The goal of this project is to test the hypothesis that short term hypoxia and reoxygenation (H/RO)-induced impairment of vascular regulation in isolated cerebral penetrating arterioles is due to specific damage of the endothelial nitric oxide synthase pathway. H/RO causes vasoconstriction in large isolated cerebral blood vessels. The mechanism for this construction it is not understood and it is not known if penetrating arterioles are directly damaged after H/RO. Since cerebral arterioles are the last regulator of blood flow to the tissue, even a small vasoconstriction would greatly increase vascular resistance. This could contribute to cerebral damage due to postischemic hypoperfusion after ischemia/ reperfusion (I/R) in vivo. Experiments will be performed in vitro. Rat cerebral arterioles and arterioles of endothelial nitric oxide synthase deficient (eNOS) mice will be isolated, cannulated, and video-microscopically observed. The vessels will be subjected to hypoxia (p02 of 1mm Hg) of up to two hours. Cell cultures of rat cerebral microvascular endothelial (EC) and smooth muscle cells (SMC) will be used in parallel experiments. (1) It will be determined if only the endothelium is impaired after H/RO. Measurements of the vessel diameter, physiological vessel responses, membrane potentials (MP) in SMC as well as intracellular calcium (Ca++) in SMC and EC will be used as indicators of injury after H/RO. Selective impairment of the endothelium will be used to investigate whether the EC is the target of H/RO induced damage. Measurement of MP and Ca++ in cultured EC and SMC will identify the cell type affected by H/RO. (2) We will determine where the eNOS dependent metabolic pathway is affected in the endothelial cell. We will test for changes in the signal transduction pathway in both isolated vessels and in cell culture using specific agonists and measuring transient changes in vessel diameter, MP, and Ca++. Detection of signal transduction proteins in cell culture will indicate the impaired step in the signal transduction chain. (3) We will demonstrate the feasibility of pharmacologically alleviating vascular impairment due to H/RO. Treatments with calcium blockers, eNOS independent vasodilators or protective flavonoid will indicate if endothelial impairment due to H/RO can be alleviated or prevented in both isolated vessel and culture ECS. These studies will give as crucial information on the cellular target of H/RO inducted vascular microvessel injury and possible treatments targeted precisely to either prevent a specific cell damage or to take advantage of using regulatory mechanisms resistant to H/RO to improve vascular perfusion in vivo.
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