The blood brain barrier (BBB), composed mostly of endothelial cells and astrocytes with contributions frommicroglia, separates the central nervous system from the cellular and molecular components of the blood,maintaining the brain's unique milieu. Injury of the BBB by ischemia and reperfusion can lead to breakdownwith hemorrhage into brain tissue. Though normally protective, during ischemia astrocytes and microglia cancontribute to injury. Hemorrhage is a complication of stroke with an incidence of about 70% in normal micefollowing 2 hr middle cerebral artery occlusion (MCAO). This model, plus in vitro models of the BBB, will beused to study the roles of astrocytes and microglial in hemorrhage with a focus on mitogen activated proteinkinase (MARK) signaling cascades and generation of oxidative stress. These intracellular signals are keydeterminants of cell fate following ischemia. How signaling changes in brain that is protected by heat shockprotein 70 (Hsp70) will be investigated. Of particular interest is apoptosis signal regulating kinase (Ask1), aMARK kinase kinase that activates the JNK and p38 pathways. Activation of Ask1 is associated withinitiation of classical mitochondrially dependent apoptosis. Ask1 is activated by oxidative stress, TNF-alpha,calcium overload, and endoplasmic reticulum stress, all conditions that are found followingischemia/reperfusion in the brain, and contribute to injury. Hsp70 can down regulate the Ask1 pathway bothat the level of Ask1 activation and downstream at the level of JNK activation, and appears to reduceoxidative stress when overexpressed. Hsp70 also reduces histological evidence of both microglial andastrocytic activation in brain following MCAO. We postulate that this pathway is central to ischemic injury,and that Hsp70 can reduce hemorrhage in part by reducing activation of this pathway. We previously foundactivation of NADPH oxidase in microglia and astrocytes by ischemia. Both in vivo and primary culturemodels will be used to improve understanding of generation of oxidative stress in glia, how intracellularsignaling responds, and how impairment of glial cells contributes to hemorrhage. Relevance to public health:Stroke, the leading cause of disability and third leading cause of death in the US, can be worsened byhemorrhage, a feared complication. These studies will improve understanding of BBB cell response, andhelp identify new targets for therapy of post ischemic hemorrhage.
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