The blood brain barrier (BBB), composed mostly of endothelial cells and astrocytes with contributions from microglia, 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 breakdown with hemorrhage into brain tissue. Though normally protective, during ischemia astrocytes and microglia can contribute to injury. Hemorrhage is a complication of stroke with an incidence of about 70% in normal mice following 2 hr middle cerebral artery occlusion (MCAO). This model, plus in vitro models of the BBB, will be used to study the roles of astrocytes and microglial in hemorrhage with a focus on mitogen activated protein kinase (MARK) signaling cascades and generation of oxidative stress. These intracellular signals are key determinants of cell fate following ischemia. How signaling changes in brain that is protected by heat shock protein 70 (Hsp70) will be investigated. Of particular interest is apoptosis signal regulating kinase (Ask1), a MARK kinase kinase that activates the JNK and p38 pathways. Activation of Ask1 is associated with initiation of classical mitochondrially dependent apoptosis. Ask1 is activated by oxidative stress, TNF-alpha, calcium overload, and endoplasmic reticulum stress, all conditions that are found following ischemia/reperfusion in the brain, and contribute to injury. Hsp70 can down regulate the Ask1 pathway both at the level of Ask1 activation and downstream at the level of JNK activation, and appears to reduce oxidative stress when overexpressed. Hsp70 also reduces histological evidence of both microglial and astrocytic 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 found activation of NADPH oxidase in microglia and astrocytes by ischemia. Both in vivo and primary culture models will be used to improve understanding of generation of oxidative stress in glia, how intracellular signaling 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 by hemorrhage, a feared complication. These studies will improve understanding of BBB cell response, and help identify new targets for therapy of post ischemic hemorrhage.
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