Stroke induced by focal occlusion of blood flow or by generalized hypoperfusion to the brain is the third leading cause of death in the United States. Neurons in various brain regions show selective vulnerability to stroke-related injury. The glial response varies from early activation and infiltration into the infarcted region to death. While the precise contribution of glial to CNS ischemic injury is unknown, recent studies suggest that microglia and astrocytes may partially mediate neuronal death following stroke via generation of cytotoxic cytokines and reactive free radicals. In vivo studies of animal tissues suggest that interleukin 1beta (IL1beta) may be an early mediator of neuronal death in stroke. The applicant's preliminary data demonstrates that IL1 is induced in human microglial cells in vitro following hypoxia and in vivo in postmortem stroke tissue. Nitric oxide generated by iNOS expressing human fetal astrocytes is a potent neurotoxin in primary human CNS cultures. IL1beta plays a central role in neurotoxicity in that iNOS is induced only in the presence of IL1beta. In addition to the cytotoxic events mediated by ischemic injury, neuroprotective mechanisms may be initiated. The induction of growth factors i.e. vascular endothelial growth factors (VEGF) following hypoxia is purported to be part of the cascade of stroke induced injury. VEGF is an angiogenic factor and has been shown to induce vascular proliferation and macrophage chemotaxis. The applicant's recent studies demonstrate that hypoxia induces VEGF expression in astrocytes. This proposal is based on the hypothesis that CNS ischemic injury is mediated by glial cells activated to generate cytokines, such as IL1 and TNF-( and subsequently nitric oxide synthase. The applicant proposes that this interaction leads to the generation of NO which in the setting of glutamate receptor activation and increased reactive oxygen species produces potent neurotoxic effects. Specifically, the applicant aims to characterize hypoxia induced gene expression in astrocytes and microglia, the potential induction of NO by hypoxia/reoxygenation from the cytotoxic actions of hypoxia-induced cytokine expression and NO, and the contribution of glutamate receptor activation and generation of reactive oxygen species to neuronal injury in vitro and in vivo. The proposed in vitro studies will utilize primary cultures of human CNS and the in vivo studies will utilize postmortem stroke tissue. Results of the proposed experiments will further the understanding of the sequelae of events leading to neuronal death following stroke induced injury.
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