Recent studies from our laboratory and others indicate that neutrophil adherence to vascular endothelium is required to produce microvascular dysfunction and myocyte necrosis in postischemic skeletal muscle. Recognition of this fact has led to a major research effort directed at evaluating the potential for inhibition of leukocyte adhesion as a novel approach to the treatment of reperfusion injury. Preliminary data from our laboratory indicates that ischemic preconditioning (IPC, a phenomenon n which a tissue is rendered resistant to the deleterious effects of prolonged ischemia and reperfusion by prior exposure to brief, repeated periods of vascular occlusion) prevents muscle necrosis induced by I/R by inhibiting leukocyte adherence and emigration during reperfusion. The overall goal of the projects outlined in this application is to determine the mechanisms by which IPC attenuates leukocyte adhesion to and emigration across postcapillary venules, microvascular barrier disruption, capillary no-reflow, and myocyte necrosis in skeletal muscles subsequently exposed to prolonged ischemia and reperfusion (I/R). Our working hypothesis is that IPC will attenuate microvascular dysfunction and myocyte necrosis in postischemic skeletal muscles via a mechanism that involves adenosine receptor activation during the period of IPC and during reperfusion after prolonged ischemia. To address this issue, we propose to determine: 1) whether IPC will attenuate leukocyte adhesion and emigration, capillary no-flow, venular protein leakage, and myocyte necrosis induced by I/R; 2) the role of adenosine produced during the preconditioning period in the protective effects of IPC that become apparent during reperfusion after prolonged ischemia; 3) whether the beneficial effects of adenosine A/1-receptor activation during the IPC period occur by a mechanism that involves activation of ATP-sensitive potassium channels; 4) the role of IPC-induced increases in 5'- nucleotidase activity during reperfusion after sustained ischemia in the protective actions of IPC; and 5) whether adenosine production is increased during reperfusion of preconditioned skeletal muscles and contributed to the protective actions afforded by IPC by activating adenosine A/2-receptors. To accomplish these aims, we will utilize intravital microscopic approaches to quantitate leukocyte adhesion and emigration, venular protein leakage, and capillary no-reflow in the mouse cremaster muscle. The influence of IPC on I/-induced myocyte necrosis will also be examined. Tissue adenosine levels will be measured by high performance liquid chromatography. The proposed studies should not only substantially improve our understanding of the mechanisms whereby IPC reduces microvascular dysfunction and myocyte necrosis in skeletal muscles subjected to subsequent prolonged periods of ischemia and reperfusion but should also provide a rationale for the pharmacologic treatment of disorders characterized by I/R.
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