(Verbatim from the application): The results of a number of recent studies indicate that brief episodes of ischemia increase the tolerance of skeletal muscle and other tissues to deleterious effects of a more prolonged exposure to ischemia and reperfusion (IR) 24 hours later, a phenomenon referred to as delayed or late phase ischemic preconditioning (delayed IPC). Although the mechanisms whereby preconditioning reduces postischemic tissue injury are not clear, preliminary data from our laboratory indicates that delayed IPC prevents muscle necrosis induced by IR by inhibiting leukocyte adherence and emigration during reperfusion after the second ischemic insult. However, the mechanisms linking events that are initiated during the period of preconditioning ischemia to the reduction in postischemic microvascular dysfunction and myocyte necrosis are unclear. Thus, the overall goal of the projects outlined in this application is to determine the mechanisms by which delayed IPC attenuates oxidant production, P-selectin expression, 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 (IR) 24 hours later. We hypothesize that nitric oxide (NO) plays a critical role in delayed IPC, acting initially as a trigger and then subsequently as the mediator of the protection. To address this issue, we propose to determine whether: (1) NO derived from endothelial NOS is produced during the period of preconditioning ischemia and triggers the protective actions of delayed IPC; (2) eNOS activity is stimulated by the increased shear stress that occurs during the repeated hyperemias that occur on release of the occlusion after each cycle of IPC or by IPC-induced bradykinin release; (3) NO produced during the period of preconditioning ischemia initiates the protective effects of delayed IPC by a mechanism that involves the generation of xanthine oxidase derived oxidant species; (4) protein kinase C (P KC) contributes to the beneficial actions of delayed precondition mg and, if so, if isoform-specific PKC translocation is induced by the NO formed during the cycles of preconditioning ischemia; and (5) NO production during reperfusion of preconditioned skeletal muscles contributes to the protective actions of delayed IPC by an iNOS dependent mechanism. To accomplish these aims, we will utilize intravital microscopic approaches to quantify oxidant production, leukocyte adhesion and emigration, microvascular protein leakage, and capillary no-reflow in cremaster muscles in wild-type control mice (C57BLl6) and in transgenic mice lacking eNOS, iNOS or nNOS. The influence of delayed IPC on xanthine oxidase activity and IR induced P-selectin expression and myocyte necrosis will also be investigated. Isoform-specific PKC translocation, NOS mRNA levels, isoform expression, and activities will be examined during IPC and hR. The proposed studies should not only substantially improve our understanding of the mechanisms whereby delayed 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 development of pharmacologic approaches that duplicate its remarkably powerful protective effects.
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