Ischemia, hypoxia and stroke are potential complications of invasive medical procedures. Exposure to focal brain ischemia may occur during neurosurgical resection, during carotid endarterectomy, and during cardiac bypass or total circulatory arrest procedures. Magnetic resonance imaging, for example, demonstrates new ischemic lesions in 58 percent of patients with valvular disease undergoing cardiopulmonary bypass. Neuropsychological impairments occur in as many as 70 percent of cardiac surgery patients. The consequences of Central Nervous System (CNS) deficits as a result of ischemic or hypoxic insult are so severe as to warrant investigation of novel methods of minimizing damage. Our long-range goal is to prevent CNS damage by the introduction of intracellular nucleic acids (DNA and mRNA) that encode neuroprotective proteins. This program of studies will test the hypothesis that delivery of a neuroprotective agent before a near-lethal stress can protect neurons. We will investigate Heat Shock Protein 70 (HSP70) as a candidate neuroprotective gene. This member of the Heat Shock Protein (HSP) family has been shown to protect against ischemic cell death in a variety of tissues and species. As a first step in evaluating the effectiveness of HSP70 in protecting neurons, we will transfect CNS neurons in vitro with mRNA or DNA encoding HSP70, subject the cells to hypoxic stress, and measure cell survival. We will evaluate: timing of transfection relative to the exposure of hypoxia, dose-response of introduced mRNA or DNA, immunohistochemical quantification of the HSP70 protein, and cellular toxicity. An essential next step in determining the potential clinical usefulness of this strategy is to evaluate the uptake, anatomic distribution and cellular localization of mRNA and DNA introduced into the cerebrospinal fluid. We will introduce mRNA or DNA encoding HSP70 and reporter enzymes into the ventricular system of rats with chronic indwelling catheters and will investigate the following questions: 1) what cell types demonstrate reporter enzyme and HSP70 mRNA and DNA uptake and expression; 2) what is the anatomical distribution of this uptake and expression; 3) what fraction of cell types are transfected, and does this vary by cell type; 4) does transfection reach deeper cell layers, specifically, does the hypoxia-sensitive CA1 cell layer of the hippocampus demonstrate expression; 5) does the distribution of expression suggest transport of mRNA; 6) do animals exhibit signs of neurological impairment; and, 7) does neuropathology demonstrate toxicity. The answers to these questions will provide baseline information for the design and implementation of future studies to determine whether intraventricularly introduced HSP70 can protect hippocampal pyramidal cells from global ischemia-induced cell death, and may lead to improved or novel therapeutic agents after CNS injury.
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