Accumulating evidence indicates that polymorphonuclear leukocytes (PMNs) contribute to the mediation of brain injury following focal ischemic stroke. However, the pathologic mechanisms involved and the cell types injured by PMNs are not known. Both free radical-derived intermediates, proteases, and the myeloperoxidase-chloride system are used by PMNs in host defense. We propose to elucidate the roles played by these chemical and enzymatic systems in vascular injury following focal ischemia. Our project has three aims to test the following hypotheses: 1) Superoxide anion and nitric oxide, separately and through the formation of peroxynitrite, modulate PMN adherence to cerebrovascular endothelium following focal ischemia, thereby affecting vascular permeability, endothelial cell injury, and secondary parenchymal injury; 2) PMN-derived elastase, acting alone and in concert with superoxide anion and peroxynitrite, contributes to microvascular and secondary parenchymal injury in focal stroke; and 3) PMN myeloperoxidase-derived oxidants (hypochlorous acid, monochloramines, and aldehydes) contribute to microvascular and secondary parenchymal cell injury following focal ischemia. Both in vivo and in vitro studies will be undertaken. In mouse models of permanent and temporary focal ischemia, we will use fluorescent videomicroscopy methods to measure PMN-endothelial adherence, loss of BBB integrity, and free radical formation in core and penumbral cortex at 1-48 h after stroke. Myeloperoxidase oxidation products will be measured by mass spectroscopy in core and penumbra and compared regionally and temporally with myeloperoxidase immunocytochemistry findings. Other measured variables will include regional blood flow, edema, and infarct size. Novel mutant mice deficient I PMN elastase and myeloperoxidase will be used in conjunction with pharmacologic interventions to elucidate causality. Injury mechanisms will also be clarified in vitro by studying PMN-mediated injury of cultured cerebrovascular endothelium. The use of PMNs derived from elastase- and MPO-deficient mice, as well as controlled and pharmacologic treatments., will allow us to identify interactions between these injury mechanisms in vitro. These studies will provide considerable mechanistic information regarding vascular injury caused by the acute inflammatory response to focal cerebral ischemia and will help in the design of therapeutics for focal stroke.
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