The goal of this project is to understand the molecular events that occur in ischemic neurons and glia in order to develop rational strategies for preventing injury. Our previous work has shown that ischemia and other types of injury induce the c-fos immediate early gene and the hsp7O heat shock gene in rat brain and in cultured brain cells, and that induction of these genes can serve as markers of the injured cells. The current proposal will determine whether the combined use of antagonists of glutamate receptors and voltage sensitive calcium channels is required to prevent induction of c-fos mRNA, FOS protein, AP1 binding and cell injury produced in vitro by glutamate and chemical ischemia in cultured neurons and in vivo by global ischemia in gerbil hippocampal pyramidal neurons. These experiments are based upon the hypothesis that calcium entry via any route may be sufficient to induce the c-fos gene and to produce cell injury. The experiments will utilize cell culture methods and the gerbil global ischemia model. c-fos mRNA is assessed using Northern blots and in situ hybridization, Fos protein via immunocytochemistry, AP-1 binding via gel shift assays,'and cell injury via LDH in vitro and cell counts in vivo. The hsp70 heat shock gene is induced in neurons located in the penumbra of focally ischemic brain and in capillary endothelial cells but not in neurons or glia in regions of infarction. The current project will examine the induction of two other hsp70 family members, hsc7O and grp78, following focal and global ischemia. In addition, two new recently cloned stress genes, called hsr1 and hsr2, will be fully sequenced and characterized, and their expression studied following focal and global ischemia. Lastly, experiments will be performed in which retroviral vectors will be used to express the hsp70, hsc7O, and grp78 stress genes in cultured astrocytes and neurons from embryonic rat brain to determine whether expression of these genes in these cells will protect them from otherwise lethal injury. Focal ischemia is produced using the suture model in the rat and global ischemia is produced by occluding both carotids in the gerbil. Stress gene mRNA expression is examined using Northern blots and in situ hybridization. Cloning and sequencing of two new stress genes will be performed.
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