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Introduction: Sirt1 is a NAD+ dependent deacetylase that is known as an important metabolic regulator for type 2 diabetes melitus. To determine the role of Sirt1 in ischemic stroke-induced metabolic imbalance, we generated the neuronal specific Sirt1 knock-in (nSirt1 KI) and knock-out (Sirt1 KO) mice and examined its effect and mechanism. Methods: To determine the role of Sirt1 in ischemic stroke-induced impaired metabolic actions including insulin and glucose abnormality, we tested the pharmacological effects of Metformin and Sirtinol as Sirt1 activator and inhibitor. To determine the genetic role of neuronal Sirt1 in the brain, we generated the nSirt1 KI mice and Sirt1 KO. We used the Ischemia/Reperfusion (I/R: 1h middle cerebral artery occlusion (MCAO)/23h reperfusion) injury model to induce the abnormal metabolic changes. Infarction volume was measured by TTC staining and we determined neurological deficit score (NDS) at 23h after 1h MCAO. The level of glucose and insulin were measured by a drop of tail blood using test-strip and ELISA Kit before, during (every 10 minutes interval), and after MCAO. Results: Metformin reduced infarction volume and Sirtinol worsened the infarction volume via Sirt1 signaling pathway. Heterozygous Sirt1 KO (Sirt1 +/-) mice showed decreased relative cerebral blood flow (rCBF) and worsen infarction volume in ischemic areas, whereas nSirt1 KI mice exhibited increased rCBF, improved NDS, regulating abnormal blood glucose and insulin, and reduced infarction volume by 42% compare to that of littermate control in I/R injury model (n=7, p Conclusion: These findings indicate that the important neuroprotective effect of neuronal cells is mediated by Sirt1 overexpression and suggest that upregulation of neuronal Sirt1 may be an important therapeutic target for ischemic stroke via regulating metabolic imbalance.

Introduction: The role of neuronal cell specific ROCK2 in ischemic stroke in vivo has been poorly investigated. We investigated the pharmacological effect of selective ROCK2 inhibition by selective ROCK2 inhibitor, KD025 and the genetic effect using neuronal specific ROCK2 knockout (ROCK2Eno2-/-) mice. Methods: Isoflurane-anesthetized littermate control (LC) and ROCK2Eno2-/- mice (20-22 g) underwent ischemia (2h middle cerebral artery occlusion (MCAO)/reperfusion (22h after 2h MCAO) injury model. An 8.0 nylon monofilament coated with a silicone was introduced into the left internal carotid artery up to the anterior cerebral artery. To assess the mechanism of ROCK2 deficiency in neuronal cells, we seeded hippocampal neuronal (HT22) cells in 6-well plates and transfected ROCK2 siRNA. Phosphorylated glycogen synthase kinase 3 beta (pGSK-3β) and Tau proteins which were involved in neuroprotection were measured by western blotting from brain lysates and neuronal cells. Infarction volume was measured by TTC staining and evaluation of behavioral was assessed by neurological deficit score (NDS) at 22h after MCAO. Results: In WT mice, ROCK2 inhibition by KD025 (100 mg/kg, p.o.) decreased infarct volumes and improved the NDS in ischemic stroke. ROCK activity (p-MYPT1 to MYPT1 ratio) of the brain lysates by KD025 was decreased dose-dependently in ischemic mice brain. In genetic modified mice, infarction volume (64.6 ± 10.4 vs. LC (102.3 ± 12.4), n=5) of ROCK2Eno2-/- mice was significantly decreased by 37% and the NDSs (1.8 ± 0.4 vs. LC (2.8 ± 0.5), n=5) were improved by 36% compared to the LC groups. Both pGSK-3β and Tau in HT22 cells with ROCK2 siRNA transfection and ROCK2Eno2-/- mice brain was significantly decreased compared to each respective controls. Conclusion: These findings indicate that neuronal ROCK2 plays important role in ischemic stroke via GSK-3β and Tau signaling pathways and inhibition of neuronal specific ROCK2 may have therapeutic benefits in ischemic stroke.

Objective Rho-associated kinase (ROCK) is a key regulator of numerous processes in multiple cell types relevant in stroke pathophysiology. ROCK inhibitors have improved outcome in experimental models of acute ischemic or hemorrhagic stroke. However, the relevant ROCK isoform (ROCK1 or ROCK2) in acute stroke is not known. Methods We characterized the pharmacodynamic and pharmacokinetic profile, and tested the efficacy and safety of a novel selective ROCK2 inhibitor KD025 (formerly SLx-2119) in focal cerebral ischemia models in mice. Results KD025 dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 h from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke. Interpretation Altogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation.

Background and Purpose: Sirt1 is a NAD+ dependent deacetylase that is known as one of important regulators for ischemic stroke. Because the role and mechanism of genetic sirt1 over-expression in ischemic brain injury has not yet been studied, we examined that brain-specific sirt1-overexpressing transgenic (BRASTO) mice might have significant beneficial effect in ischemic stroke through regulation of sirt1-Rho kinase (ROCK) signaling pathway. Methods: We induced a middle cerebral artery occlusion model in mice and tested the effect of resveratrol (100 mg/kg, i.p.) in ischemia/reperfusion (2h/22h) injury and tested the role and mechanism with BRASTO mice in photothrombotic stroke. Protein levels in mice brain tissue lysates were determined using western blotting. Results: Sirt1 activation by resveratrol treatment reduced infarction volume and improved neurological deficit score in transient focal cerebral ischemia. BRASTO mice did not show any obvious phenotypic abnormalities and BRASTO mice improved relative cerebral blood flow and reduced infarction volume by 68.4% compare to that of WT in photothrombotic stroke. Sirt1 over-expression in BRASTO mice was significantly increased by 4.2 fold higher than that of WT mice and Rho-kinase activity was significantly decreased by 54.6% in BRASTO mice. Conclusions: These findings indicate that neuronal specific sirt1 activation in BRASTO mice reduced infarction volume in photothrombotic stroke via down-regulation of ROCK signaling pathway. These results suggest that effect of neuronal sirt1 activation may have therapeutic benefits in ischemic stroke via ROCK signaling pathway.

Background and Purpose: Thrombolysis remains the only effective and approved medical treatment of acute ischemic stroke. Nattokinase is a potent fibrinolytic enzyme purified from the fermented soybean cheese Natto. The fibrinolytic activity and thrombolytic effects of nattokinase have been observed both in vitro and in vivo. Here, we determined whether nattokinase may have a beneficial effect in photothrombotic stroke. Methods: The plasma fibrinolytic activity of nattokinase was measured by euglobulin clot lysis times (ECLT). Arterial thrombus formation was investigated using in vivo ferric chloride induced thrombosis model. To determine the effect of nattokinase in ischemic brain injury, we treated nattokinase at 1h, 2h, and 4h before operation and induced a photothrombotic stroke model in mice. Results: The ECLT was significantly shortened by administration of nattokinase. Nattokinase inhibited the ferric chloride induced arterial thrombosis. Nattokinase improved relative cerebral blood flow in both ischemic region and non-ischemic region. Nattokinase significantly reduced infarction volume at 4h treatment (26.2 ± 6.5 mm3, n=5, p=0.019) compared to that of vehicle control (42.9 ± 2.5 mm3, n=7) in photothrombotic stroke. Conclusions: These findings indicate that nattokinase improved blood flow by inhibiting platelet aggregation and thrombus formation and significantly induced neuroprotective effect in ischemic brain damage. These results suggest that nattokinase may have therapeutic benefits in ischemic brain injury.

Objectives: Vascular injury and thrombosis are main leading causes of cardiovascular diseases. Tuberous sclerosis complex (TSC) is a genetic disorder caused by heterozygous mutations in either of two genes, TSC1 and TSC2. Although role of TSCs has been implicated in cardiovascular diseases, the tissue- and isoform-specific roles of TSCs in the vascular response to injury are not known. Methods and Results: To determine the role of TSC1 in arterial injury and thrombosis, we generated vascular smooth muscle cell-specific TSC1 conditional knockout mice (TSC1SM22-/-) by crossing vascular smooth muscle cell-specific Cre (SM22Cre) mice with TSC1flox/flox mice and performed carotid artery ligation in haploinsufficient TSC1 conditional knockout mice (TSC1SM22+/-) compared with that of WT or haploinsufficient TSC2 knockout mice (TSC2+/-). Acute carotid artery occlusion was investigated by 5% ferric chloride injury. Arterial thrombosis and neointima formation were measured at 14 days after arterial ligation. Expression of proteins was observed by immunoblot analysis. The neointima formation was significantly increased in TSC1SM22+/- mice (intimal thickness/medial thickness ratio; 1.14 ± 0.14, p Conclusion: These findings suggest that regulation of TSC1 and mTOR might be useful for therapeutic intervention in vascular injury and thrombosis.