Products of cellular lipid metabolism have been implicated in the pathophysiology of ischemic brain injury. Cell culture models of both hypoxic and ischemic brain injury will be used to determine the role of lipid metabolites in the destructive processes initiated by hypoxia and ischemia. Primary cultures of both glial cells and neurons will be studied to determine the pathophysiological processes occurring in each cell type. Hypoxia will be produced by incubating cell cultures in an anaerobic environment whereas ischemia will be modeled by incubating cells anaerobically in a glucose-free media. Reoxygenation will be produced by returning cells to an incubator containing air and 5% carbon dioxide. Reperfusion will be modeled by returning cells to a media containing glucose and an incubator containing air and 5% carbon dioxide. Cell cultures will be exposed to varying durations of hypoxia and ischemia and cellular injury and viability will be quantitated. Next, cell cultures will be exposed to varying durations of reoxygenation and reperfusion following several different durations of hypoxia and ischemia, respectively. Based on these initial studies, appropriate durations of hypoxia, hypoxia and reoxygenation, ischemia, and ischemia and reperfusion win be selected to study the effect of these conditions on lipid metabolism of glial cells and neurons. Studies of lipid metabolism will include free fatty acid release, eicosanoid production, phospholipid composition, and diacylglycerol production. Next, free radical production and production of lipid peroxides during reoxygenation and reperfusion will be determined. Mechanisms leading to the production of lipid metabolites during and after hypoxia and ischemia will then be delineated. Subsequent studies will ascertain mechanisms of cellular injury resulting from these metabolites. Based on the understanding gained by studying the mechanisms of injury, pharmacological interventions will be selected and studied in this model.
