Background and Purpose-Enhancing collateral artery growth is a potent therapeutic approach to treat cardiovascular ischemic disease from occlusive artery. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has gained attention for its ability to promote arteriogenesis, ameliorating brain damage, by the mechanisms involving monocyte upregulation. However, the recent clinical study testing its efficacy in myocardial ischemia has raised the question about its safety. We tested alternative colony-stimulating factors for their effects on collateral artery growth and brain protection. Methods-Brain hypoperfusion was produced by occluding the left common carotid artery in C57/BL6 mice. After the surgery, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, or GM-CSF (100 g/kg/day) was administered daily for 5 days. The angioarchitecture for leptomeningeal anastomoses and the circle of Willis were visualized after the colony-stimulating factor treatment. Circulating blood monocytes and Mac-2-positive cells in the dorsal surface of the brain were determined. A set of animals underwent subsequent ipsilateral middle cerebral artery occlusion and infarct volume was assessed. Results-Granulocyte colony-stimulating factor as well as GM-CSF promoted leptomeningeal collateral growth after common carotid artery occlusion. Both granulocyte colony-stimulating factor and GM-CSF increased circulating blood monocytes and Mac-2-positive cells in the dorsal brain surface, suggesting the mechanisms coupled to monocyte upregulation might be shared. Infarct volume after middle cerebral artery occlusion was reduced by granulocyte colony-stimulating factor, similarly to GM-CSF. Macrophage colony-stimulating factor showed none of theses effects. Conclusions-Granulocyte colony-stimulating factor enhances collateral artery growth and reduces infarct volume in a mouse model of brain ischemia, similarly to GM-CSF. (Stroke. 2011;42:770-775.)

Summary:Several studies have suggested that cyclooxygenase-2 (COX-2) plays a role in ischemic neuronal death. Genetic disruption of COX-2 has been shown to reduce susceptibility to focal ischemic injury and N-methyl-D-aspartate-mediated neurotoxicity. The purpose of this study was to examine the effects of COX-2 deficiency on neuronal vulnerability after transient forebrain ischemia. Marked upregulation of COX-2 immunostaining in neurons was observed at the early stage and prominent COX-2 staining persisted in the CA1 medial sector and CA2 sector over 3 days after ischemia. The immunohistologic pattern of COX-2 staining in these sectors gradually condensed to a perinuclear location. The degree of hippocampal neuronal injury produced by global ischemia in COX-2-deficient mice was less than that in wild-type mice, coincident with attenuation of DNA fragmentation in the hippocampus. Also, treatment with a selective COX-2 inhibitor, nimesulide, after ischemia decreased hippocampal neuronal damages. These results of genetic disruption and chemical inhibition of cyclooxygenase-2 show that inhibition of COX-2 ameliorates selective neuronal death after transient forebrain ischemia in mice.

Background and Purpose-Recent studies have demonstrated that neurotrophic factors promote neurogenesis after cerebral ischemia. However, it remains unknown whether administration of genes encoding those factors could promote neural regeneration in the striatum and functional recovery. Here, we examined the efficacy of intraventricular injection of a recombinant adenovirus-expressing heparin-binding epidermal growth factor-like growth factor (HB-EGF) on neurogenesis, angiogenesis, and functional outcome after focal cerebral ischemia. Methods-Transient focal ischemia was induced by middle cerebral artery occlusion (MCAO) for 80 minutes with a nylon filament in Wistar rats. Three days after MCAO, either adenovirus-expressing HB-EGF (Ad-HB-EGF) or Ad-LacZ, the control vector, was injected into the lateral ventricle on the ischemic side. Bromodeoxyuridine (BrdU) was injected intraperitoneally twice daily on the sixth and seventh days. On the eighth or 28th day after MCAO, we evaluated infarct volume, neurogenesis, and angiogenesis histologically. Neurological outcome was serially evaluated by the rotarod test after MCAO. Results-There was no significant difference in infarct volume between the 2 groups. Treatment with Ad-HB-EGF significantly increased the number of BrdU-positive cells in the subventricular zone on the 8th day. In addition, on the 28th day, BrdU-positive cells differentiated into mature neurons in the striatum on the ischemic side but seldom the cells given Ad-LacZ. Enhancement of angiogenesis at the peri-infarct striatum was also observed on the eighth day in Ad-HB-EGF-treated rats. Treatment with Ad-HB-EGF significantly enhanced functional recovery after MCAO. Conclusions-Our data suggest that gene therapy using Ad-HB-EGF contributes to functional recovery after ischemic stroke by promoting neurogenesis and angiogenesis. (Stroke. 2005;36:859-864.)

Background and Purpose-Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to accelerate collateral growth (arteriogenesis) at the circle of Willis in rat brain. However, the effect of GM-CSF on leptomeningeal collateral growth has not been established. We examined the effect of unilateral common carotid artery (CCA) occlusion and GM-CSF treatment on leptomeningeal collateral growth in mice. Methods-Adult mice were subjected to unilateral CCA occlusion or sham surgery followed by an alternate-day regimen of GM-CSF (20 g/kg) or saline injection. On day 7, latex perfusion was performed in 1 set of mice to visualize the leptomeningeal vessels, and the number of Mac-2 ϩ monocytes/macrophages on the dorsal surface of the brain was counted. In another set of mice, on day 7, permanent ipsilateral middle cerebral artery (MCA) occlusion was performed, and infarct volume was measured. Results-Leptomeningeal collateral growth was observed after CCA occlusion, and that was enhanced by GM-CSF treatment. An increase in the number of Mac-2 ϩ cells on the surface of the brain occurred after CCA occlusion and was enhanced by GM-CSF treatment. Seven days after CCA occlusion, GM-CSF treatment decreased the infarct size attributable to subsequent MCA occlusion. Conclusion-After

Global ischemia promotes neurogenesis in the dentate gyrus of the adult mouse hippocampus. Cyclooxygenase (COX)-2, the principal isoenzyme in the brain, modulates inflammation, glutamate-mediated cytotoxicity, and synaptic plasticity. We demonstrated that delayed treatment with different classes of COX inhibitor significantly blunted enhancement of dentate gyrus proliferation of neural progenitor cells after ischemia. COX-2 immunoreactivity was observed in both neurons and astrocytes in the dentate gyrus, but not in neural progenitor cells in the subgranular zone. Moreover, in the postischemic dentate gyrus of heterozygous and homozygous COX-2 knockout mice, proliferating bromodeoxyuridine-positive cells were significantly fewer than in wild-type littermates. These results demonstrate that COX-2 is an important modulator in enhancement of proliferation of neural progenitor cells after ischemia.

Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen-glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

Background and Purpose-It is well-established that hypertension leads to endothelial dysfunction in the cerebral artery.Recently, cilostazol has been used for the secondary prevention of ischemic stroke. Among antiplatelet drugs, phosphodiesterase inhibitors including cilostazol have been shown to have protective effects on endothelial cells. The aim of the present study is to investigate the effects of cilostazol and aspirin on endothelial nitric oxide synthase (eNOS) phosphorylation in the cerebral cortex, endothelial function, and infarct size after brain ischemia in spontaneously hypertensive rats (SHR). Methods-Five-week-old male SHR received a 5-week regimen of chow containing 0.1% aspirin, 0.1% cilostazol, 0.3% cilostazol, or the vehicle control. The levels of total and Ser 1177 -phosphorylated eNOS protein in the cerebral cortex were evaluated by Western blot. To assess the contribution of eNOS in maintaining cerebral blood flow, we monitored cerebral blood flow by laser-Doppler flowmetry after L-N 5 -(1-iminoethyl)ornithine infusion. Additionally, we evaluated residual microperfusion using fluorescence-labeled serum protein and infarct size after transient focal brain ischemia. Results-In SHR, the blood pressure and heart rate were similar among the groups. Cilostazol-treated SHR had a significantly higher ratio of phospho-eNOS/total eNOS protein than vehicle-treated and aspirin-treated SHR. Treating with cilostazol, but not aspirin, significantly improved cerebral blood flow response to L-N 5 -(1-iminoethyl)ornithine. Cilostazol also increased residual perfusion of the microcirculation and reduced brain damage after ischemia compared to vehicle control and aspirin. Conclusions-These findings indicate that cilostazol, but not aspirin, can attenuate ischemic brain injury by maintaining endothelial function in the cerebral cortex of SHR. (Stroke. 2011;42:2571-2577.)Key Words: brain ischemia Ⅲ endothelial function Ⅲ hypertension Ⅲ phosphodiesterase-3 inhibitor H ypertension is one of the most important risk factors for cerebrovascular disease and is closely associated with endothelial dysfunction. Some studies observed endothelial dysfunction in patients with hypertension or cerebrovascular disease. 1,2 In hypertensive animal models, hypertension impairs endothelium-dependent vasodilatation, cerebrovascular autoregulation, and cerebral blood flow (CBF) responses, and it exacerbates ischemic brain damage. [3][4][5][6] Endothelial dysfunction is often characterized by a decrease in the bioavailability of endothelium-derived nitric oxide (NO). In endothelial cells, NO is produced by endothelial nitric oxide synthase (eNOS), and eNOS activity is regulated primarily by calcium-calmodulin activation and multisite phosphorylation of specific serine or threonine residues. Most importantly, phosphorylation of eNOS-Ser 1177 is thought to play a crucial role in eNOS activation. In a cerebral ischemia model, modification of the eNOS-Ser 1177 phosphorylation state modulated CBF and the outcome of ischemic injury. 7 Th...

Microcirculatory disturbances contribute to the expansion of infarct lesions after focal cerebral ischemia. Recently, it was shown that Rho-kinase involves in endothelial dysfunction via down-regulation of endothelial nitric oxide synthase function in a rodent stroke model. However, it is not clear whether endothelial Rho-kinase is activated in vivo or Rho-kinase activation contributes to microcirculatory disturbances after cerebral ischemia. In this study, we assessed the temporal and spatial profiles of Rho-kianse activity and the effect of the Rho-kinase inhibitor fasudil on microcirculatory disturbances in the focal brain ischemia. Rho-kinase activation was evaluated by analyzing the phosphorylation of adducin, a substrate of Rho-kinase, by immunohistochemistry. Staining for p-adducin was found in endothelia in the ischemic area 6 hr after induction of ischemia. Microcirculatory disturbances and increased endothelial cell staining for von Willebrand factor (vWF) were observed in the same area. Postischemic treatment with fasudil suppressed endothelial Rho-kinase activation, preserved microcirculation, and inhibited endothelial cell vWF staining. These effects resulted in inhibition of infarct expansion and improvement of neurologic deficits. These findings indicate that Rho-kinase is activated in the endothelial cells and contributes to microcirculatory disturbances in cerebral ischemia. The vascular protective effect of Rho-kinase inhibitors may be useful in the treatment of the acute phase of ischemic stroke.