This proposal is a competing renewal application of our current project to study a role of Na+-K+-Cl- cotransporter isoform1 (NKCC1) in cerebral ischemic damage. The long-term goal of the research is to understand ischemia-induced cell death and develop a more effective approach to ischemia treatment. NKCC1 is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system (CNS). In the initial funding period, we have investigated NKCC1 activity under several conditions that are associated with ischemic insults. We found the NKCC1 activity in cultured cortical astrocytes was significantly stimulated under high extracellular K+ ([K+]0). Pharmacological inhibition or genetic ablation of NKCC1 abolished high [K+]0-induced astrocyte swelling and decreased glutamate release. In cultured neurons, activation of both ionotropic and mGluR group l glutamate receptors stimulated NKCC1 activity in a Ca++-dependent manner. Infarct volume and cerebral edema were significantly reduced by bumetanide, a potent inhibitor for NKCC1. Lour preliminary study revealed a neuroprotection in NKCC1 knockout mice following focal ischemia. These data strongly suggest that NKCC1 has an important role in cerebral ischemic damage. However, the cellular mechanisms underlying the role of NKCC1 in ischemic cell damage have not been fully understood. We hypothesize that NKCC1 contributes to perturbation in ion homeostasis and necrotic ischemic cell death. We will test the hypothesis by following Specific Aims:
Aim 1 : Determine the role of NKCC1 in intracellular Na+ and Cl- overload, swelling, and the swelling-mediated glutamate release from cortical astrocytes in an in vitro oxygen and glucose deprivation (OGD) model of ischemia.
Aim 2 : Investigate the contribution of NKCC1 to OGD-mediated ischemic cell death in cortical neurons.
Aim 3 : Investigate whether genetic ablation of NKCC1 reduced brain damage (gray and white matter) in NKCC1-/- mice following transient focal ischemia.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BDCN-3 (01))
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Golanov, Eugene V
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University of Wisconsin Madison
Schools of Medicine
United States
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