Hypoxic-ischemic brain injury from stroke and other cerebral vascular diseases is the leading cause of neurologic disability and mortality. Although no clinical therapy has yet been proven effective in limiting neuronal loss from of acute cerebral ischemia, improved understanding of the cellular mechanisms underlying hypoxic-ischemic brain injury may provide opportunities for rational therapy. Recent evidence supports the hypothesis that a failure of intracellular calcium homeostasis may be central to the development of hypoxic-ischemic neuronal degeneration. The present proposal utilizes two research tools to examine this hypothesis and its consequences. First, a cortical cell culture model of hypoxic-ischemic neuronal loss has been characterized in the sponsor's laboratory; preliminary observations indicate this injury is associated with intracellular calcium accumulation. Second, ratio imaging techniques using the fluorescent calcium indicator, fura-2, will allow direct observation of cytosolic free calcium alterations in hypoxic neurons. Calcium measurements will be paired with assessments of neuronal survival under hypoxic conditions to identify differentially vulnerable cell populations. Pharmacological studies will assess major routes of intracellular calcium entry and clearance, to determine how these are altered by energy depletion and to examine potential therapeutic interventions. Confocal fluorescence microscopy will be utilized to examine the subcellular distribution of calcium changes in hypoxic neurons. Finally, attention will turn to cellular ionic events which occur in concert with calcium alterations, using whole cell patch-clamp and perforated-patch microelectrode physiology and fluorescent indicators for pH, magnesium, and other ions. In addition to research training in the neurosciences, the CIDA program will provide focused experience in teaching, general neurology, and clinical investigation in cerebral vascular disease.
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