Cerebral ischemia is followed by an inflammatory reaction that involves the ischemic brain. Although it is well established that this inflammation contributes to the progression of ischemic damage, the mechanisms of its pathogenic role have not been fully elucidated. One of the gene products expressed during inflammation is cyclo-oxygenase-2 (COX2). COX2 is a rate limiting enzyme for prostanoid synthesis that also generates toxic free radicals. COX2 upregulation has been shown to be responsible for many of the cytotoxic effects of inflammation. In this proposal, molecular and biochemical techniques, as well as methods for assessing tissue outcome, will be used to test the hypothesis that COX2 is expressed after cerebral ischemia and that its reaction products contribute to ischemic brain damage. Focal cerebral ischemia will be produced by occlusion of the middle cerebral artery in rats and mice. First, RT-PCR will be used to determine whether ischemia upregulates COX2 MRNA. Second, using western blotting and immunocytochemistry, we will study whether induction of COX2 message corresponds to an increase in the synthesis of COX2 protein and, if so, we will identify the cell type(s) in which COX2 is unregulated. Third, by measuring prostanoid concentration in the post-ischemic brain, we will determine whether COX2 upregulation leads to increased synthesis of biologically active end-products. Fourth, we will begin to define whether COX2 expression contributes to cerebral ischemic damage. In particular, we will determine whether treatment with COX2 inhibitor NS-398 ameliorates focal cerebral ischemic damage. In addition, COX2 knockout mice will be used. These mice do not express COX2 during inflammation. If COX2 upregulation contributes to cerebral ischemic damage, we would expect that COX2 knockouts will be relatively protected from ischemic damage. Preliminary results suggest that this is a most promising area of research that will advance our understanding of the fundamental mechanisms of cerebral ischemia and may unveil new avenues for stroke treatment.
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