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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.)

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.

Background and Purpose-Brain ischemia stimulates neurogenesis. However, newborn neurons show a progressive decrease in number over time. Under normal conditions, the cAMP-cAMP responsive element binding protein (CREB) pathway regulates the survival of newborn neurons. Constitutive activation of CREB after brain ischemia also stimulates hippocampal neurogenesis. Thus, activation of cAMP-CREB signaling may provide a promising strategy for enhancing the survival of newborn neurons. We examined whether treatment of mice with the phosphodiesterase-4 inhibitor rolipram enhances hippocampal neurogenesis after ischemia. Methods-Both common carotid arteries in mice were occluded for 12 minutes. Bromodeoxyuridine (BrdU) was used to label proliferating cells. Mice were perfused transcardially with 4% paraformaldehyde, and immunohistochemistry was performed. To evaluate the role of CREB in the survival of newborn neurons after ischemia, intrahippocampal injection of a CRE-decoy oligonucleotide was delivered for 1 week. We examined whether the activation of cAMP-CREB signaling by rolipram enhanced the proliferation and survival of newborn neurons. Results-Phospho-CREB immunostaining was markedly upregulated in immature neurons, decreasing to low levels in mature neurons. The number of BrdU-positive cells 30 days after ischemia was significantly less in the CRE-decoy treatment group than in the vehicle group. Rolipram enhanced the proliferation of newborn cells under physiologic conditions but not under ischemic conditions. Rolipram significantly increased the survival of nascent BrdU-positive neurons, accompanied by an enhancement of phospho-CREB staining and decreased newborn cell death after ischemia. Conclusions-CREB phosphorylation regulates the survival of newborn neurons after ischemia. Chronic pharmacological activation of cAMP-CREB signaling may be therapeutically useful for the enhancement of neurogenesis after ischemia.

Background and Purpose-Neurons acquire tolerance to ischemic stress when preconditioning ischemia occurs a few days beforehand. We focused on collateral development after mild reduction of perfusion pressure to find an endogenous response of the vascular system that contributes to development of ischemic tolerance. Methods-After attachment of a probe, the left common carotid artery (CCA) of C57BL/6 mice was occluded. The left middle cerebral artery (MCA) was subsequently occluded permanently on days 0, 1, 4, 14, and 28 (nϭ8 each). The change in cortical perfusion during MCA occlusion was recorded. A sham group of mice received only exposure of the CCA and MCA occlusion 14 days later. In apoE-knockout mice, the MCA was occluded 14 days after CCA occlusion or sham surgery. Infarct size and neurologic deficit were determined 4 days after MCA occlusion. Results-Mice that had 45% to 65% of baseline perfusion after CCA occlusion were used. Cortical perfusion after MCA occlusion was significantly preserved in day 14 (47Ϯ16%) and day 28 (46Ϯ7%) groups compared with day 0 (28Ϯ8%), day 1 (33Ϯ19%), day 4 (29Ϯ16%), and sham groups (32Ϯ9%). Infarct size and neurologic deficits were also attenuated in day 14 and day 28 groups compared with other groups. In apoE-knockout mice, there was no significant difference in perfusion, neurologic deficits, or infarction size between groups with and without CCA occlusion. Conclusion-Chronic mild reduction of perfusion pressure resulted in preservation of cortical perfusion and attenuation of infarct size after MCA occlusion. These responses of collaterals were impaired in apoE-knockout mice. Key Words: chronic ischemia Ⅲ chronic perfusion Ⅲ collateral circulation Ⅲ focal ischemia Ⅲ ischemic tolerance C ollateral circulation through leptomeningeal vessels may determine the severity of ischemic injury after occlusion of middle cerebral artery (MCA) in patients with stroke. 1 Although chronic hypoperfusion is believed to be critical for collateral development, 2 factors leading to it are uncertain. For better understanding of collateral circulation in cerebral ischemia, an animal model in which cerebral collaterals can be investigated is needed. Recently, Busch et al 3 demonstrated that 3-vessel (one carotid plus both vertebral arteries) occlusion induced arteriogenesis at the circle of Willis in hypoperfused rat brain. They found that the diameter of the posterior cerebral artery enlarged significantly 1 week after 3-vessel occlusion, 3 but the distal leptomeningeal collaterals that determine stroke outcome did not change. Although morphologic assessment of leptomeningeal collaterals requires multivessel angiography 4 or latex perfusion methods, 5 the level of collateral circulation after cerebral ischemia can be functionally examined by measuring the change of cerebral perfusion at the time of MCA occlusion. In this study, we examined the effect of a mild reduction of cerebral perfusion pressure by occlusion of the left common carotid artery (CCA) on collateral development and infarct siz...

Neuronal progenitors in the adult hippocampus continually proliferate and differentiate to the neuronal lineage, and ischemic insult promotes hippocampal neurogenesis. However, newborn neurons show a progressive reduction in numbers during the initial few weeks, therefore, enhanced survival of newborn neurons seems to be essential for therapeutic strategy. Bcl-2 is a crucial regulator of programmed cell death in CNS development and in apoptotic and necrotic cell death. Therefore, we tested whether Bcl-2 overexpression enhances survival of newborn neurons in the adult mouse hippocampus under normal and ischemic conditions. Many newborn neurons in the hippocampal dentate gyrus undergo apoptosis. Human Bcl-2 expression in NSE-bcl-2 transgenic mice began at the immature neuronal stage and remained constant in surviving mature neurons. Bcl-2 significantly increased survival of newborn neurons under both conditions, but particularly after ischemia, with decreased cell death of newborn neurons in NSE-bcl-2 transgenic mice. We also clarified the effect by Bcl-2 overexpression of enhanced survival of newborn neurons in primary hippocampal cultures with BrdU labeling. These findings suggest that Bcl-2 plays a crucial role in adult hippocampal neurogenesis under normal and ischemic conditions.

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