This proposed research project is designed to assess the involvement of oxygen-derived free radicals in ischemia-reperfusion (I/R) injury in skeletal muscle. The proposed studies will focus on the contribution of neutrophils to I/R-induced vascular injury. Our working hypothesis is that ischemia (4 hours inflow occlusion) results in the conversion of xanthine dehydrogenase to the oxy-radical producing xanthine oxidase. Concomitantly, ATP is catabolized under the hypoxic conditions to hypoxanthine. Upon reperfusion (reoxygenation), a burst of superoxide, hydrogen peroxide, and hydrdoxyl radical production occurs as a result of the oxidation of hypoxanthine by xanthine oxidase. The oxy-radicals formed by xanthine oxidase mediate tissue injury via two mechanisms: 1) by directly reacting with cellular and interstitial components to initiate lipid peroxidation and other processes that lead to cellular necrosis, and 2) by triggering the infiltration of neutrophils which, in turn, mediate injury by both oxidative and non-oxidative (eg, proteases) processes. The proposed studies will utilize microvascular permeability and evaluation of the no-reflow phenomenon as indicies of tissue injury and changes in tissue myeloperoxidase activity to monitor neutrophil infiltration. We propose to determine: (1) the extent to which xanthine dehydrogenase is converted to xanthine oxidase in ischemic skeletal muscle; (2) whether xanthine oxidase inhibition or inactivation modifies ischemia-induced neutrophil infiltration; (3) whether administration of calcium channel inhibitors will ameliorate tissue injury and limit the neutrophil infiltration associated with I/R; (4) whether superoxide-dependent chemoattractants initiate neutrophil infiltration; (5) whether depletion of circulating neutrophils (with antineutrophil serum) ameliorates I/R-induced tissue injury; and (6) whether neutrophil adherence to microvascular endothelium plays a role in I/R-induced tissue injury. We also propose to develop a model that will allow for assessment of neutrophil-mediated injury in the absence of ischemia. The proposed studies should improve our understanding of the role of oxidants as mediators of cell injury in skeletal muscle.
