a shift in research to understand how all the manifestations connect, interact, and further contribute to this pathology. Many strides have been made to understand the common secondary complications that occur after SAH, especially focusing on complications that occur early on, often known as early brain injury (EBI) [9, 10]. Some of the complications that EBI encompasses are delayed neuronal injury/death (DND), oxidative stress and inflammatory destruction of the parenchyma, and ischemic deficits leading to cortical spreading depression (CSD). These complications have been theorized to play a major role in the pathogenesis and may contribute significantly to poor morbidity and outcome after SAH.

d neuronal injury/death (DND), oxidative stress and inflammatory destruction of the parenchyma, and ischemic deficits leading to cortical spreading depression (CSD). These complications have been theorized to play a major role in the pathogenesis and may contribute significantly to poor morbidity and outcome after SAH. Individual studies on several secondary complications have shed light on shared mechanisms and pathways that may be activated after or during or even before the hemorrhage, which may explain a number of these secondary manifestations. Research has also shifted from considering primary angiographic vasospasm as a major contributor to poor outcome to other secondary mechanisms that may also occur early on during the hemorrhage and interact with angiographic vasospasm and predispose the brain to significant delayed injury and poor outcome [10–13].

ons. Research has also shifted from considering primary angiographic vasospasm as a major contributor to poor outcome to other secondary mechanisms that may also occur early on during the hemorrhage and interact with angiographic vasospasm and predispose the brain to significant delayed injury and poor outcome [10–13]. Recent research has proposed additional mechanisms behind brain predisposition to injury and poor outcome, some of which include global ischemia, delayed cerebral ischemia (DCI), and cortical spreading depression (CSD) [14–16]. Recent work has also focused on trying to delineate the fundamental differences between ischemic deficits and hemorrhagic insult and how early brain injury (EBI) after SAH may be linked to transient global ischemia or may be actually a result of an ischemic deficit introduced early on by the hemorrhage. Does transient global ischemia occur before or during the hemorrhage, and thus predisposing the brain to the secondary complications mentioned? Or is transient global ischemia a separate entity that has its own manifestations, mechanisms, and complications, separate from those pertaining to SAH? In this paper we discuss the secondary complications that arise after SAH, its relationship to the pathogenesis, and recent work that has been done to decipher their triggers and roles in poor outcome. Additionally, we will discuss the similarities in pathogenesis between global ischemia and SAH.

Recent research has proposed additional mechanisms behind brain predisposition to injury and poor outcome, some of which include global ischemia, delayed cerebral ischemia (DCI), and cortical spreading depression (CSD) [14–16]. Recent work has also focused on trying to delineate the fundamental differences between ischemic deficits and hemorrhagic insult and how early brain injury (EBI) after SAH may be linked to transient global ischemia or may be actually a result of an ischemic deficit introduced early on by the hemorrhage. Does transient global ischemia occur before or during the hemorrhage, and thus predisposing the brain to the secondary complications mentioned? Or is transient global ischemia a separate entity that has its own manifestations, mechanisms, and complications, separate from those pertaining to SAH? In this paper we discuss the secondary complications that arise after SAH, its relationship to the pathogenesis, and recent work that has been done to decipher their triggers and roles in poor outcome. Additionally, we will discuss the similarities in pathogenesis between global ischemia and SAH. 2. Global Cerebral Ischemia and Stroke Ischemia is generally defined as a diminution of cerebral blood flow (CBF) below critical thresholds, resulting in a damage to the entire brain (global ischemia which is necessarily transient if the patient is to survive, and thus it is this type of global ischemia that is often investigated in animal models) or a focal region to which perfusion is relatively low [17, 18]. Global cerebral ischemia occurs when the blood supply to the entire or large part of the brain is impeded [19]. Global cerebral ischemia may also arise from a number of clinical conditions such as cardiac arrest that lasts more than about 10 minutes [19]. This transient insult may result in permanent brain damage and other parenchymal changes that are not completely understood. Since the majority of global cerebral ischemic insults occur due to cardiac arrest, a substantial effort has been allotted to establish protocols for proper management and efficient resuscitation protocols for cardiac arrest patients [19]. Despite optimal resuscitation and adequate ongoing supportive measures, the postarrest period is often accompanied by ongoing cerebral ischemia or no reflow to multiple regions in the brain. This phase of cerebral ischemia is followed by a short phase of cerebral hyperaemia and a prolonged phase of hypoperfusion that lasts from several hours to days and which correlates with significant neurocognitive, behavioural, sensory, and motor deficits [19].

al ischemia or no reflow to multiple regions in the brain. This phase of cerebral ischemia is followed by a short phase of cerebral hyperaemia and a prolonged phase of hypoperfusion that lasts from several hours to days and which correlates with significant neurocognitive, behavioural, sensory, and motor deficits [19]. Other types of stroke including SAH are associated with a similar pattern of ischemic insult to the brain and may share similarities in cellular pathophysiology. SAH in rats was associated with an upregulation of vasoconstriction-mediating receptors, endothelin B (ETB), and serotonin receptors (5-HT1B) and with reductions in vasodilators like nitric oxide (NO) [20, 21]. Similarly, in a model of transient global ischemia in rats, Johansson and colleagues demonstrated that animals had prolonged neurological deficits as well as functional upregulation of the same ETB and 5-HT1B receptors in forebrain cerebral arteries. These findings suggest the contribution of cerebral artery vasoconstriction, cerebral hypoperfusion, and neuronal damage to transient global ischemia, which mimics similar findings in SAH [22].

neurological deficits as well as functional upregulation of the same ETB and 5-HT1B receptors in forebrain cerebral arteries. These findings suggest the contribution of cerebral artery vasoconstriction, cerebral hypoperfusion, and neuronal damage to transient global ischemia, which mimics similar findings in SAH [22]. 3. Secondary Complications after SAH 3.1. Early Brain Injury: Delayed Neuronal Injury Cells die after stroke primarily by apoptosis or necrosis [23]. Both are thought to occur after global cerebral ischemia and SAH. The exact pathways activated in these types of stroke are not entirely worked out and there may be different contributions of apoptotic and necrotic cell death. It is documented that transient global cerebral ischemia can trigger multiple cellular events and activate pathways which lead to both apoptotic and necrotic cell death in endothelial, glial, and neuronal cells [24].

not entirely worked out and there may be different contributions of apoptotic and necrotic cell death. It is documented that transient global cerebral ischemia can trigger multiple cellular events and activate pathways which lead to both apoptotic and necrotic cell death in endothelial, glial, and neuronal cells [24]. During aneurismal rupture causing SAH, the intracranial pressure can increase enough to cause global cerebral ischemia. In some cases, if the bleeding continues and the intracranial pressure does not decrease, then the patient dies immediately, probably secondary to acute cardiac changes secondary to the increased intracranial pressure and near-instantaneous brain death. In survivors, however, the contribution of transient global ischemia to brain injury is variable. Some patients have very small hemorrhages, do not loose consciousness, and thus do not have transient global ischemia. They are still at risk for DCI [25]. Interestingly, patients who become transiently unconscious at the time of their SAH and then awaken have likely had a transient global cerebral ischemic event and may have been at a higher risk of developing DCI [26]. Patients also only develop acute focal cerebral ischemia immediately after SAH in about 3% of cases [27].

erestingly, patients who become transiently unconscious at the time of their SAH and then awaken have likely had a transient global cerebral ischemic event and may have been at a higher risk of developing DCI [26]. Patients also only develop acute focal cerebral ischemia immediately after SAH in about 3% of cases [27]. Cellular apoptosis is reported to be a mechanism of EBI after the SAH and has been investigated in several studies. These studies focused on large cerebral arteries and found endothelial cell apoptosis after SAH [28, 29]. Neuronal apoptosis in the cortex and hippocampus has been detected after SAH in humans [30]. In animal studies, neurons, astrocytes, and oligodendroglia also exhibited apoptosis after SAH [31]. In some studies, there were fewer neurons in the hippocampus and inner cortical layers 5 days after SAH in rats [32]. The pathways involved in apoptosis after SAH have not been widely investigated. The apoptotic pathways include intrinsic (caspase-independent and mitochondrial) and extrinsic (cell-death receptor) pathways [33–35].

Cellular apoptosis is reported to be a mechanism of EBI after the SAH and has been investigated in several studies. These studies focused on large cerebral arteries and found endothelial cell apoptosis after SAH [28, 29]. Neuronal apoptosis in the cortex and hippocampus has been detected after SAH in humans [30]. In animal studies, neurons, astrocytes, and oligodendroglia also exhibited apoptosis after SAH [31]. In some studies, there were fewer neurons in the hippocampus and inner cortical layers 5 days after SAH in rats [32]. The pathways involved in apoptosis after SAH have not been widely investigated. The apoptotic pathways include intrinsic (caspase-independent and mitochondrial) and extrinsic (cell-death receptor) pathways [33–35]. Ischemia caused by increased intracranial pressure (ICP) is probably the first process that activates apoptosis. Apoptosis was observed within minutes of SAH in a rat endovascular perforation model of SAH and persisted for at least 24 hours [12, 35]. Ischemia following a SAH causes apoptotic cell death within the brain through several pathways such as induction of heat shock protein 70 (HSP70) [36]. HSP70 is a sensitive biomarker, which is activated diffusely throughout the brain one day after SAH is induced by endovascular perforation in rats. It continues to be activated 5 days after the SAH [36]. Ischemia also is associated with excitotoxic mechanisms that are mediated through the efflux of the amino acid glutamate. Glutamate activates the n-methyl-d-aspartate (NMDA) receptor following ischemia, resulting in an influx of sodium and calcium into neurons and subsequent neuronal death [37]. This mechanism has been suggested to cause neuronal apoptosis in vitro and in vivo [38].

mediated through the efflux of the amino acid glutamate. Glutamate activates the n-methyl-d-aspartate (NMDA) receptor following ischemia, resulting in an influx of sodium and calcium into neurons and subsequent neuronal death [37]. This mechanism has been suggested to cause neuronal apoptosis in vitro and in vivo [38]. The death receptor pathway has been implicated in apoptosis after cerebral ischemia and SAH. This pathway is activated by multiple cell membrane receptors, including the tumor necrosis factor receptor (TNFR), Fas, and DR3-5 [6, 29]. The ligands for these receptors include TNF-α, TNF-related apoptosis-inducing ligand, and Fas ligand. This pathway is activated by cerebral ischemia [23]. It has been shown that TNF-α is upregulated in the endothelium of dog basilar artery after SAH, and the inhibition of this with broad spectrum apoptosis inhibitors prevented vasospasm [39]. The dogs also had improved neurological outcomes [39]. TNF-α binding to TNFR activates caspase 8 and in some cases caspase 10. Downstream caspases are then activated, including caspases 3 and 9. Caspase 3 is a common essential component in the apoptotic pathway [39]. Cleaved caspase 3, a component of the intrinsic, caspase-dependent pathway, was detected in hippocampus and cortex after experimental SAH [12, 40]. The mitochondrial apoptotic pathway is likely involved in cerebral ischemia. Akt (protein kinase B) and mitogen-activated protein kinase (MAPK) are protein kinases that, when activated, inhibit apoptosis by interacting with Bax, Bad, glycogen synthase kinase-3, apoptosis signal-regulating kinase 1, and caspase 9. Akt activity is reduced after cerebral ischemia and its prevention reduced ischemic neuronal death [35]. Inhibiting Akt phosphorylation, which activates it, was associated with EBI after experimental SAH, and overexpression of Akt reduced brain injury [34, 41]. The MAPK may also be involved in EBI [35].

and caspase 9. Akt activity is reduced after cerebral ischemia and its prevention reduced ischemic neuronal death [35]. Inhibiting Akt phosphorylation, which activates it, was associated with EBI after experimental SAH, and overexpression of Akt reduced brain injury [34, 41]. The MAPK may also be involved in EBI [35]. Other mechanisms include caspase-independent intrinsic cell death pathway involved mitochondrial apoptosis-inducing factor (AIF), endonuclease G, and Bcl2/adenovirus E1B 19 kDa-interacting protein (BNIP3) [35]. Nuclear translocation of AIF was found after cerebral ischemia, suggesting the activation of this pathway; however, its role in SAH is less well studied [42].

insic cell death pathway involved mitochondrial apoptosis-inducing factor (AIF), endonuclease G, and Bcl2/adenovirus E1B 19 kDa-interacting protein (BNIP3) [35]. Nuclear translocation of AIF was found after cerebral ischemia, suggesting the activation of this pathway; however, its role in SAH is less well studied [42]. Autophagy is a process where cells form a multimembrane bound structure called the autophagosome, which sequesters cytoplasm and cell organelles in order to degrade them and recycle cytoplasm [43]. It occurs at basal levels in many tissues and is important in development, differentiation, and remodeling of organs and tissues. Autophagy is linked to apoptosis, but it is unclear if it causes cell death or is activated by some apoptotic pathways [44]. Autophagy has been suggested to provide a neuroprotective role in maintaining cellular homeostasis [43]. On the other hand, under certain conditions, it can have deleterious neurodegenerative effects [45]. After experimental SAH, autophagy has been observed in neurons and astrocytes of the basal frontal cortex on electron microscopy and in brain homogenates by an increase in the amount of membrane-bound microtubule-associated protein I light chain 3 [46]. Cathepsin D, an enzyme associated with degradation of damaged proteins and beclin-1, is also associated with autophagy and also is significantly higher after SAH [46]. Beclin-1 is a protein that interacts with Bcl-2 which is integral in the autophagic process [47]. Activation of autophagy with rapamycin reduced brain injury markers after SAH, whereas inhibition of autophagy with 3-methyladenine aggravated brain injury. This suggests that autophagy plays a neuroprotective role following SAH [44, 47].

otein that interacts with Bcl-2 which is integral in the autophagic process [47]. Activation of autophagy with rapamycin reduced brain injury markers after SAH, whereas inhibition of autophagy with 3-methyladenine aggravated brain injury. This suggests that autophagy plays a neuroprotective role following SAH [44, 47]. As discussed above, all of the apoptosis pathways are also likely important in global ischemic deficits after ischemic stroke [19] and may indeed account for some of the EBI after SAH. While neurons and other brain cells die by apoptosis after cerebral ischemia, the predominant mechanism of cell death is caused by necrosis, especially in the core of the ischemic brain [24]. Furthermore, activation of the death receptor pathway in apoptotic-deficient situations causes a sort of a combined form of cell death called necroptosis. Reports have demonstrated that neurons in the core tend to demonstrate liquefaction necrosis, while neurons in the penumbra tend to undergo apoptosis [23, 48, 49]. Apoptosis after cerebral ischemia occurs through intrinsic and extrinsic pathways [48, 50]. In the intrinsic pathway, ischemia results in the generation of permeability pores in the inner mitochondrial membrane, which results in the release of a number of proapoptotic factors and ultimately results in deoxyribonucleic acid (DNA) fragmentation and necrosis [51, 52]. The mitochondrial independent pathways after global ischemia tend to activate death receptors such as TNFR and Fas. Caspases also tend to play a major role in apoptotic activation in both cerebral ischemia and SAH [52, 53].

ctors and ultimately results in deoxyribonucleic acid (DNA) fragmentation and necrosis [51, 52]. The mitochondrial independent pathways after global ischemia tend to activate death receptors such as TNFR and Fas. Caspases also tend to play a major role in apoptotic activation in both cerebral ischemia and SAH [52, 53]. 4. Nitric Oxide and Nitric Oxide Synthases (NOS) Nitric oxide has a physiological role as a vasodilator and inhibitor of platelet activation and inflammation [54]. Reduction in NO is thought to contribute to angiographic vasospasm after SAH [55, 56] as well as to EBI [57, 58]. Within 10minutes of SAH in rats, there is acute vasoconstriction probably due to scavenging of NO [58]. NO concentrations subsequently increase above basal levels at 24 hours after SAH [59]. Another mechanism by which NO and NO synthases (NOS) can cause angiographic vasospasm and brain injury is by endothelial NOS uncoupling. This was demonstrated in the brain tissue of mice with SAH, in which there also was increased superoxide and nitrotyrosine production, and significant reduction in NO formation due to the dysfunction of eNOS [60]. Thus, while NO from eNOS might cause vasodilatation and reduce angiographic vasospasm and brain injury, under some conditions it could also be detrimental [61].

SAH, in which there also was increased superoxide and nitrotyrosine production, and significant reduction in NO formation due to the dysfunction of eNOS [60]. Thus, while NO from eNOS might cause vasodilatation and reduce angiographic vasospasm and brain injury, under some conditions it could also be detrimental [61]. While most of the studies in SAH rely on pharmacological manipulations, the contribution of NO to ischemic stroke and transient global ischemia has been assessed in genetically manipulated mice [62, 63]. Mice with reduced neuronal or inducible NOS have reduced infarct sizes, whereas those with eNOS reduction have more [63]. 5. Oxidative Stress Reactive oxygen and nitrogen (such as peroxynitrite) species are hypothesized to be important in brain injury after cerebral ischemia and SAH. Multiple studies show that there is release of reactive oxygen species (ROS) after experimental and human SAH [64–69]. Reactive oxygen species can exacerbate inflammation and generalized oxidative stress after SAH by increasing lipid peroxidation, causing direct DNA damage and protein oxidation. These processes in turn activate apoptotic signals and inflammatory cascades that further damage the brain [66]. A major source of ROS after SAH is thought to be oxidation reactions catalyzed by the heme groups of hemoglobin that are obviously abundant in the subarachnoid space after SAH [67].

ge and protein oxidation. These processes in turn activate apoptotic signals and inflammatory cascades that further damage the brain [66]. A major source of ROS after SAH is thought to be oxidation reactions catalyzed by the heme groups of hemoglobin that are obviously abundant in the subarachnoid space after SAH [67]. Reactive oxygen species can be generated by NOS isoforms (endothelial, neuronal, and inducible NOS). Multiple reports have demonstrated that under oxidative environments, NOS, particularly eNOS, can contribute to overproduction of peroxynitrite due to the reaction of NO and superoxide anion radicals [70]. Peroxynitrite oxidizes tetrahydrobiopterin, a cofactor for eNOS, and the zinc-thiolate complex in eNOS, which can uncouple eNOS and lead to generation of superoxide anion radicals [70]. Another source of superoxide anion radicals in the cerebral vasculature is the membrane-bound enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [71]. NADPH oxidase transfer electrons from NADH or NADPH to molecular oxygen through flavins that are present in the protein structure of the enzyme. This also generates superoxide anion radical which seems to be produced continuously at a low level in cerebral arteries. Structurally, NADPH oxidase has both membrane bound and cytosolic subunits. Functionally, it is a constitutively active enzyme that can mediate vasodilatation, for example, in rabbit cerebral arterioles in vivo [72]. The role of NADPH oxidase in the pathophysiology of SAH has not been widely investigated. In one study, inhibition of NADPH oxidase with diphenyleneiodonium reduced middle cerebral artery vasospasm after SAH in rats [73]. Vascular production of superoxide anion radical and NADPH oxidase activity were increased 24 hours after SAH in this model, and this was associated with membrane translocation of p47phox, one of the NADPH oxidase subunits.

oxidase with diphenyleneiodonium reduced middle cerebral artery vasospasm after SAH in rats [73]. Vascular production of superoxide anion radical and NADPH oxidase activity were increased 24 hours after SAH in this model, and this was associated with membrane translocation of p47phox, one of the NADPH oxidase subunits. Another source of oxidative stress after SAH may be xanthine dehydrogenase. This enzyme is found in endothelial cells where it produces uric acid from purines [74]. Ischemia can convert it to xanthine oxidase, which produces uric acid, superoxide anion radical, and hydrogen peroxide. This is involved in the pathophysiology of brain injury after cerebral ischemia. After SAH, Marklund and colleagues found that delayed cerebral ischemia was associated with increased concentrations of hypoxanthine, allantoin, and uric acid in cerebral microdialysis samples, possibly due to xanthine oxidase activity [75]. However, after experimental SAH in dogs, uric acid was increased in the cerebrospinal fluid, and this was inhibited with allopurinol [76]. This did not reduce angiographic vasospasm.

ncentrations of hypoxanthine, allantoin, and uric acid in cerebral microdialysis samples, possibly due to xanthine oxidase activity [75]. However, after experimental SAH in dogs, uric acid was increased in the cerebrospinal fluid, and this was inhibited with allopurinol [76]. This did not reduce angiographic vasospasm. Reactive oxygen species are also postulated to be important in cerebral ischemia and infarction. Nitric oxide also can be beneficial after ischemia by mediating vasodilation. However, it can also have toxic effects by, for example, inhibiting complexes I and II in the mitochondrial transport chain [19]. Also, as mentioned above, it can react with superoxide anion radical to produce peroxynitrite. Peroxynitrite generation promotes formation of other ROS such as hydroxyl-free radical and nitrogen dioxide. These nitrosylate tyrosine residues in proteins can result in further structural parenchymal damage [77]. NO has also been shown to upregulate the activity of poly (ADP-ribose) polymerase which leads to neuronal death through ATP consumption [77]. Additionally, in cerebral ischemia, constitutive NOS activity from the endothelial and neuronal NOS isoforms can be increased due to activation of various glutamate receptors, resulting in increased intracellular calcium and cytotoxicity [19]. In general, infarct volume and outcomes are worse in mice lacking eNOS, supporting the beneficial role of vascular endothelial NO [62]. On the other hand, genetic deletion of nNOS or iNOS tends to improve outcomes.

ation of various glutamate receptors, resulting in increased intracellular calcium and cytotoxicity [19]. In general, infarct volume and outcomes are worse in mice lacking eNOS, supporting the beneficial role of vascular endothelial NO [62]. On the other hand, genetic deletion of nNOS or iNOS tends to improve outcomes. 6. Inflammation Inflammation is hypothesized to mediate brain injury and angiographic vasospasm after SAH [78, 79]. There is an increase in proinflammatory cytokines, including TNF-α, interleukin 1-β (IL1-β), and IL6 acutely after experimental SAH [80–82]. Additionally, it has been demonstrated that pharmacologic inhibition of TNF-α or IL1-β attenuated EBI and improved blood-brain barrier (BBB) function after SAH [80, 81]. Another protein involved in proinflammatory cascade activation is NF-κB, a transcription factor in endothelial cells, that becomes phosphorylated resulting in the subsequent inactivation of IκB-α [83]. When NF-κB was activated in the arterial wall, there was an increase in TNF-α, IL1-β, and adhesion molecules. Pyrrolidine dithiocarbamate, an inhibitor of NF-κB, reduced vasospasm and the increase in the inflammatory cytokines and adhesion molecules. Leukocytes play a role in the immune response following SAH through their role in activating cytokines such as endothelin-1, a power vasoconstrictor that becomes elevated in experimental and clinical SAH [84]. In a study of 224 patients with SAH, a leukocyte count of greater than 15 × 109/L was associated with a 3.3-fold increase in the probability of developing angiographic vasospasm [85].

role in activating cytokines such as endothelin-1, a power vasoconstrictor that becomes elevated in experimental and clinical SAH [84]. In a study of 224 patients with SAH, a leukocyte count of greater than 15 × 109/L was associated with a 3.3-fold increase in the probability of developing angiographic vasospasm [85]. Selectins are from a family of cellular adhesion molecules that play a role in the inflammatory response [79]. They are categorized into leukocyte (L) selectin, platelet (P) selectin, and endothelial (E) selectin, which together mediate the capture, rolling, and adhesion of leukocytes in blood vessels [79]. Functionally, E selectin acts through binding to a carbohydrate site on the leukocytes that helps leukocytes target the site of inflammation. An increase in selectins in cerebrospinal fluid of patients with SAH and in animal models of SAH supports their role in the recruiting of leukocytes to cerebral vessels and brain after SAH [86]. Immunohistochemistry of ruptured cerebral aneurysms found increased E selectin in the aneurysm wall, which could also be a contributor [87].

selectins in cerebrospinal fluid of patients with SAH and in animal models of SAH supports their role in the recruiting of leukocytes to cerebral vessels and brain after SAH [86]. Immunohistochemistry of ruptured cerebral aneurysms found increased E selectin in the aneurysm wall, which could also be a contributor [87]. Brain damage after transient global ischemia involves similar pathways to those activated after SAH [19]. Cerebral ischemia leads to migration of peripheral neutrophils and monocytes into the brain. Multiple proinflammatory cytokines are released by neurons and glia, leading to increased selectin and adhesion molecules on cerebral blood vessels, similar to what is observed after SAH. Cytokines are also similarly involved in the pathogenesis of brain injury due to ischemia. Interleukin-1 beta (IL-1β) has been reported to play a detrimental role in brain injury, while proinflammatory IL-6 and anti-inflammatory cytokine IL-10 have uncertain roles [19]. Additionally, TNF-α has been found to either aggravate ischemic brain injury or to promote development of ischemic tolerance [19, 88].

emia. Interleukin-1 beta (IL-1β) has been reported to play a detrimental role in brain injury, while proinflammatory IL-6 and anti-inflammatory cytokine IL-10 have uncertain roles [19]. Additionally, TNF-α has been found to either aggravate ischemic brain injury or to promote development of ischemic tolerance [19, 88]. 7. Blood-Brain Barrier Disruption and Brain Edema Brain edema is a well-documented phenomenon that occurs days after experimental SAH in multiple animal models [6, 89]. Claassen et al. also concluded, based on interpretation of CT scans, that about 10% of patients had global cerebral edema within 24 hours of SAH [90]. Global cerebral edema was an independent risk factor for poor outcome and mortality. Brain edema may develop due to BBB dysfunction, which is also documented after acute experimental SAH [7, 8, 29]. Multiple processes may contribute to BBB breakdown after SAH, including endothelial cell apoptosis [29]. Blood breakdown products such as oxyhemoglobin and oxidative stress caused by hemoglobin can contribute to BBB disruption [91]. Additionally, proinflammatory cytokines like TNF-α and thromboxane A2 cause endothelial cell apoptosis and contribute to BBB dysfunction [92]. Inflammatory cytokines increase matrix metalloproteinases (MMP) that also disrupt the BBB. Yan et al. reported that inhibition of p53 ameliorated endothelial cell apoptosis and attenuated BBB disruption and brain edema after SAH in rats [93].

e A2 cause endothelial cell apoptosis and contribute to BBB dysfunction [92]. Inflammatory cytokines increase matrix metalloproteinases (MMP) that also disrupt the BBB. Yan et al. reported that inhibition of p53 ameliorated endothelial cell apoptosis and attenuated BBB disruption and brain edema after SAH in rats [93]. Accumulating evidence suggests a role for MMP-9 in the early disruption of the BBB after SAH [94]. MMP-9 degrades the extracellular matrix of the cerebral microvessel basal lamina, which includes collagen IV, laminin, fibronectin, and interendothelial tight junction proteins such as zona occludens-1 [95–97]. Basal lamina degradation starts as early as 6 hours and peaks 48 hours after experimental SAH created by endovascular perforation in rats [98]. Similar to after SAH, in after cerebral ischemia there is a release of proinflammatory cytokines like TNF-α and IL-1β from glia, leading to generation of adhesion molecules in the vasculature which can result in the weakening of the BBB [99, 100]. Thus, BBB disruption occurs after both a SAH and cerebral ischemia and predisposes to fluid/protein extravasation into the interstitial space resulting in cerebral edema.

ike TNF-α and IL-1β from glia, leading to generation of adhesion molecules in the vasculature which can result in the weakening of the BBB [99, 100]. Thus, BBB disruption occurs after both a SAH and cerebral ischemia and predisposes to fluid/protein extravasation into the interstitial space resulting in cerebral edema. 8. Excitotoxic Amino Acids Excitatory amino acids may play a role in the pathogenesis of SAH. Germanò et al. reported that the NMDA receptor antagonist, felbamate, attenuated BBB disruption 48 hours after SAH [101]. In view of the known action of felbamate, this suggests a role for NMDA receptor activation in BBB disruption after SAH. Additionally, Unterberg et al. found elevated brain glutamate by intracerebral microdialysis in patients with delayed ischemic deficits after SAH in humans [102]. Similar findings are observed in cerebral ischemia, where glutamate and other excitatory amino acids are increased in brain tissue [19]. The glutamate excitotoxicity hypothesis of brain injury after cerebral ischemia may not be proven, but the process likely occurs after SAH, especially in patients who develop focal ischemia due to delayed angiographic vasospasm or other complications or those with reduced cerebral perfusion pressure from brain swelling and edema. In the excitotoxicity hypothesis, there is brain energy depletion, like in the case with hypoxia-ischemia. Glutamate, one of the most abundant excitatory amino acids, is rapidly effluxed into the extracellular compartment due to neuronal depolarization. It activates NMDA receptors which causes increased intracellular calcium and sodium [19]. Increased calcium activates catabolic enzymes and cell death signaling pathways [38]. Blockade or retardation in the reuptake of excitotoxic amino acids like cysteine results in the depletion of antioxidant intracellular glutathione stores, purportedly causing neuronal injury and death [38]. Furthermore, the use of antiexcitotoxic agents such as NMDA and AMPA-R antagonists conferred neuroprotection through the amelioration of glutamate-induced excitotoxicity caused by hypoxic ischemic injury [38]. This success has not been translated into human ischemic stroke, however. These drugs also are not widely tested in clinical SAH, in part because they failed in human ischemic stroke.

tagonists conferred neuroprotection through the amelioration of glutamate-induced excitotoxicity caused by hypoxic ischemic injury [38]. This success has not been translated into human ischemic stroke, however. These drugs also are not widely tested in clinical SAH, in part because they failed in human ischemic stroke. 9. Summary The pathophysiology of SAH and cerebral ischemia share some common mechanisms. Cerebral ischemia is often seen as a complication of SAH as well. Early brain injury is also emerging as a key complication and a cause of morbidity and mortality after SAH. Again, common mechanisms may be involved in EBI and cerebral ischemia. Indeed part of EBI may be transient global cerebral ischemia, or at least a common hypoperfusion mechanism that acts between both cerebral insults (Figure 1). Further research is required to help elucidate the differences between EBI in a SAH and ischemic brain injury. Acknowledgments M. Sabri and E. Lass have no disclosures. R. L. Macdonald receives grant support from the Physicians' Services Incorporated Foundation, The Brain Aneurysm Foundation, Canadian Stroke Network, and the Heart and Stroke Foundation of Ontario. Figure 1 Some hypothetical relationships between cerebral ischemia due to cardiac arrest (CA) and subarachnoid hemorrhage.

1. Introduction Stroke imposes an enormous economic and human burden. Despite some spontaneous recovery observed during the first 3 months, around half of stroke patients are left with permanent disability, in which upper extremity motor impairment is the most prominent. Most hemiplegic patients also have a gait abnormality including decreased velocity, cadence, stride length, and prolonged swing phase on the affected side [1]. In addition to reduced ambulation, this could impair balance and lead to falls [2]. Perhaps the most common experimental stroke model is transient middle cerebral artery occlusion (MCAO) [3], which reproduces many features of human stroke. Numerous tests are available to assess behavioral impairment in MCAO rats, varying from simple tasks measuring general severity of neurological impairment to more demanding reaching tasks that measure upper extremity function [3–5]. Versatile analysis of gait and ambulation has been limited in stroke animals until the CatWalk system was recently introduced as an automated and quantitative gait analysis tool. It is based on video analysis of light reflected by the paws as they contact the glass floor. This represents a rapid way to objectively quantify several gait parameters such as position, pressure and surface area of each paw, which are used to calculate spatial paw statistics, the relative positions between paws, temporal parameters of gait, and interlimb coordination. This system has clinical relevance because the principle is very similar to the GAITRite system that can assess gait in stroke patients [1].

ure and surface area of each paw, which are used to calculate spatial paw statistics, the relative positions between paws, temporal parameters of gait, and interlimb coordination. This system has clinical relevance because the principle is very similar to the GAITRite system that can assess gait in stroke patients [1]. Recently, four papers have described the use of CatWalk in experimental stroke models. Wang et al. [6] studied gait 4 days and 5 weeks after cortical lesion (pMCAO model). Four days after ischemia, the intensity and maximal area of the affected forepaw were significantly decreased. They also found impairment of interlimb coordination. Most of these impairments persisted for 5 weeks. Vandeputte et al. [7] showed reduced intensity, print area, and width max area of the contralateral hindlimbs one day after cortical photothrombosis (Rose Bengal model). Encarnacion et al. [8] tested two rat strains after transient MCAO (filament model). They showed short-term deficits in intensity, stride length, stand index, duty cycle, and placement time of the impaired forelimb. In this study, deficits in hindlimb swing speed and placement time were more long lasting. Hetze et al. [9] showed decreases in maximum contact area, stride length, and swing speed in the impaired hindlimb following transient MCAO in mice.

de length, stand index, duty cycle, and placement time of the impaired forelimb. In this study, deficits in hindlimb swing speed and placement time were more long lasting. Hetze et al. [9] showed decreases in maximum contact area, stride length, and swing speed in the impaired hindlimb following transient MCAO in mice. MCAO rats usually develop compensatory strategies to overcome motor deficits. This has not been evaluated in the aforementioned studies although there is data available for all limbs. Another issue that has not been discussed is the difficulty to motivate the rats to cross the runway in a consistent manner without stopping and turning around. To overcome this, a goal box can be mounted at the end of the runway in the CatWalk XT version 9.1. Here we report our experiences in using CatWalk XT with a goal box in rats subjected to transient MCAO. 2. Material and Methods 2.1. Animals Male Wistar rats (BE Harlan Laboratories Ltd., Israel), 3 months old, weighing 350–400 g at the beginning of the study were used. The rats were housed individually under 12 h/12 h day and night cycles in a temperature-controlled environment (20 ± 1°C). All animal procedures were approved by the Animal Ethics Committee (Hämeenlinna, Finland) and conducted in accordance with the guidelines set by the European Community Council Directives 86/609/EEC. All efforts were made to minimize the number of animals used and to ensure their welfare throughout.

ironment (20 ± 1°C). All animal procedures were approved by the Animal Ethics Committee (Hämeenlinna, Finland) and conducted in accordance with the guidelines set by the European Community Council Directives 86/609/EEC. All efforts were made to minimize the number of animals used and to ensure their welfare throughout. 2.2. Middle Cerebral Artery Occlusion Focal cerebral ischemia was induced by the intraluminal filament technique (n = 7) [10]. Under halothane anesthesia, the right common carotid artery was exposed through a midline cervical incision. A heparinized nylon filament (diameter 0.25 mm, rounded tip) was inserted into the stump of the external common carotid artery. The filament was advanced 1.8–2.1 cm into the internal common carotid artery until resistance was felt. After 60 minutes of occlusion, the filament was removed and the external carotid artery was permanently closed by electrocoagulation. The sham-operated rats (n = 6) were treated in a similar manner, except that the filament was not placed into the internal carotid artery. The neurological impairment was assessed 24 hours after MCAO using a modified version of the limb-placing test [11] and animals with no behavioral impairment were excluded from the study.

am-operated rats (n = 6) were treated in a similar manner, except that the filament was not placed into the internal carotid artery. The neurological impairment was assessed 24 hours after MCAO using a modified version of the limb-placing test [11] and animals with no behavioral impairment were excluded from the study. 2.3. Magnetic Resonance Imaging Quantification of MR images was performed 24 hours after MCAO with a Bruker 7 T horizontal scanner to exclude the animals with no cortical damage or those with signs of hemorrhage. Based on MRI images, 3 animals were excluded from the study. For determination of the infarct volume, the rats were anesthetized with 5% isoflurane in a gas mixture of 30% O2/70% N2O. After induction, anesthesia was maintained throughout the imaging with 2.5% isoflurane inhaled through a nose mask. T2-weighted multislice images were acquired using a RARE sequence with the following parameters: time-to-repetition TR = 2.5 s, effective time-to-echo effTE = 40 ms, RARE factor 8, matrix size of 256 × 256, field-of-view of 30 mm × 30 mm, and BR 15 slices with a slice thickness of 1 mm. Infarct volumes were analyzed by using ImageJ. Areas of surviving gray matter in the cortex and striatum were outlined for each hemisphere. The difference between the size of an intact area in the contralateral hemisphere and the respective residual area in the ipsilateral hemisphere was recorded as the infarcted area. The total infarct volume was calculated by multiplying the infarct area by the distance between the slices and summing together the volumes.

he difference between the size of an intact area in the contralateral hemisphere and the respective residual area in the ipsilateral hemisphere was recorded as the infarcted area. The total infarct volume was calculated by multiplying the infarct area by the distance between the slices and summing together the volumes. 2.4. CatWalk Test CatWalk XT 9 (Noldus, The Netherlands), a quantitative gait analysis system, was used for this study. An enclosed glass walkway is illuminated from the long edge with a green light that is completely internally reflected. The light reflected by the paws as they contact the glass floor is captured by a high-speed video camera, which is then transformed into a digital image. The walkway was fixed to 90 mm wide. The camera was positioned 40 cm below the walkway and automatic detection settings were applied. An intensity threshold was set to 0.11, the camera gain was set to 18, and the maximum allowed speed variation was set to 50%. The animals were trained for three days. On the first day of training the lights in the room were on and food pellets were placed in the goal box to motivate the animals to complete the task. On the second and third days, the training took place in the dark with the only light source coming from the computer screen, where the CatWalk system was activated, but the program was not used. Rats were subjected to gait assessment at days 0 (baseline), 6, 21, and 42 after MCAO or sham surgery. Tests were performed in the same conditions as the training sessions, with the only exception that another male Wistar rat (non-testing) was systematically put in the goal box to motivate the trial rats to run towards it. The same motivator rat was used for all animals and in all test sessions. In case the animal was not motivated by the goal box, alternative positive motivators were used such as noise and food reward. Otherwise, animals were allowed to run back and forth on the walkway until 4 accepted runs were collected. An observer blind to the experimental groups performed the behavioral analysis and the data analysis.

mal was not motivated by the goal box, alternative positive motivators were used such as noise and food reward. Otherwise, animals were allowed to run back and forth on the walkway until 4 accepted runs were collected. An observer blind to the experimental groups performed the behavioral analysis and the data analysis. During the data analysis the steps were automatically labeled as right fore paw (RF), right hind paw (RH), left fore paw (LF), and left hind paw (LH), in which the right stands for the nonimpaired side and the left for the impaired side. Faulty labels caused by tail, whiskers, or genitalia were removed. After identification of individual footprints, we performed an automated analysis of a wide range of parameters. Data were classified as follows: (1) individual paw statistics; (2) comparative paw statistics; (3) interlimb coordination; (4) temporal parameters. In addition to the automatic values we also analyzed a package of individual footprint parameters such as the paw angle, toe spread, print length, and intermediate toe spread (Table 1). One trial consisted of four runs and at least one successfully recorded print from each paw was counted in from each run. Toe spread was determined as the distance between the innermost and outermost toes of the foot. Intermediate toe spread in the four-toed forepaw print was the distance between the innermost toe and the toe next to the outermost toe. In the five-toed hindpaws the intermediate toe spread was determined as the distance between the second and the third toes. The paw angle was determined simultaneously with print length by drawing a line from the back of the palm and following the pads of the paw to the second toe from the centre.

to the outermost toe. In the five-toed hindpaws the intermediate toe spread was determined as the distance between the second and the third toes. The paw angle was determined simultaneously with print length by drawing a line from the back of the palm and following the pads of the paw to the second toe from the centre. 2.5. Statistics All statistical tests were performed with GraphPad Prism5 statistical software (La Jolla, CA, USA). CatWalk data for the overall group effect and group × time interaction were analyzed using two-way repeated measures ANOVA followed by Bonferroni's post hoc comparison tests when appropriate. Linear correlations between body weight, speed, and gait parameters were evaluated by the Pearson's product-moment correlation coefficient. All values are presented as mean ± standard error of mean (SD). P values <0.05 were considered significant.

OVA followed by Bonferroni's post hoc comparison tests when appropriate. Linear correlations between body weight, speed, and gait parameters were evaluated by the Pearson's product-moment correlation coefficient. All values are presented as mean ± standard error of mean (SD). P values <0.05 were considered significant. 3. Results 3.1. Corticostriatal Infarction Affects Gait Bilaterally Transient MCAO resulted in variable cortical infarction and included most of the parietal sensorimotor cortex. Typically the striatum was completely damaged (Figure 1). Quantification of MR images made 24 hours after surgery showed a severe corticostriatal infarct in all the included MCAO rats (cortex 77.7 ± 15.4; striatum 32.9 ± 0.55 mm3). This was associated with severe, long-lasting behavioral impairment compared to sham-operated rats at the end of the followup when assessed by the cylinder test (35.6 ± 4.7% reduction of the impaired forelimb use), sticky label test (719 ± 120% increase in time to remove the label), and Montoya's staircase (43.6 ± 3.2% reduction in the number of eaten pellets). Gait parameters were first analyzed to compare impairment between the contralateral (impaired) versus ipsilateral (non-impaired) paws within groups (Figure 2). Prior to their operation, both sham-operated and MCAO rats showed no bias in their use of impaired and non-impaired fore or hindpaws. Interestingly, although MCAO animals displayed functional deficits in some of the CatWalk parameters, we did not find significant differences when comparing the contralateral and ipsilateral sides.

eir operation, both sham-operated and MCAO rats showed no bias in their use of impaired and non-impaired fore or hindpaws. Interestingly, although MCAO animals displayed functional deficits in some of the CatWalk parameters, we did not find significant differences when comparing the contralateral and ipsilateral sides. 3.2. Temporal Parameters Are Affected by MCAO Cadence describes the number of steps per second that the animal makes along the walking path. Similar to that reported in stroke patients, MCAO rats showed significant decrease in cadence (Figure 3(a); group effect F1,33 = 9.611, P < 0.05; and time effect F1,33 = 3.173, P < 0.05). This parameter is directly affected by the total run duration and the speed of the animal. The stance duration (average time in seconds that the paw is in contact with the glass plate for each step) in MCAO rats was increased for the LF (group effect F1,33 = 6.95, P < 0.05), RF (group effect F1,33 = 8.69, P < 0.05, time effect F1,33 = 2.91, P < 0.05; and interaction effect F1,33 = 3.15, P < 0.05), LH (group effect F1,33 = 5, P < 0.05), and RH (group effect F1,33 = 7.53, P < 0.05). Post hoc analysis of the RF values showed that MCAO animals had increased stance duration postoperative day 6 (P < 0.05) and 42 (P < 0.05) (Figure 4(a)). However, when we compared the contralateral versus ipsilateral sides of the animals, we did not find significant differences.

H (group effect F1,33 = 7.53, P < 0.05). Post hoc analysis of the RF values showed that MCAO animals had increased stance duration postoperative day 6 (P < 0.05) and 42 (P < 0.05) (Figure 4(a)). However, when we compared the contralateral versus ipsilateral sides of the animals, we did not find significant differences. The swing duration (average time in seconds in which the paw is not in contact with the glass plate) was significantly increased only in the LF (group effect F1,33 = 6.51, P < 0.05) (Figure 4(b)). As we found that the run speed slightly decreased and stance duration increased in MCAO animals, the total run duration was consequently significantly longer (group effect F1,33 = 5.28, P < 0.05), and the swing speed was also greater for all four paws of MCAO rats (Table 1). Taken together, our data from gait analysis during the 42-day followup after MCAO demonstrate that ischemia affected both contralateral and ipsilateral paws and also affected both forepaws and hindpaws. These results confirmed that the deficit was stable during the followup.

The swing duration (average time in seconds in which the paw is not in contact with the glass plate) was significantly increased only in the LF (group effect F1,33 = 6.51, P < 0.05) (Figure 4(b)). As we found that the run speed slightly decreased and stance duration increased in MCAO animals, the total run duration was consequently significantly longer (group effect F1,33 = 5.28, P < 0.05), and the swing speed was also greater for all four paws of MCAO rats (Table 1). Taken together, our data from gait analysis during the 42-day followup after MCAO demonstrate that ischemia affected both contralateral and ipsilateral paws and also affected both forepaws and hindpaws. These results confirmed that the deficit was stable during the followup. 3.3. Comparative Paw Statistics Consistent with data in stroke patients [12], we found that MCAO rats presented significantly shorter stride length (distance between successive placements of the same paw during maximal contact) in the LH (group effect F1,33 = 7.28, P < 0.05; and interaction effect F1,33 = 3.05, P < 0.05) at postoperative day 42 (P < 0.05) (Figure 4(c)). This was also true for the RF (group effect F1,33 = 4.85, P < 0.05; and interaction effect F1,33 = 3.24, P < 0.05) and for the RH (group effect F1,33 = 3.06, P < 0.05; and interaction effect F1,33 = 3.05, P < 0.05). Post hoc analysis showed that the differences were present at both postoperative day 6 (P < 0.05) and day 42 (P < 0.05). Statistical analysis showed no significant differences for the LF (group effect F1,33 = 2.582, P = 0.13).

the RH (group effect F1,33 = 3.06, P < 0.05; and interaction effect F1,33 = 3.05, P < 0.05). Post hoc analysis showed that the differences were present at both postoperative day 6 (P < 0.05) and day 42 (P < 0.05). Statistical analysis showed no significant differences for the LF (group effect F1,33 = 2.582, P = 0.13). MCAO rats showed significantly longer step cycles (the time in seconds between two consecutive contacts of the same paw) for the RF (group effect F1,33 = 9.02, P < 0.05), RH (group effect F1,33 = 9.65, P < 0.01), LF (group effect F1,33 = 8.68, P < 0.05), and LH (group effect F1,33 = 6.57, P < 0.05; and interaction time × group effect F1,33 = 2.91, P < 0.05). Detailed post hoc analysis of the LH values showed significant differences at day 6 (P < 0.05) and day 42 (P < 0.05) after reperfusion (Table 1). However, we did not find significant differences between the contralateral and ipsilateral sides of the animal.

; and interaction time × group effect F1,33 = 2.91, P < 0.05). Detailed post hoc analysis of the LH values showed significant differences at day 6 (P < 0.05) and day 42 (P < 0.05) after reperfusion (Table 1). However, we did not find significant differences between the contralateral and ipsilateral sides of the animal. When we analyzed duty cycles (the percentage of time the paw accounts for the total step cycle of the paw), we found that this only varied in the RF of MCAO animals (interaction effect F1,33 = 3.91, P < 0.05; and time effect F1,33 = 4.30, P < 0.05) at postoperative day 6 (P < 0.05) (Figure 4(d)). Moreover, the base of support of the hindpaws significantly increased at postoperative day 42 (time effect F1,33 = 5.62, P < 0.01; and interaction time × group effect F1,33 = 3.78, P < 0.05) (Figure 3(b)). As the base of support is the average width between either the front paws or the hindpaws; MCAO animals showed wider steps with their hindpaws, most likely as a mechanism to compensate for their unsteady gait.

time effect F1,33 = 5.62, P < 0.01; and interaction time × group effect F1,33 = 3.78, P < 0.05) (Figure 3(b)). As the base of support is the average width between either the front paws or the hindpaws; MCAO animals showed wider steps with their hindpaws, most likely as a mechanism to compensate for their unsteady gait. 3.4. Interlimb Coordination or Individual Footprint Parameters Were Not Affected by Ischemia No differences between the sham-operated and MCAO group were detected when interlimb coordination parameters were analyzed. The overall interlimb coordination during gait (known as gait regularity index of step sequence) was not affected, and all values remained above 95% for both groups during the followup. Another parameter to assess interlimb coordination is phase dispersion (the temporal relationship between placements of two paws within a step cycle). Phase dispersion between diagonal limb pairs (i.e., LF to RH and RF to LH) showed no differences during the 42-day followup after MCAO (Table 1). Interlimb coordination in ipsilateral pairs (i.e., LF to LH and RF to RH) as well as between front paws was not altered by MCAO (data not shown). In line with previous results in phase dispersion, we did not observe irregularities in the overall step sequence, but we found the alternative pattern AB in majority of sham-operated and MCAO rats (data not shown).

rs (i.e., LF to LH and RF to RH) as well as between front paws was not altered by MCAO (data not shown). In line with previous results in phase dispersion, we did not observe irregularities in the overall step sequence, but we found the alternative pattern AB in majority of sham-operated and MCAO rats (data not shown). Unlike the findings reported by Wang et al. [6], we did not find significant differences between sham-operated and MCAO rats when the maximum paw contact area, maximum intensity, print area, print width, print length, and toe spread were analyzed. However, a strong time effect was observed during the followup of both MCAO and sham-operated animals (Table 1), which led us to believe that body weight had influenced all these gait parameters. 3.5. Body Weight Affects CatWalk Parameters Animals were 358 ± 10 g at the beginning of the study. Due to the surgical procedures, all the animals lost some body weight after their operation. Particularly, the MCAO group showed severe loss of weight (11 ± 5%) during the first week after ischemia, but this was followed by progressive weight gain during the rest of the followup (time effect F1,33 = 107.9, P < 0.001; and interaction effect F1,33 = 4.42, P < 0.01).

s lost some body weight after their operation. Particularly, the MCAO group showed severe loss of weight (11 ± 5%) during the first week after ischemia, but this was followed by progressive weight gain during the rest of the followup (time effect F1,33 = 107.9, P < 0.001; and interaction effect F1,33 = 4.42, P < 0.01). CatWalk uses the light reflected by the paws as they contact the glass of the apparatus. As explained above, some gait parameters showed a characteristic feature, in which the values increased during the followup, and this seemed to be intimately related to weight recovery. Therefore, to assess whether body weight could affect CatWalk parameters, we conducted Pearson's correlation analysis (Figure 5). As described previously [13, 14], we found a strong negative correlation between the body weight loss and the severity of the lesion (sham Pcc = −0.89, P < 0.05; MCAO Pcc = −0.85, P < 0.05), whereby those animals with larger infarcts suffered more acute weight loss. Conversely, body weight did not affect run duration, speed, or stride length (Table 2). In addition, we also found that body weight was positively influencing swing speed and the maximum intensity of sham-operated animals, but weight did not influence the values for forepaws (Table 2) or hindpaws (data not shown) of MCAO rats. This was probably due to the differences in the weight recovery rates of both groups within the first week after ischemia. Individual paw parameters such as maximum paw contact area (Figure 5(a)), paw print area, paw print width, and toe spread (Figure 5(b)) from both sham and MCAO groups were also affected by body weight for the forepaws (Table 2) and the same was true for hindpaws (data not shown). Therefore, we here showed that some of the parameters measured by the CatWalk system were interdependent on weight gain in both the fore and hindpaws. Thus, in cases where the body weight of the animals is affected by the surgical procedure (i.e., cerebral ischemia) the results can become biased.

paws (data not shown). Therefore, we here showed that some of the parameters measured by the CatWalk system were interdependent on weight gain in both the fore and hindpaws. Thus, in cases where the body weight of the animals is affected by the surgical procedure (i.e., cerebral ischemia) the results can become biased. 3.6. Temporal Parameters Can Also Directly Affect Other Gait Parameters The motivation of the animals to walk towards the goal box is very important for the generation of reliable data. Unmotivated animals had difficulties completing their runs, which affect their speed and consequently cadence, and also other temporal parameters. Interestingly, speed showed strong negative correlation with maximum contact area in MCAO animals (Figure 5(c)), indicating that when there is a decrease in walking speed due to infarct mediated impairment, rats increased the contact area of the paw with the walking surface. Also, stride length and stance (Figure 5(d)) showed an intimate interdependency with speed in the fore- and hindpaws of both sham-operated and MCAO rats. When the speed increased the animals had longer stride lengths and decreased stance duration. There was no correlation between temporal parameters and individual paw or interlimb coordination (Table 2). As cadence and speed showed very similar behaviors, when we correlated cadence with gait parameters, we observed that cadence was influencing the same parameters that speed did (data not shown).

stance duration. There was no correlation between temporal parameters and individual paw or interlimb coordination (Table 2). As cadence and speed showed very similar behaviors, when we correlated cadence with gait parameters, we observed that cadence was influencing the same parameters that speed did (data not shown). 4. Discussion The CatWalk system was initially designed to measure gait in models of spinal cord injury, neuropathic pain, and peripheral nerve injury [15]. In stroke animals, previous studies have shown transient, variable, and/or minor gait impairment [6–9], which may be partly due to the different models and time points selected for testing. In the present study, rats subjected to transient MCAO showed a severe and long-lasting impairment when sensitive sensorimotor tests were applied (e.g., cylinder test, sticky label test, and Montoya's staircase). In contrast, only minor impairment was observed in gait after MCAO, which is consistent with behavioral observation—gross locomotion is seemingly normal a few weeks after MCAO.

e and long-lasting impairment when sensitive sensorimotor tests were applied (e.g., cylinder test, sticky label test, and Montoya's staircase). In contrast, only minor impairment was observed in gait after MCAO, which is consistent with behavioral observation—gross locomotion is seemingly normal a few weeks after MCAO. 4.1. Body Weight Contributes to a Variety of Gait Parameters The body weight of the animal is considered as a possible confounder of gait data [16, 17], and measures such as intensity and maximum contact area are obviously affected by the weight of the animals. Thus, to assess whether body weight was affecting gait parameters, we measured the animal weight every day after ischemia induction. Consistent with the findings by Koopmans et al. [17], we hence found an interdependency of body weight with contact area, print area, and print width. Particularly in the MCAO model, this interdependency may have an impact on gait results, since there is a severe initial loss of body weight after surgery, related to lesion size and neurological impairment [14, 18, 19]. More importantly, this might also be relevant in other models involving long-term followup with progressive gain of body weight.

l, this interdependency may have an impact on gait results, since there is a severe initial loss of body weight after surgery, related to lesion size and neurological impairment [14, 18, 19]. More importantly, this might also be relevant in other models involving long-term followup with progressive gain of body weight. 4.2. Walking Speed and Motivation Are Key Factors Affecting CatWalk Data Data acquisition on the CatWalk system depends on the velocity of the animals when walking across the runway. In line with previous studies [6, 20], we showed that both walking speed and cadence were directly associated with stride length and temporal parameters such as stance and swing speed. The CatWalk system allows setting up a maximum speed variation within runs. This value is a compromise between obtaining easy runs but reliable data as well. Based on a pilot study, we here selected a maximum speed variation of 50%. Although the differences in temporal parameters between MCAO animals and sham-operated animals are most likely due to the impairment caused by ischemia, they can also be affected by the speed, which intimately depends on the motivation of the animal to walk towards the goal box. Consequently, these interdependencies may lead to misinterpretation of experimental data.

etween MCAO animals and sham-operated animals are most likely due to the impairment caused by ischemia, they can also be affected by the speed, which intimately depends on the motivation of the animal to walk towards the goal box. Consequently, these interdependencies may lead to misinterpretation of experimental data. In addition, as explained above, the motivation of animals to complete the task plays a crucial role in data acquisition. In order to improve the motivation and in turn to prevent stopping and turning around, the runway is connected to a goal box with a cage underneath. However, this did not help and we noted that animals showed reduced motivation to cross the test runway upon repeated testing (habituation). Due to technical reasons, the runs have to be carried out in a dark environment in which the rats are more likely to display exploratory behavior [21]. Thus, it is important to motivate rats properly to complete a run with appropriate speed in a dark and relatively safe environment. Alternative positive motivators to be considered are smell, noise, and food reward [22]. On the other hand, gait impairment may completely disappear when the animals are performing a highly motivated behavior [22]. And lastly, we are not sure whether the other behavioral tests (i.e., cylinder test, sticky label test, and Montoya's staircase) to which the rats were subjected might have interfered with their CatWalk performance.

it impairment may completely disappear when the animals are performing a highly motivated behavior [22]. And lastly, we are not sure whether the other behavioral tests (i.e., cylinder test, sticky label test, and Montoya's staircase) to which the rats were subjected might have interfered with their CatWalk performance. 4.3. Impact of Ischemic Damage on Gait Impairment in MCAO Rats MCAO rats showed a large corticostriatal lesion followed by secondary damage/pathology in the thalamus [23], which may contribute to late impairment. Indeed, delayed worsening in several gait parameters was observed at postoperative day 42. Previously, the impact of cortical and subcortical lesions on gait is suggested to be transient and minor [24]. In line with this, there is evidence that the corticospinal tract does not contribute to overground locomotion, but is required for skilled locomotion such as ladder walking [25–27]. Interestingly, this indicates that in the absence of supraspinal input, the spinal cord is able to generate basic stepping and modulate locomotor activity [28].

e is evidence that the corticospinal tract does not contribute to overground locomotion, but is required for skilled locomotion such as ladder walking [25–27]. Interestingly, this indicates that in the absence of supraspinal input, the spinal cord is able to generate basic stepping and modulate locomotor activity [28]. Much to our surprise some gait parameters were altered bilaterally in MCAO rats. Having a closer look on previous studies, bilateral impairment (e.g., print area, max area) was also observed by Wang et al. [6]. Unilateral lesions are known to result in impairments of both sides of the body [29]. However, the bilateral gait adjustment we observed here is most likely related to compensation, which may help stabilize the animal during voluntary locomotion [22]. It seems that ischemic rats are partially able to compensate by postural adjustments and shifting body weight to the intact limbs but also by adjusting the gait of the ipsilateral limb according to the contralateral side. The proper gait for terrestrial walking is needed for escape. Thus, bilateral gait adjustment/compensation may be an evolutionary mechanism needed for survival.

y postural adjustments and shifting body weight to the intact limbs but also by adjusting the gait of the ipsilateral limb according to the contralateral side. The proper gait for terrestrial walking is needed for escape. Thus, bilateral gait adjustment/compensation may be an evolutionary mechanism needed for survival. At first, gait analysis seemed to be an attractive and feasible approach in MCAO rats. However, a high sample size (15–20 animals/group) is needed to detect a therapeutic effect large enough to notice a gait improvement [9]. Particularly, to observe a treatment effect in our study and by using the stride length as an example, we would only need 8 animals per group to obtain a relatively large effect size (d = 1.4 and α = 0.05; β = 0.08). However, we would need to increase the number of animals per group to n = 25–30 to observe what is considered to be a medium size effect (d = 0.75). Thus our data suggests that CatWalk can be a helpful complementary tool when testing therapeutic compounds in experimental models of stroke if combined with other tests that are not dependent on weight, speed, or compensatory strategies.

to n = 25–30 to observe what is considered to be a medium size effect (d = 0.75). Thus our data suggests that CatWalk can be a helpful complementary tool when testing therapeutic compounds in experimental models of stroke if combined with other tests that are not dependent on weight, speed, or compensatory strategies. 4.4. How Similar Is Gait Impairment in Rats versus Humans after Stroke? The gait impairments in MCAO animals were subtle, but persistent, and resembled those of patients with stroke such as the decreased cadence and in the increase in the base of support. Both of these alterations in gait have been observed in hemiplegic stroke patients [12, 30]. The general markers of interlimb coordination were unaltered in MCAO animals, which again suggests similarity with the gait of stroke patients, namely, that the central nervous system controls impaired gait by controlling the speed performance within the limits of the available compensatory behavior between affected and unaffected sides [31, 32].

limb coordination were unaltered in MCAO animals, which again suggests similarity with the gait of stroke patients, namely, that the central nervous system controls impaired gait by controlling the speed performance within the limits of the available compensatory behavior between affected and unaffected sides [31, 32]. 5. Conclusions CatWalk produces an exhaustive number of gait parameters that are potentially useful in the assessment of motor behaviors in MCAO rats. Some parameters are affected by body weight, speed, and motivation, even when a goal box is used, which may confound the data interpretation. In addition, compensatory adjustments develop to stabilize locomotion after a severe ischemic lesion. Although bipedal versus quadrupedal gait impairment after stroke seems to share some similarities, the translational applicability of CatWalk data remains open and further work is needed to explore this issue. Conflict of Interests The authors declare that they have no conflict of interests. Authors' Contribution S. Parkkinen and F. J. Ortega shared first authorship. Acknowledgments This study was supported by the strategic funding of the University of Eastern Finland (UEF-Brain) and Biocenter Finland. Figure 1 Corticostriatal lesion. Representative T2-weighted magnetic resonance images of coronal sections acquired 24 hours after MCAO.

Authors' Contribution S. Parkkinen and F. J. Ortega shared first authorship. Acknowledgments This study was supported by the strategic funding of the University of Eastern Finland (UEF-Brain) and Biocenter Finland. Figure 1 Corticostriatal lesion. Representative T2-weighted magnetic resonance images of coronal sections acquired 24 hours after MCAO. Figure 2 Graphical representation of selected gait parameters. The animal is walking towards the left. Black and white boxes represent time fractions where the paw is in contact with the surface or lifted at walking. RF: right forelimb; LF: left forelimb; RH: right hindlimb; LH: left hindlimb. Figure 3 Focal cerebral ischemia significantly affected cadence and base of support (BOS). (a) MCAO animals showed a decreased number of steps per second (cadence) when walking along the CatWalk runway at postoperative days 6 and 42. (b) Ischemic animals showed significantly larger hindlimb BOS at postoperative day 42. All values are given as mean ± SD. Statistics: **P < 0.01 versus sham-operated group. Figure 4 Effect of focal cerebral ischemia on temporal and comparative paw parameters. (a) Cerebral ischemia increased the duration of the stance phase of all paws at postoperative days 6 and 42. (b) Swing duration was only significantly different in the left forelimb (LF) at the acute phase after ischemia. (c) Stride length of MCAO animals was generally shorter. (d) Only the right forelimb (RF) of MCAO animals denoted longer duty cycle at postoperative day 6. All values are given as mean ± SD. Statistics: *P < 0.05 versus sham-operated group.

ficantly different in the left forelimb (LF) at the acute phase after ischemia. (c) Stride length of MCAO animals was generally shorter. (d) Only the right forelimb (RF) of MCAO animals denoted longer duty cycle at postoperative day 6. All values are given as mean ± SD. Statistics: *P < 0.05 versus sham-operated group. Figure 5 Gait parameter correlations. Scatter plots showing the correlations between gait parameters and body weight ((a), (b)) and locomotor speed ((c), (d)) of the left and right forepaws. Linear regression lines were plotted in each group (solid lines for sham-operated and dotted lines for MCAO rats). The values of Pearson's Products Moment Correlations Coefficients (Pcc) and P-values are shown for each group. Maximum contact area (a) and toe spread (b) showed positive correlation with body weight. By contrast, the maximum contact area (c) and stance (d) showed negative correlations with locomotor speed. Values for the left and right hindpaws followed the same correlation patterns (data not shown). Table 1 CatWalk gait parameter statistics. MCAO Time Interaction F value P value F value P value F value P value Temporal parameters

Figure 5 Gait parameter correlations. Scatter plots showing the correlations between gait parameters and body weight ((a), (b)) and locomotor speed ((c), (d)) of the left and right forepaws. Linear regression lines were plotted in each group (solid lines for sham-operated and dotted lines for MCAO rats). The values of Pearson's Products Moment Correlations Coefficients (Pcc) and P-values are shown for each group. Maximum contact area (a) and toe spread (b) showed positive correlation with body weight. By contrast, the maximum contact area (c) and stance (d) showed negative correlations with locomotor speed. Values for the left and right hindpaws followed the same correlation patterns (data not shown). Table 1 CatWalk gait parameter statistics. MCAO Time Interaction F value P value F value P value F value P value Temporal parameters Cadence 9.611 0.010* 3.173 0.037* 2.012 0.131 Stance duration RF 8.692 0.013* 2.917 0.048* 3.015 0.043* RH 7.538 0.019* 2.553 0.072 2.268 0.098 LF 6.951 0.023* 2.591 0.069 2.605 0.068 LH 5.004 0.047* 2.615 0.067 2.818 0.054 Swing duration RF 2.226 0.163 0.088 0.966 0.229 0.875 RH 3.851 0.075 0.875 0.463 0.707 0.554 LF 6.512 0.026* 2.313 0.094 2.379 0.087 LH 3.805 0.077 1.386 0.264 0.583 0.629 Speed 3.667 0.081 2.259 0.099 2.533 0.073 Run duration 5.28 0.042* 1.521 0.227 2.814 0.054 Swing speed RF 5.069 0.045* 0.872 0.465 1.991 0.134 RH 9.524 0.010* 1.43 0.251 2.822 0.053 LF 9.858 0.009** 2.406 0.085 2.966 0.046* LH 7.993 0.016* 1.6 0.208 3.51 0.026* Comparative paws

Cadence 9.611 0.010* 3.173 0.037* 2.012 0.131 Stance duration RF 8.692 0.013* 2.917 0.048* 3.015 0.043* RH 7.538 0.019* 2.553 0.072 2.268 0.098 LF 6.951 0.023* 2.591 0.069 2.605 0.068 LH 5.004 0.047* 2.615 0.067 2.818 0.054 Swing duration RF 2.226 0.163 0.088 0.966 0.229 0.875 RH 3.851 0.075 0.875 0.463 0.707 0.554 LF 6.512 0.026* 2.313 0.094 2.379 0.087 LH 3.805 0.077 1.386 0.264 0.583 0.629 Speed 3.667 0.081 2.259 0.099 2.533 0.073 Run duration 5.28 0.042* 1.521 0.227 2.814 0.054 Swing speed RF 5.069 0.045* 0.872 0.465 1.991 0.134 RH 9.524 0.010* 1.43 0.251 2.822 0.053 LF 9.858 0.009** 2.406 0.085 2.966 0.046* LH 7.993 0.016* 1.6 0.208 3.51 0.026* Comparative paws Stride length RF 4.855 0.049* 1.079 0.371 3.242 0.034* RH 7.452 0.019* 0.848 0.477 3.058 0.041* LF 2.582 0.136 0.436 0.728 1.704 0.185 LH 7.228 0.021* 0.799 0.503 3.05 0.042* Step cycle RF 9.026 0.012* 1.465 0.242 2.146 0.113 RH 9.658 0.01** 2.3 0.095 2.621 0.067 LF 8.684 0.013* 2.375 0.087 2.432 0.082 LH 6.571 0.026* 1.987 0.135 2.914 0.048* Duty cycle RF 4.166 0.066 4.305 0.011* 3.91 0.017* RH 1.296 0.279 2.794 0.055 1.252 0.306 LF 2.713 0.127 1.936 0.143 0.601 0.618 LH 1.164 0.334 0.197 0.659 1.224 0.312 Base of support Forepaw 3.547 0.086 1.319 0.284 1.584 0.211 Hindpaw 3.73 0.079 5.626 0.003** 3.785 0.019* Interlimb Coordination Regularity index 0.124 0.730 0.123 0.945 0.146 0.931 Phase dispersion (Diagonal) LF → RH 1.842 0.202 0.634 0.598 0.219 0.881 RF → LH 3.84 0.075 0.949 0.428 0.391 0.759 Individual paw

Stride length RF 4.855 0.049* 1.079 0.371 3.242 0.034* RH 7.452 0.019* 0.848 0.477 3.058 0.041* LF 2.582 0.136 0.436 0.728 1.704 0.185 LH 7.228 0.021* 0.799 0.503 3.05 0.042* Step cycle RF 9.026 0.012* 1.465 0.242 2.146 0.113 RH 9.658 0.01** 2.3 0.095 2.621 0.067 LF 8.684 0.013* 2.375 0.087 2.432 0.082 LH 6.571 0.026* 1.987 0.135 2.914 0.048* Duty cycle RF 4.166 0.066 4.305 0.011* 3.91 0.017* RH 1.296 0.279 2.794 0.055 1.252 0.306 LF 2.713 0.127 1.936 0.143 0.601 0.618 LH 1.164 0.334 0.197 0.659 1.224 0.312 Base of support Forepaw 3.547 0.086 1.319 0.284 1.584 0.211 Hindpaw 3.73 0.079 5.626 0.003** 3.785 0.019* Interlimb Coordination Regularity index 0.124 0.730 0.123 0.945 0.146 0.931 Phase dispersion (Diagonal) LF → RH 1.842 0.202 0.634 0.598 0.219 0.881 RF → LH 3.84 0.075 0.949 0.428 0.391 0.759 Individual paw Max contact area RF 0.737 0.408 5.338 0.004** 0.186 0.905 RH 0.807 0.388 6.443 <0.001*** 0.767 0.520 LF 0.179 0.680 3.397 0.029* 0.185 0.905 LH 2.558 0.138 10.29 <0.0001*** 0.515 0.674 Max intensity RF 0.377 0.551 6.462 <0.001*** 1.786 0.169 RH 2.694 0.129 3.256 0.033* 0.102 0.958 LF 0.3162 0.585 3.479 0.026* 0.883 0.459 LH 0.271 0.612 6.399 <0.001*** 0.408 0.748 RF: right forepaw; LF: left forepaw; RH: right hindpaw; LH: left hindpaw. *P < 0.05. **P < 0.01. ***P < 0.001. Table 2 Relationships between body weight and speed with gait parameters. Body weight   Speed Sham MCAO Sham MCAO P cc P value P cc P value P cc P value P cc

Max contact area RF 0.737 0.408 5.338 0.004** 0.186 0.905 RH 0.807 0.388 6.443 <0.001*** 0.767 0.520 LF 0.179 0.680 3.397 0.029* 0.185 0.905 LH 2.558 0.138 10.29 <0.0001*** 0.515 0.674 Max intensity RF 0.377 0.551 6.462 <0.001*** 1.786 0.169 RH 2.694 0.129 3.256 0.033* 0.102 0.958 LF 0.3162 0.585 3.479 0.026* 0.883 0.459 LH 0.271 0.612 6.399 <0.001*** 0.408 0.748 RF: right forepaw; LF: left forepaw; RH: right hindpaw; LH: left hindpaw. *P < 0.05. **P < 0.01. ***P < 0.001. Table 2 Relationships between body weight and speed with gait parameters. Body weight   Speed Sham MCAO Sham MCAO P cc P value P cc P value P cc P value P cc P value Infarct volume — — −0.85 0.015* Body weight −0.01 0.95 −0.02 0.90 Run duration         Stride length LF −0.04 0.83 0.08 0.68  LF 0.86 <0.0001*** 0.77 <0.0001*** RF −0.05 0.84 0.07 0.65  RF 0.73 <0.0001*** 0.75 <0.0001*** Swing speed         Stance LF 0.50 0.012* 0.09 0.62  LF −0.92 <0.0001*** −0.87 <0.0001*** RF 0.42 0.042* 0.10 0.61  RF −0.93 <0.0001*** −0.87 <0.0001*** Max intesity         Max contact area LF 0.42 0.042* 0.20 0.28  LF −0.03 0.88 −0.49 0.0086** RF 0.40 0.049* 0.23 0.23  RF −0.24 0.26 −0.49 0.0083** Max contact area         Max intensity LF 0.45 0.02* 0.40 0.05*  LF 0.18 0.40 −0.33 0.09 RF 0.49 0.014* 0.46 0.012*  RF −0.19 0.36 −0.20 0.30 Print width         Print width LF 0.53 0.007** 0.54 0.003**  LF 0.33 0.12 0.11 0.59 RF 0.38 0.07 0.58 0.0013**  RF 0.05 0.80 0.10 0.63 Toe spread         Toe spread LF 0.62 0.008** 0.53 0.0005***  LF 0.18 0.40 0.11 0.59 RF 0.40 0.05* 0.56 0.002**  RF 0.31 0.14 0.27 0.17 Stride length         Base of support LF 0.10 0.62 0.11 0.56  Forelimb 0.08 0.71 0.15 0.46 RF 0.18 0.40 0.21 0.29  Hindlimb 0.08 0.70 0.28 0.15 RF: right forepaw; LF: left forepaw.

.63 Toe spread         Toe spread LF 0.62 0.008** 0.53 0.0005***  LF 0.18 0.40 0.11 0.59 RF 0.40 0.05* 0.56 0.002**  RF 0.31 0.14 0.27 0.17 Stride length         Base of support LF 0.10 0.62 0.11 0.56  Forelimb 0.08 0.71 0.15 0.46 RF 0.18 0.40 0.21 0.29  Hindlimb 0.08 0.70 0.28 0.15 RF: right forepaw; LF: left forepaw. *P < 0.05. **P < 0.01. ***P < 0.001.

1. Introduction Stroke represents the third most common cause of death in developed countries, following only coronary heart diseases and cancer [1]. It is frequently associated with higher risk for a wide range of physical and neuropsychological consequences [2, 3]. Although the importance of poststroke psychiatric comorbidity is currently well documented, it had been previously underestimated [4]. In the 1970s, the identification of mood disorders, especially depression, as specific complications following stroke introduced the concept that clinical depression after stroke could be an organic consequence of the brain damage rather than an understandable psychological reaction to motor disability [5, 6]. Since then, research on depression after stroke has gained momentum [7].

epression, as specific complications following stroke introduced the concept that clinical depression after stroke could be an organic consequence of the brain damage rather than an understandable psychological reaction to motor disability [5, 6]. Since then, research on depression after stroke has gained momentum [7]. However, despite the large bulk of the literature which has been published on this topic, there is still uncertainty in relation to depression after stroke prevalence, etiology, and management. Although the risk of all depressive disorders was reported ranging from 25% to 79% among people suffering from a stroke [21], poststroke major depression prevalence ranged from 3% to 40% [22]. Data available from 51 studies that have been run between 1977 and 2002 confirmed that depressive symptoms were assessed in 33% (29–36%) among all stroke survivors at any time during followup [23]. Similar estimates were described by more recent studies [24, 25]. Variations in depression after stroke prevalence rates across studies seem to arise from differences in criteria that are used to define depression, stroke and patients' characteristics and timing of mood detection, as well as the complexity in recognition, assessment, and diagnosis of this disorder in post-stroke settings [23]. Risk of inappropriate diagnosis is high [26, 27] because of the difficulties in the assessment of mood abnormalities in patients with neurological deficits, particularly associated with dysphasia and dementia, who often experience many concurrent and “overlapping” somatic, cognitive, and affective symptoms [28].

[23]. Risk of inappropriate diagnosis is high [26, 27] because of the difficulties in the assessment of mood abnormalities in patients with neurological deficits, particularly associated with dysphasia and dementia, who often experience many concurrent and “overlapping” somatic, cognitive, and affective symptoms [28]. However, early screening and diagnosis of depressed mood might be relevant, since depression after stroke is related to poorer outcomes. Based on main available evidence, together with worse long term functional outcomes [29], depression after stroke is associated with reduction in rehabilitation treatment efficacy [30], limitations in daily living activities [29, 31], cognitive impairment [32, 33], and a higher risk of recurrent stroke [34]. Furthermore, depression after stroke was reported to be related to a high mortality risk [35]. Some reviews examined prevalence rates and clinical correlates of depression [7, 23, 36], but data on the impact of depression after stroke on survival need to be clarified and systematically analyzed. Therefore, we performed a systematic review and meta-analysis in order to explore the relationship between depression and subsequent mortality in the poststroke population. 2. Methods The present paper was conducted according to the Meta-Analyses of Observational Studies in Epidemiology (MOOSE) guidelines [37].

However, early screening and diagnosis of depressed mood might be relevant, since depression after stroke is related to poorer outcomes. Based on main available evidence, together with worse long term functional outcomes [29], depression after stroke is associated with reduction in rehabilitation treatment efficacy [30], limitations in daily living activities [29, 31], cognitive impairment [32, 33], and a higher risk of recurrent stroke [34]. Furthermore, depression after stroke was reported to be related to a high mortality risk [35]. Some reviews examined prevalence rates and clinical correlates of depression [7, 23, 36], but data on the impact of depression after stroke on survival need to be clarified and systematically analyzed. Therefore, we performed a systematic review and meta-analysis in order to explore the relationship between depression and subsequent mortality in the poststroke population. 2. Methods The present paper was conducted according to the Meta-Analyses of Observational Studies in Epidemiology (MOOSE) guidelines [37]. 2.1. Search Strategy We used PubMed (date range: January, 1, 1990 to November, 25, 2012) and Web of Science (date range: January, 1, 2002 to November, 25, 2012) electronic databases for search purposes. No restrictions of language were set. We used the following terms for the PubMed search strategy: (1) “Depression” [Mesh]; (2) “depression” [all fields]; (3) “Stroke” [Mesh]; (4) “post-stroke” [title/abstract]; (5) “post stroke” [title/abstract]; (6) “Mortality” [Mesh]; (7) mortal* [title/abstract]; and (8) death* [title/abstract]. We combined the terms as follows: (1 or 2) and (3 or 4 or 5) and (6 or 7 or 8). A similar search strategy was used for Web of Science database, combining the following topic terms: (1) depress*; (2) mood*; (3) affective*; (4) stroke*; (5) post-stroke*; (6) poststroke*; (7) mortal*; and (8) death*. The search phrase was built as follows: (1 or 2 or 3) and (4 or 5 or 6) and (7 or 8).

(6 or 7 or 8). A similar search strategy was used for Web of Science database, combining the following topic terms: (1) depress*; (2) mood*; (3) affective*; (4) stroke*; (5) post-stroke*; (6) poststroke*; (7) mortal*; and (8) death*. The search phrase was built as follows: (1 or 2 or 3) and (4 or 5 or 6) and (7 or 8). 2.2. Eligibility Criteria We included studies with the following characteristics:assessment of depression in a sample of people suffering from a previous stroke; estimation of the association between depression after stroke at baseline and subsequent mortality at followup; additional available data on mortality also in a comparison group suffering from stroke but without depression. Exclusion criteria were based on the following: depression diagnosed exclusively before stroke admission; results shown as continuous or quantitative scores based on psychometric scales without any dichotomization around a standardized cut-off value for depression; data replicated in multiple works whose inclusion would involve duplication of data. 2.3. Data Collection Process A preliminary screening (reading of titles and, if needed, of abstracts) was performed in order to include all potentially relevant articles. After the first screening, the final eligibility was assessed retrieving papers in full text. Two investigators (F. Bartoli and N. Lillia) independently performed both first and final screenings of papers. When differences of opinion between reviewers occurred, these were resolved by discussion with a third member (A. Lax) of the research team, and consensus was thereby reached.

retrieving papers in full text. Two investigators (F. Bartoli and N. Lillia) independently performed both first and final screenings of papers. When differences of opinion between reviewers occurred, these were resolved by discussion with a third member (A. Lax) of the research team, and consensus was thereby reached. 2.4. Data Extraction We developed a specific data extraction sheet. One author (F. Bartoli) extracted data from the included studies and another (C. Crocamo) checked the accuracy for the inclusion in statistical software. Any disagreement was resolved by discussion with the other authors. We extracted the following information from each included study: year of publication, country, study design, sample size, depression definitions and measures, duration of followup, reported association measure (e.g., hazard ratio, relative risk), main results. When there was any uncertainty about the data, we contacted the corresponding author for clarification. We collected also information suitable for a basic quality evaluation of studies included, based on the comparability between exposed and nonexposed groups, the risk of selection bias, the evaluation of representativeness of recruited samples, and the reliability of depression assessment.

ding author for clarification. We collected also information suitable for a basic quality evaluation of studies included, based on the comparability between exposed and nonexposed groups, the risk of selection bias, the evaluation of representativeness of recruited samples, and the reliability of depression assessment. 2.5. Data Analysis We analyzed data using the Review Manager (RevMan) 5.1 software [38] and STATA statistical software package, version 10 [39]. For articles providing both major and minor depression data, we analyzed only specific data of subgroups with major depression, excluding patients with minor depression. For studies showing results at different followup periods, we included just the results at the longer followup. We performed two different pooled analyses based on two different association measures, odds ratio (OR) and hazard ratio (HR) with related 95% confidence intervals, according to the available data from the included papers. The HR is commonly used in the medical literature when describing survival data, and it is defined as the estimate of the ratio of the probability that if the analyzed event has not already occurred, it will occur in the next time interval, divided by the length of that interval in the index group versus the control group. HR is the risk at any instance of followup, whereas OR quantifies the association at the end of followup. For the HRs, we estimated log hazard ratios and standard errors obtained from Cox proportional hazards regression models. Results were summarized using conventional forest plots. Random-effects models for estimating pooled effects were considered preferable rather than fixed-effect models because high variability across the included studies was expected, for example, in relation to followup duration, recruitment inclusion/exclusion criteria, setting, and depression after stroke definition. We performed subgroups analysis based on the followup duration of the included studies. The results are structured by three followup periods, short term (<2 years), medium term (2–5 years), and long term (>5 years). We performed sensitivity analyses excluding studies with potential methodological issues based on the low representativeness of recruited samples or on the lack of reliability of methods assessing depression. The presence and the level of heterogeneity were assessed using Q test and I2 statistic, respectively. A funnel plot was created in order to visually inspect the risk of publication bias.

gical issues based on the low representativeness of recruited samples or on the lack of reliability of methods assessing depression. The presence and the level of heterogeneity were assessed using Q test and I2 statistic, respectively. A funnel plot was created in order to visually inspect the risk of publication bias. We performed Egger's test for the statistical estimation of publication bias. 3. Results 3.1. Study Selection 261 and 769 records were generated from PubMed and Web of Science databases, respectively. The preliminary screening based on titles and, where needed, abstracts identified 40 papers as potentially relevant. These papers were retrieved in full text. Among these, 27 were excluded because they did not meet the inclusion criteria. Detailed reasons for ineligibility are shown in flow diagram (Figure 1). 13 studies [8–20] were included for meta-analysis, all had data suitable for pooled estimation of the OR and four for pooled analysis of the HR mortality. 3.2. Study Characteristics All articles were in English. The year of publication ranged from 1993 to 2011. Seven papers were from USA, two from Australia, and four from Europe. There is a high variability among studies in terms of study design, sample size, male/female ratio, methods and time of detection of depression, and followup length. The followup period ranged between 12 months and 10 years. Detailed characteristics of the included papers are described in Table 1.

and four from Europe. There is a high variability among studies in terms of study design, sample size, male/female ratio, methods and time of detection of depression, and followup length. The followup period ranged between 12 months and 10 years. Detailed characteristics of the included papers are described in Table 1. 3.3. Odds Ratio of Mortality among People with Depression after Stroke The studies (n.13) involved 59,598 subjects: 6,052 with depression after stroke and 53,546 from comparison groups. 1,544 cases of death at followup were detected from depression after stroke sample and 18,216 from people without depression. The pooled OR (95% CI) for mortality at followup in people with depression was 1.22 (1.02–1.47) (Figure 2). Heterogeneity was high (χ2 = 28.59; P = 0.005; I2 = 58%). According to subgroups analyses, a statistically significant association between depression after stroke and mortality was found exclusively for studies with medium term followup. However, this result was influenced by the large size study by Williams and colleagues [20] that accounts for most of the weight of the overall effect. Funnel plot for publication bias is shown in Figure 3. Egger's test was not statistically significant (coefficients = 0.68 (−0.53–1.90); P = 0.241). 3.4. Hazard Ratio of Mortality among People with Depression after Stroke The pooled HR (95% CI) for mortality at followup in people with depression after stroke was 1.52 (1.02–2.26) (Figure 4). Test for heterogeneity showed an I2 = 53% (χ2 = 6.42; P = 0.09).

Funnel plot for publication bias is shown in Figure 3. Egger's test was not statistically significant (coefficients = 0.68 (−0.53–1.90); P = 0.241). 3.4. Hazard Ratio of Mortality among People with Depression after Stroke The pooled HR (95% CI) for mortality at followup in people with depression after stroke was 1.52 (1.02–2.26) (Figure 4). Test for heterogeneity showed an I2 = 53% (χ2 = 6.42; P = 0.09). 3.5. Quality Assessment and Sensitivity Analyses There was no risk of poor comparability between depressed and not depressed subjects, since all studies recruited both exposed and nonexposed cohorts from the same sources. However, most of the included papers were prone to some methodological issues and potential risk of bias. In one study [9], the presence or the absence of a history of stroke in the selected population was ascertained on the basis of self-report information rather than on clinical diagnosis or medical records, introducing a potential risk of selection bias. Furthermore, data from two studies [10, 12] were based on participants of clinical trials, who, unlike those derived from purely observational studies, may not be equally representatives of the reference population. The difference between individuals allocated for receiving a specific treatment and individuals treated with placebo or other active controls might actually introduce a potential performance bias. Finally, three studies [11, 18, 20] investigated the effect of depression after stroke in special populations (veterans), not representatives of general population suffering from stroke. The sensitivity analysis excluding the studies based on samples of veterans [11, 18, 20], individuals recruited from clinical trials [10, 12], and self-report stroke patients [9] highlighted a pooled OR of 1.61 (1.01–2.55).

special populations (veterans), not representatives of general population suffering from stroke. The sensitivity analysis excluding the studies based on samples of veterans [11, 18, 20], individuals recruited from clinical trials [10, 12], and self-report stroke patients [9] highlighted a pooled OR of 1.61 (1.01–2.55). As regards the methods to assess depression after stroke, we found several quality issues. Five studies [8, 11, 13, 18, 20] collected relevant information from clinical records and/or administrative data. These sources may have lower sensitivity than rating scales or structured clinical interviews, since the risk of an inadequate diagnosis of depression after stroke in clinical practice is often high [40]. Furthermore, eight studies [9, 10, 12, 14–17, 19] used psychometric scales (e.g., Hospital Anxiety and Depression Scale; Hamilton Rating Scale for Depression) to evaluate depressive symptoms. Among these, four studies [10, 12, 14, 15] combined the psychometric evaluation with structured (Composite International Diagnostic Interview) or semistructured (Present State Examination) interviews to assess depression. However, the specific risk of developing the mortality outcome among individuals suffering from major depression, and not from other depressive disorders (e.g., “minor” depression or dysthymia), was available from just three studies (two out of these were based on the DSM-III criteria) [10, 14, 15]. The sensitivity analysis including only these studies showed a pooled OR of 2.75 (1.14–6.65).

iduals suffering from major depression, and not from other depressive disorders (e.g., “minor” depression or dysthymia), was available from just three studies (two out of these were based on the DSM-III criteria) [10, 14, 15]. The sensitivity analysis including only these studies showed a pooled OR of 2.75 (1.14–6.65). 4. Discussion 4.1. Main Findings This paper on the mortality risk in subjects suffering from depression after stroke identified 13 studies with data suitable for meta-analysis. There are three main findings. First, suffering from depression after stroke has an important bearing on the chances of death, with an OR of 1.22 (1.02–1.47) (95% CI). The sensitivity analyses based on specific characteristics of recruited samples and on the methods to detect depression confirm the statistical significance of the relationship between baseline depression and risk of subsequent mortality among people with stroke.

ances of death, with an OR of 1.22 (1.02–1.47) (95% CI). The sensitivity analyses based on specific characteristics of recruited samples and on the methods to detect depression confirm the statistical significance of the relationship between baseline depression and risk of subsequent mortality among people with stroke. Second, the relationship between depression and mortality seems to be related to the duration of observation. Subgroup analysis of short term studies (<2 years) did not show a statistically significant association between depression after stroke and mortality, whereas subgroup analysis of long term studies (>5 years) showed some trend. On the other hand, subgroup analysis of medium term studies (range: 2–5 years) showed results above the threshold of statistical significance. It should be noted that the studies with followups longer than 5 years, actually considered really long intervals, ranging from 7 to 10 years. Therefore, we can assume that the burden of depression on the risk of mortality among stroke patients emerges only after 2–5 years from the index stroke, whereas, afterwards, this difference may be mitigated by the aging of stroke survivors.

years, actually considered really long intervals, ranging from 7 to 10 years. Therefore, we can assume that the burden of depression on the risk of mortality among stroke patients emerges only after 2–5 years from the index stroke, whereas, afterwards, this difference may be mitigated by the aging of stroke survivors. However, we need to point out that there are some alternative explanations to this result. Long term followup subgroup included studies for an overall size of 362 patients suffering from depression, so the lack of a statistical significant association may be due to the small sample sizes of the included papers. Furthermore, the study of Jorge and colleagues [12], a placebo-controlled trial of antidepressants with prospective design, represents an outlier within the long term followup subgroup (OR: 0.74 (0.34–1.61)) that may have consistently influenced the pooled OR in this subgroup. Lastly, we need to highlight that the study of Williams and colleagues [20] on a National Cohort of Veterans hospitalized following an ischemic stroke accounted for a large proportion of the overall weight of medium term followup subgroup analysis. Therefore, the overall effect of medium term subgroup is consistently influenced by this large size study. Furthermore, studies from this subgroup showed a high heterogeneity in terms of time of depression detection and several other characteristics, for example, recruited population and sample size. Paolucci and colleagues [17] evaluated depression within the first nine months after stroke. Both House et al. [10] and Willey et al. [19] assessed depression up to 30 days after stroke, whereas Williams et al. [20] collected information on depression in the first 3 years, excluding those who die within 30 days after stroke. Therefore, it is important to highlight the low degree of comparability between studies. Further studies exploring the influence of duration of followup on the depression mortality after stroke association are probably needed.

tion on depression in the first 3 years, excluding those who die within 30 days after stroke. Therefore, it is important to highlight the low degree of comparability between studies. Further studies exploring the influence of duration of followup on the depression mortality after stroke association are probably needed. Finally, also the HR pooled analysis showed a significant relationship between depression after stroke and mortality. There was a high level of heterogeneity among the included studies in terms of results, recruited population, sample size, and duration of followup. However, the lack of included papers for this analysis did not allow for better exploring the variations of HR through subgroups or sensitivity analyses. It should be noted that the study of Williams and colleagues [20] explored the HR of mortality, but using adjusted measures not suitable for our meta-analysis. The study found a significant HR (HR = 1.13; 95% CI: 1.06–1.21). This result is in line with the overall effect of our meta-analysis and might be taken in consideration when we consider the HR of mortality in subjects suffering from both stroke and depression.

using adjusted measures not suitable for our meta-analysis. The study found a significant HR (HR = 1.13; 95% CI: 1.06–1.21). This result is in line with the overall effect of our meta-analysis and might be taken in consideration when we consider the HR of mortality in subjects suffering from both stroke and depression. 4.2. Strengths and Limitations To our knowledge, this is the first meta-analysis that systematically synthesizes data from studies comparing the mortality among individuals with and without depression after a stroke. The well-known advantage of a meta-analysis of observational studies is that it allows the synthesis of the results of a large amount of studies, providing findings more robust than those deriving from data of individual studies.

dies comparing the mortality among individuals with and without depression after a stroke. The well-known advantage of a meta-analysis of observational studies is that it allows the synthesis of the results of a large amount of studies, providing findings more robust than those deriving from data of individual studies. Observational studies are an important source in epidemiological research, but they are prone to many methodological issues [41, 42]. Therefore, we paid critical attention to the quality of evaluated papers. According to quality assessment, we found methodological issues and potential risk of bias related to selection of sample or depression assessment in some studies. However, the sensitivity analyses did not show significantly different results than the overall pooled analysis. Therefore, lower quality related to poor level of representativeness of selected samples, either lack of reliability or specificity of methods to detect depression, did not seem consistently affecting and influencing the association between depression after stroke and subsequent mortality. However, we need to highlight the low degree of comparability between studies. Studies differ for several important characteristics, other than followup duration, such as study design, source of recruitment, and time of depression assessment.

ing the association between depression after stroke and subsequent mortality. However, we need to highlight the low degree of comparability between studies. Studies differ for several important characteristics, other than followup duration, such as study design, source of recruitment, and time of depression assessment. Our paper included only published studies with sufficient data, excluding conference abstracts because these often cannot give reliable information on patients' characteristics, inclusion criteria, exposure detection, and other relevant issues. Furthermore, hand searching and searching of the grey literature were not conducted, and possibly some relevant studies could not be included. Therefore, we need to consider the risk that an amount of negative or uncertain results remained unpublished or, at least, were not available from databases that we have explored. However, Egger's test showed the lack of risk of publication bias. 4.3. Clinical Perspectives The nature of the relationship between depression and mortality remains unknown. Depression may affect prognosis and risk of mortality after stroke because stroke patients suffering from depression may be less compliant to treatment. When mental health disorders cooccur with other medical conditions, this cooccurrence tends to reduce adherence to interventions [43, 44].

d mortality remains unknown. Depression may affect prognosis and risk of mortality after stroke because stroke patients suffering from depression may be less compliant to treatment. When mental health disorders cooccur with other medical conditions, this cooccurrence tends to reduce adherence to interventions [43, 44]. On the other hand, the relationship may be explained by the fact that depression could be more frequent in people vulnerable to physical disability and a higher stroke severity [45]. In that case, depression may be not an independent factor, but simply a mediator variable for severe physical damage related to a higher likelihood of mortality. Emerging line of evidence highlighted that the relationship between depression and stroke and other severe illnesses, for example, myocardial infarction, heart disease, and cancer, is bidirectional and, at least in part, is driven by several biological processes, including immune dysregulation [46]. Depression may lead to dysregulation of immunologic mechanisms, coagulation abnormalities, and vascular endothelial dysfunction, which are associated with an increased risk of cardiovascular disease and mortality [47].

nd, at least in part, is driven by several biological processes, including immune dysregulation [46]. Depression may lead to dysregulation of immunologic mechanisms, coagulation abnormalities, and vascular endothelial dysfunction, which are associated with an increased risk of cardiovascular disease and mortality [47]. Clinicians should regularly assess symptoms of depression in people who report a stroke in their clinical history. Sensitivity and specificity of assessment and screening of depression among people with stroke represent often an important issue. Frequently, a mood disorder remains undetected and, therefore, undertreated [28, 48]. Depressive disorders among patients with anosognosia, neglect, or aprosody, who deny symptoms of depression, may also be underdiagnosed, although symptoms related to specific physical disease, such as changes in appetite or insomnia, may be overestimated by clinicians [26].

tected and, therefore, undertreated [28, 48]. Depressive disorders among patients with anosognosia, neglect, or aprosody, who deny symptoms of depression, may also be underdiagnosed, although symptoms related to specific physical disease, such as changes in appetite or insomnia, may be overestimated by clinicians [26]. Equally, treatment of depression is often complicated as people with stroke are often more prone to side effects and interactions among different drugs rather than general population [40], likewise similar comorbidities in mental health disorders [44]. Main data on drugs therapy showed the importance of antidepressant medications, particularly with SSRI, as this may improve not only the life expectancy of poststroke patients but also their quality of life. Trials are limited and focused mainly on antidepressant agents such as Fluoxetine [49, 50], Citalopram [51, 52], and Reboxetine [52, 53]. Systematic reviews found that antidepressants usage may reduce symptoms of depression, but it also pointed out that clinicians should use these drugs with caution in people with persistent depression, as little is known about the risks, especially of seizures, falls, and delirium [54]. Furthermore, there was no clear effect of pharmacological therapy on the prevention of depression after stroke [55].

on, but it also pointed out that clinicians should use these drugs with caution in people with persistent depression, as little is known about the risks, especially of seizures, falls, and delirium [54]. Furthermore, there was no clear effect of pharmacological therapy on the prevention of depression after stroke [55]. 4.4. Conclusions Despite some limitations, the results of this meta-analysis confirm the potential role of depression on poststroke mortality. Regular screening might help in detecting prevalent cases [48]. Further research is needed in order to clarify the nature of depression poststroke/mortality association and related pathophysiological processes. Secondly, effectiveness of pharmacotherapy and psychotherapy for preventing and treating depression after stroke should be explored. Before any recommendation on their routine use, given the well-known implementation issues of the even more robust guidelines [56], further randomized controlled trials are needed to estimate effectiveness of antidepressants for depression treatment and their potential benefits in terms of life expectancy. Conflict of Interests The authors declared that they have no conflict of interests. Acknowledgments The authors thank Allan House, Huanguang Jia, Claudia Kemper, Peter Knapp, Halvor Naess, Joshua Willey, and Linda Williams for providing them important information and for clarification on relevant data in their articles, and they all also thank the authors of the included papers. Figure 1 Flowchart: search results and excluded/included studies.

Acknowledgments The authors thank Allan House, Huanguang Jia, Claudia Kemper, Peter Knapp, Halvor Naess, Joshua Willey, and Linda Williams for providing them important information and for clarification on relevant data in their articles, and they all also thank the authors of the included papers. Figure 1 Flowchart: search results and excluded/included studies. Figure 2 OR of mortality among subjects with depression after stroke. Figure 3 Funnel Plot. Egger's test: bias = 0.68 (−0.53–1.90); P = 0.241. Figure 4 HR of mortality among subjects with depression after stroke. Table 1 Articles suitable for meta-analysis. Study Country Participants Recruitment Depression after stroke assessment Followup Reported results on mortality Almeida and Xiao, 2007 [8] Australia 574 males 55% Patients with first-ever diagnosis of stroke from January to December 1990 First diagnosis of ICD-9 and ICD-10 depressive disorders recorded during the 24 months following the stroke 10 years RR: 1.72 (0.98–3.01) HR: 1.26 (0.71–2.23) (depression versus control without mental disorder) Ellis et al., 2010 [9] USA 124 males 48% Participants between 25 and 74 years with stroke diagnosis from NHANES I Epidemiologic Followup Study (NHEFS) interviewed in 1982 Center for Epidemiologic Studies Depression Scale (CES-D) ≥ 16 8 years Mortality rate (per 1000): 105.1 versus 84.7 (depression versus no depression)

Ellis et al., 2010 [9] USA 124 males 48% Participants between 25 and 74 years with stroke diagnosis from NHANES I Epidemiologic Followup Study (NHEFS) interviewed in 1982 Center for Epidemiologic Studies Depression Scale (CES-D) ≥ 16 8 years Mortality rate (per 1000): 105.1 versus 84.7 (depression versus no depression) House et al., 2001 [10] UK 448 males 54% Patients with definite clinical diagnosis of stroke (not subarachnoid hemorrhage) from a randomized controlled trial ICD-10 major depression at 1 month after stroke, according to Present State Examination 12 and 24 months OR at 12 months: 1.3 (0.65–2.7) (major depression) OR at 24 months: 1.7 (0.95–3.0) (major depression) Jia et al., 2006 [11] USA 5825 males 98% Patients with stroke diagnosis between October 2000 and September 2001 from a cohort of veterans, who survived 60 days or more after stroke, and with an index length of stay less than 365 days Depression (primary or secondary diagnosis) according to ICD-9 codes and antidepressant medication dispensing within 12 months of the index stroke 12 months Crude death rate: 11.0% versus 12.0% (depression versus no depression)

o survived 60 days or more after stroke, and with an index length of stay less than 365 days Depression (primary or secondary diagnosis) according to ICD-9 codes and antidepressant medication dispensing within 12 months of the index stroke 12 months Crude death rate: 11.0% versus 12.0% (depression versus no depression) Jorge et al., 2003 [12] USA 104 males Patients between ages 25 and 89 years with acute stroke within the previous 6 months, between June 1991 and June 1997 and from double-blind, placebo-controlled trial DSM-IV depression due to stroke, with “major depressive-like episode” or “minor depressive disorder,” according to the Present State Examination and Hamilton Depression Rating Scale 9 years Prevalence of mortality: 25/56 (45%) versus 25/48 (52%) (depression versus no depression) Kemper et al., 2011 [13] Germany 977 males 71% Patients aged 50 years and older with first ischemic stroke in 2005, without previous aphasia, dementia, depression, or nursing care dependency Diagnosis of depression within the year after stroke, according to ICD-10 codes 12 months after stroke Adjusted OR: 0.91 (0.55–1.52) (depression versus no depression)

males 71% Patients aged 50 years and older with first ischemic stroke in 2005, without previous aphasia, dementia, depression, or nursing care dependency Diagnosis of depression within the year after stroke, according to ICD-10 codes 12 months after stroke Adjusted OR: 0.91 (0.55–1.52) (depression versus no depression) Morris et al., 1993a [14] Australia 84 males 54% Patients with stroke undergoing rehabilitation consecutively enrolled from 1986 to 1987 examined approximately two months after stroke (mean 7.6 weeks) DSM-III major depression approximately 2 weeks after stroke according to Composite International Diagnostic Interview (CIDI) and Montgomery and Asberg Depression Rating Scale (MADRS) 15 months after the initial evaluation (mean 59 weeks) Prevalence of mortality: 3/13 (23%) versus 1/48 (2%) (major depression versus no depression) Morris et al., 1993b [15] USA 91 males: 59% Patients consecutively admitted to a university hospital stroke unit between 1979 and 1981 with either thromboembolic cerebral infarction or intracerebral hemorrhage DSM-III major depression 1–3 weeks after stroke according to Present State Examination and Hamilton Depression Rating Scale 10 years Prevalence of mortality: 26/37 (70%) versus 22/54 (41%) (major depression versus no depression)

en 1979 and 1981 with either thromboembolic cerebral infarction or intracerebral hemorrhage DSM-III major depression 1–3 weeks after stroke according to Present State Examination and Hamilton Depression Rating Scale 10 years Prevalence of mortality: 26/37 (70%) versus 22/54 (41%) (major depression versus no depression) Naess et al., 2010 [16] Norway 771 (376 returning questionnaire) males 60% Patients with acute stroke consecutively admitted to the Stroke Unit, Haukeland University Hospital, Norway, from February 2006 to November 2008 Hospital Anxiety and Depression Scale (HADS-D) ≥ 11 (at least 6 months after stroke) Mean followup: 382 days (range 185–756) HR: 4.4 (P = 0.002) (depression versus no depression) Paolucci et al., 2006 [17] Italy 1064 males 60% Patients with ischemic or hemorrhagic stroke (first or subsequent event) confirmed by neuroimaging (CT or MRI), consecutively admitted to one of the study centers between June 2000 and July 2001 (DESTRO study) Depression within the first 9 months after the stroke according to a Beck Depression Inventory (BDI) ≥ 10 2 years Prevalence of mortality: 5.48 % versus 4.85% (depression versus no depression) Ried et al., 2011 [18] USA 790 males 98% Patients with a stroke diagnosis between July 2000 and September 2001, from a cohort of veterans Major depressive disorder or depressive disorder NOS according to ICD-9 codes during the 12 months after stroke 7-year follow-up period (maximum follow-up time: 2465 days) HR: 1.28 (0.96 to 1.71) (depression versus no depression)

ts with a stroke diagnosis between July 2000 and September 2001, from a cohort of veterans Major depressive disorder or depressive disorder NOS according to ICD-9 codes during the 12 months after stroke 7-year follow-up period (maximum follow-up time: 2465 days) HR: 1.28 (0.96 to 1.71) (depression versus no depression) Willey et al., 2010 [19] USA 340 males 42% Patients with first-ever ischemic stroke between July 1993 and July 1997, aged >39 years (data deriving from the Northern Manhattan Stroke Study (NOMASS)) First question on the Hamilton Depression Rating Scale regarding their mood in the week after the onset of the stroke (assessment within 30 days of their stroke) 5 years from initial stroke Adjusted HR: 1.15 (0.76–1.75) (depression versus no depression) Williams et al., 2004 [20] USA 51119 males 98% Patients with a first ischemic stroke from a cohort of veterans who survived beyond 30 days afterward, from October 1990 to September 1998 Diagnosis of depression in the first 3 years after stroke according to ICD-9 codes 3 years after stroke Adjusted HR: 1.13 (1.06–1.21) (depression versus no depression)

1. Introduction miRNAs are approximately 20-nucleotide, single-stranded RNA molecules that target mRNA through partial complementarity and they can regulate gene expression through inhibition of translation or transcript degradation [1]. It is now predicted that 40% to 50% of mammalian mRNAs could be regulated at the translational level by miRNAs [2]. In mammals, specific miRNAs are known to control processes including development, neuronal cell fate, apoptosis, proliferation, adipocyte differentiation, hematopoiesis, and exocytosis as well as in diseases [3–5] and possibly neuronal disorders [6]. miRNA expression has been detected in stroke [2, 7], Alzheimer's disease [8], Parkinson's disease [9], Down's syndrome [10], and schizophrenia [11]. These miRNAs expression profiles may be as diagnostically useful as mRNA expression profiles [12]. In the nucleus, miRNAs are transcribed as hairpin clusters of primary miRNAs (pri-miRNAs; 5′-capped polyadenylated transcripts), which is converted to 70-nt stem loop structures (pre-miRNAs) by Drosha (a type-III RNase) in association with a cofactor Pasha (aka DiGeorge syndrome critical region gene 8) [13]. pre-miRNAs are transported from nucleus to cytosol by exportin-5 and acted on by another type-III RNase known as Dicer that deletes the terminal loop of pre-miRNAs to form mature miRNAs [14].

e-miRNAs) by Drosha (a type-III RNase) in association with a cofactor Pasha (aka DiGeorge syndrome critical region gene 8) [13]. pre-miRNAs are transported from nucleus to cytosol by exportin-5 and acted on by another type-III RNase known as Dicer that deletes the terminal loop of pre-miRNAs to form mature miRNAs [14]. 2. miRNA Expression and Its Functions in the Brain miRNAs serve important roles in the development and function of the brain [15–19]. Studies support that tissue-specific miRNAs contribute to establish and maintain protein expression profiles underlying distinct cellular phenotypes. The discovery of seven brain-specific miRNAs (miR-9, miR-124a, miR-124b, miR-135, miR-153, miR-183, and miR-219) in mouse and human differentiating neurons implicated these miRNAs as effectors in mammalian neuronal processes [20]. Further studies showed that expression levels of the brain-specific miR-124 are 100 times higher in mouse central nervous system than in other organs, whereas levels of muscle-specific miR-1 are 100 to 1000 times lower in mouse central nervous system than in heart and skeletal muscles [21]. Transfection of brain-specific miR-124 into HeLa cells shifted the expression profile toward that of the brain's, whereas transfection of the heart and skeletal muscle-specific miR-1 into HeLa cells shifted the expression profile toward that of the muscle's [22]. Among neural-derived cells, integrated mRNA-miRNA functional analyses of mature neurons (MNs), neural progenitor cells (NPCs), and neuroblastoma cells (NBCs) revealed that several very highly expressed genes (e.g., Robo1, Nrp1, Epha3, Unc5c, Dcc, Pak3, and Limk4) and a few underexpressed miRNAs (e.g., miR-152, miR-146b, and miR-339-5p) in MNs are associated with one important cellular process-axon guidance; some very highly expressed mitogenic pathway genes (e.g., Map2k1, Igf1r, Rara, and Runx1) and underexpressed miRNAs (e.g., miR-370, miR-9, and miR-672) in NBCs are associated with cancer pathways [23].

NAs (e.g., miR-152, miR-146b, and miR-339-5p) in MNs are associated with one important cellular process-axon guidance; some very highly expressed mitogenic pathway genes (e.g., Map2k1, Igf1r, Rara, and Runx1) and underexpressed miRNAs (e.g., miR-370, miR-9, and miR-672) in NBCs are associated with cancer pathways [23]. 2.1. The Function of miRNAs in Cerebral Ischemia Several reports have demonstrated the effects of specific miRNAs in neuronal differentiation, neurogenesis, neural cell specification, and neurodevelopmental function [6, 24]. In stroke etiology, miRNAs have distinct expression patterns that modulate pathogenic processes, including atherosclerosis (miR-21 and miR-126), hyperlipidemia (miR-33 and miR-125a-5p), hypertension (miR-155), and plaque rupture (miR-222 and miR-210) [25]. miRNA profiling (screening) was performed on rat brains subjected to middle cerebral artery occlusion (MCAO) and reperfusion for 24 or 48 hours. They identified the expression of 114 miRNAs in ischemic brain samples. Among them, 106 and 82 transcripts were detected in the 24-hour and 48-hour reperfusion brain samples, respectively [2]. To understand miRNAs' functional significance in ischemic pathophysiology, Dharap et al. reported the level of miRNAs in adult rat brain as a function of reperfusion time after transient MCAO [7]. Of the 238 miRNAs evaluated, 8 showed increased expressions and 12 showed decreased ones at least at 4 out of 5 reperfusion time points studied between 3 hours and 3 days compared with sham [7]. The differentially expressed miRNAs and their protein kinase c-(PKC) isoform specific gene network in mouse brain after HPC (hypoxic pre-conditioning) and 6 h MCAO are determined [26]. Moreover, anti-miR-320a could bring about a reduction of infarct volume in cerebral ischemia with a concomitant increase in aquaporins-1 and 4 mRNA and protein expression [27]. Tan and colleagues carried out miRNA profiling from peripheral blood of young stroke patients aged 18–49 years, and identified characteristic patterns in ischemic stroke [28].

ing about a reduction of infarct volume in cerebral ischemia with a concomitant increase in aquaporins-1 and 4 mRNA and protein expression [27]. Tan and colleagues carried out miRNA profiling from peripheral blood of young stroke patients aged 18–49 years, and identified characteristic patterns in ischemic stroke [28]. 2.2. Neuroprotection miR-497 promoted ischemic neuronal death by repressing expression of Bcl-2 and Bcl-w, supporting the role of apoptosis in the pathogenesis of ischemic brain injury [29]. Knockdown of cerebral miR-497 in mice attenuated brain infarction, protected neuron, and improved neurological outcome after focal ischemia [29]. In rats subjected to transient cerebral ischemia, the brain-specific miR-134 and miR-124, involved in brain and neural tube development, respectively, are upregulated [2, 24, 30]. This process may be related to regeneration during the rest 24 hours of reperfusion in the injured brain cells. Anti-miR-1 treatment, as late as 4 hours following ischemia, significantly reduced cortical infarct volume in adult female rats, while anti-Let7 robustly reduced both cortical and striatal infarcts, and preserved sensorimotor function and interhemispheric neural integration. Antagomirs to miR-1 and Let7f, with consensus binding sites in the 3 UTRs of multiple IGF signaling pathway components confer neuroprotection, while antagomir to a brain-specific miRNA not associated with IGF signaling, was not neuroprotective [31]. Moreover, miR-34a was significantly upregulated at 1, 7, and 14 days after status epilepticus and at 2 months after temporal lobe epilepsy. Experiments with the miR-34a antagomir revealed that targeting miR-34a led to an inhibition of activated caspase-3 protein expression, which may contribute to increased neuronal survival and reduced neuronal death or apoptosis [32]. Besides, in astrocyte, miR-181 regulation of Bcl-2 and Mcl-1 contributes to mitochondrial dysfunction observed with in vitro ischemic injury, in this case glucose deprivation [33]. Increased miR-181a exacerbated injury both in vitro and in the mouse ischemia model [34].

val and reduced neuronal death or apoptosis [32]. Besides, in astrocyte, miR-181 regulation of Bcl-2 and Mcl-1 contributes to mitochondrial dysfunction observed with in vitro ischemic injury, in this case glucose deprivation [33]. Increased miR-181a exacerbated injury both in vitro and in the mouse ischemia model [34]. 2.3. Angiogenesis miR-126 is recognized as the most important miRNA for maintaining vascular integrity during ongoing angiogenesis, as it targets SPRED1 and PIK3R2, two negative regulators of VEGFs signaling [35]. Growth factors increase the expression of the proangiogenic miR-130a and miR-296 in endothelia cells [36]. miR-130a stimulates angiogenesis by inhibiting GAX and HOXA5, while, miR-296 acts through the inhibition of hepatocyte growth factor-(HGF-) regulated tyrosine kinase [36]. miR-210 is induced by hypoxia in endothelial cells [37]. miR-210 overexpression enhances the formation of capillary-like structures and VEGF-driven migration of normoxic endothelial cells, whereas inhibition of miR-210 decreases tube formation and migration [37]. The modulation of endothelial cell responses to hypoxia is mediated via the regulation of the receptor tyrosine-kinase ligand EphrinA3 [37]. Animal experiments demonstrated that miR-210 was elevated after one day of MCAO and gradually decreased after 7 and 14 days of MCAO [38]. miR-424 promotes angiogenesis by inhibiting cullin 2 (CUL2), thereby increasing HIF-1α levels [39]. Recently, the miR-23-27-24 cluster has also been reported to have a prominent role in angiogenesis [40]. miR-378 promotes angiogenesis by targeting tumor suppressor candidate 2 (Fus-1) and suppressor of fused (Sufu), thus inducing indirect upregulation of VEGF and angiopoietin-1/2 [41].

y increasing HIF-1α levels [39]. Recently, the miR-23-27-24 cluster has also been reported to have a prominent role in angiogenesis [40]. miR-378 promotes angiogenesis by targeting tumor suppressor candidate 2 (Fus-1) and suppressor of fused (Sufu), thus inducing indirect upregulation of VEGF and angiopoietin-1/2 [41]. 2.4. Remyelination In recent years, specific miRNAs such as miR-219, miR-138, miR-9, miR-23, and miR-19b have been found to participate in the regulation of oligodendrocyte differentiation and myelin maintenance, as well as in the pathogenesis of demyelination-related diseases (e.g., multiple sclerosis, ischemic stroke, and leukodystrophy) [42]. miR-19b, especially, plays essential roles in increasing the number of oligodendroglial cells [43]. The overexpression of miR-19b downregulates PTEN protein levels in Oligodendrocyte precursor cells (OPCs) by activating its downstream targets of the Akt signaling (PI3sOPCs) and the Akt signaling (PI3K/Akt/mTOR) pathway [44]. miR-19b increases the phosphorylation of Akt, but it does not affect its overall levels. The Akt1/2 kinase inhibitor cancels miR-19b-mediated OPC proliferation [43]. miR-145, -132, -200, and -182 are critical in the pathogenesis of ischemic stroke. The antagomir-mediated prevention of significantly upregulated miR-145 expression has been found to lead to an increased protein expression of its downstream target, superoxide dismutase-2 (SOD2), in the postischemic brain [45]. miR-132 regulates MeCP2 (methyl-CpG binding protein 2, also expressed in glial cells) expression, which is decreased in the preconditioned cortex. The down-regulation of miR-132 induces a rapid increase in the MeCP2 protein levels, but not the mRNA levels, in the mouse cortex [42]. The early activation of miR-200 family members improved neural cell survival via PHD2 mRNA silencing and subsequent HIF-1α (hypoxia-inducible factors-1α, a well-established transcription factor rapidly induced by hypoxia) stabilization [42].

n the MeCP2 protein levels, but not the mRNA levels, in the mouse cortex [42]. The early activation of miR-200 family members improved neural cell survival via PHD2 mRNA silencing and subsequent HIF-1α (hypoxia-inducible factors-1α, a well-established transcription factor rapidly induced by hypoxia) stabilization [42]. 3. miRNAs as Biomarkers of Cerebral Ischemia Serum/plasma miRNAs derived from various tissues/organs are stable and resistant to nuclease digestion as well as other harsh conditions, including boiling, low/high pH, extended storage, freeze-thaw cycles [46]. Expression levels of miRNAs in blood have been found to be reproducible and indicative of the disease state [46]. Furthermore, miRNAs also exist in other body fluids, including urine, tear, ascetic fluid, and amniotic fluid [46]. 20 and 25 miRNA transcripts were detected in the blood of MCAO rats reperfused for 24 and 48 hours, respectively. Transcripts that were common to both the blood and brain at 24-hour reperfusion included rno-miR-16, -23a, -103, -107, -150, -185, -191, -292-5p, -320, -451, -494, and let-7 (a, d, f, and i). miRNAs found at 48-hour reperfusion in both the blood and brain were miR-26a, -26b, -103, -107, -140*, -150, -185, -195, -191, -214, -320, -328, -352, -494, and let-7 (a, c, and i) [2].

at 24-hour reperfusion included rno-miR-16, -23a, -103, -107, -150, -185, -191, -292-5p, -320, -451, -494, and let-7 (a, d, f, and i). miRNAs found at 48-hour reperfusion in both the blood and brain were miR-26a, -26b, -103, -107, -140*, -150, -185, -195, -191, -214, -320, -328, -352, -494, and let-7 (a, c, and i) [2]. The correlation between blood and brain miR-210 in ischemic mice was positive. Compared to healthy controls, blood miRNA-210 was significantly decreased in stroke patients, especially at 7 days and 14 days of stroke onset. The cut off point of miR-210 in diagnosis was 0.505 with 88.3% sensitivity. MiR-210 level in stroke patients with good outcomes was significantly higher than patients with poor outcomes. Therefore blood miR-210 is a novel sensitive biomarker for clinical diagnosis and prognosis in acute cerebral ischemia [38]. miRNA profile of small artery (SA) stroke peripheral blood samples showed a distinctly different pattern from that of the large artery (LA) stroke samples [28]. Hence, the subtypes of stroke could be predicted using the microRNA profiling. miR-320 has been observed to be marginally down-regulated in all stroke patients with especially good outcome. The down-regulation of miR-320 could also lead to antiapoptotic processes [47] that could be useful in the restoration of normal cell or endovascular activities. Consequently it could be predictive of a favorable outcome via activation of angiogenesis in stroke patients [28].

roke patients with especially good outcome. The down-regulation of miR-320 could also lead to antiapoptotic processes [47] that could be useful in the restoration of normal cell or endovascular activities. Consequently it could be predictive of a favorable outcome via activation of angiogenesis in stroke patients [28]. 3.1. miRNAs as Emerging Therapeutic Targets in Ischemic Diseases miRNAs have been investigated as mediators of ischemic tissue damage. miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) and miR-182 family (miR-182, miR-183, and miR-96) were upregulated early after ischemic preconditioning. Among them miR-200b, miR-200c, and miR-429 targeted PHD2 and had the best neuroprotective effect [48]. In the ischemia of cardiac myocytes, miR-199a was acutely downregulated as early as 30 minutes after ischemia, leading to rapid upregulation of its target HIF-1α [49]. Angiopoietin-1 is a vascular strengthening factor during vascular development and a protective factor for pathological vascular inflammation and leakage. The TT genotype (rs2507800) in the 3′-UTR of angiopoietin-1 may reduce the risk of stroke by interfering with miR-211 binding [50]. 3.2. Clinical Implications and Problems With the growing evidence for the involvement and the regulatory function of miRNAs in many pathophysiological processes, these small regulatory RNAs are evolving as promising therapeutic targets [51]. Whereas miRNAs as an treatment application in cerebrovascular diseases remain experimental so far and several limitations need to be solved before clinical practice.

nd the regulatory function of miRNAs in many pathophysiological processes, these small regulatory RNAs are evolving as promising therapeutic targets [51]. Whereas miRNAs as an treatment application in cerebrovascular diseases remain experimental so far and several limitations need to be solved before clinical practice. 3.3. miRNA-Replacement Therapy After passing the cellular membrane, miRNA-mimicking oligonucleotides (miR-mimics) need to be integrated into the RNA-induced silencing complex [52] and induce translational inhibition of the degradation of their mRNA targets. Hence, possible chemical modifications that can increase the resistance to degradation of these oligonucleotide chemistries or facilitate their cellular uptakes are limited. Although overexpression of miRNAs by pre-miR-oligonucleotides or miR-mimics is a well-established method for the characterization of miRNA-function in vitro, only a few successful applications for the in vivo treatment of mammals exist so far [51]. 3.4. miRNA Inhibition AntagomiRs are single-stranded RNA molecules, modified by 2′-O-methyl and phosphorothioate substitution for stability. To enable cellular uptake, they are conjugated with cholesterols. These compounds can achieve significant miRNA knockdown [53] and have been used successfully to treat experimentally induced diseases in different organs and tissues [54, 55]. Clinical trials have successfully tested LNA-based drugs for the treatment of hepatitis C [56, 57] and several other LNA-based therapeutics are under development [58].

ieve significant miRNA knockdown [53] and have been used successfully to treat experimentally induced diseases in different organs and tissues [54, 55]. Clinical trials have successfully tested LNA-based drugs for the treatment of hepatitis C [56, 57] and several other LNA-based therapeutics are under development [58]. 4. Future Prospects Identification of specific miRNAs as key regulators of the response to ischemia has opened new clinical avenues. Figure 1 summarized the function of microRNAs in cerebral ischemia. Circulation miRNAs may be qualified as excellent non-invasive clinical biomarkers. During pathological processes, the expression of miRNAs is different in various cell types. Individual miRNAs can regulate the expression of multiple target genes, and manipulating miRNAs expression can influence an entire gene network and thereby modify complex disease pathologies [59]. Moreover, innovative strategies targeting miRNAs have been developed and could be applied in the treatment of ischemic diseases. Further studies on miRNAs are expected to shed new light in stroke therapy and management in the future. Figure 1 Regulation of miRNAs in ischemic stroke. Bracket indicates reference cited.

1. Background Between 20% and 60% of stroke patients are diagnosed with depression/anxiety [1], and these are often newly diagnosed in stroke patients both during hospitalization and up to 3 years after discharge [1–20]. Depression is associated with longer institutionalization and poorer rehabilitation outcomes [21, 22]. Further, depression is more often diagnosed for females and white stroke patients [23, 24], and it is correlated with higher rates of suicidal ideation and stroke mortality [25–27]. Depression increases the risk of stroke [28] as well as increased healthcare costs [29–34]. As these and other stroke related factors are evaluated, understanding their impact on healthcare cost is necessary for better management, improved therapeutic outcomes, and reduced healthcare cost. 2. Depression and Healthcare Cost Several studies have reported the effect of depression/anxiety on healthcare costs. For example, while female Medicare patients had a higher prevalence of depression and higher use of outpatient services, inpatient hospital costs for male patients were 47% higher compared to females ($15,060 versus $10,240, P < 0.001) [30]. In another study, the medical cost of depressed patients was 54% higher compared to nondepressed patients [34]. While higher cost among stroke patients is associated with greater number of readmissions, longer hospitalizations, and greater number of outpatient visits compared to a control group without depression, evidence is sparse about whether these costs vary by race and gender.

higher compared to nondepressed patients [34]. While higher cost among stroke patients is associated with greater number of readmissions, longer hospitalizations, and greater number of outpatient visits compared to a control group without depression, evidence is sparse about whether these costs vary by race and gender. In this study of Tennessee stroke patients (n = 17, 010), we examine two issues: (1) prevalence of depression among stroke patients by race and gender and (2) the effect of depression on total hospitalization cost in 2008 by race and gender.