The inflammatory response accompanying cerebral ischemia is increasingly appreciated to be involved in potentiating injury. In the intact brain this response is mediated by both the resident microglia and invading peripheral leukocytes and macrophages, with possible contributions from activating astrocytes. Among the various substances secreted by inflammatory cells are reactive oxygen and nitrogen species, cytokines, chemokines, glutamate, and potentially damaging proteases. Using mixed cultures of neurons, astrocytes, and microglia, this project will examine the contribution of inflammatory cells to injury in neurons and astrocytes, with emphasis on reactive oxygen species (ROS), production and scavenging, and cell-cell interaction. We will first establish whether microglia potentiate damage to neurons and/or astrocytes in ischemia, and which toxic factor(s) are involved. Inflammatory cytokines, proteases and iNOS will be measured, and the effect of blockade tested. We will then determine whether inhibiting ROS release from microglia, or over-expressing endogenous antioxidants in neurons or astrocytes is protective. We will examine the role of NADPH oxidase in ROS generation by non-neuronal cells. We will assess whether reduced astrocyte uptake of glutamate, or inhibition of mitochondrial function by ROS contribu7tes to injury. We will then study the participation of creating strokes in a bone marrow chimera model, where animals transplanted with marrow cells lacking a functional NADPH oxidase will be studied. This will allow us to begin dissecting out the contributions of different inflammatory cells using cells of different genotypes in vivo. These studies should provide unique insights into the participation of different cell types in mediating oxidative damage in ischemic and ischemia-like insults.
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