Our major objective is to evaluate systematically the role of oxygen-derived free radicals in reperfusion injury. The generation of free radicals during regional ischemia, cardioplegic arrest and reperfusion in the pig heart model will be studied using measurements of lipid peroxidation and gluathione release from the heart. Two different sources of free radicals will be examined. To evaluate polymorphonuclear neutrophils as a potential source, (1) neutrophil depleted medium and (2) anti-inflammatory agents will be used in the perfusion medium followed by quantitation of neutrophil influx by labeling with Indium-111 or 3H-thymidine. A second source of free radicals, the combined presence of hypoxanthine, xanthine oxidase and molecular 02 will be examined by studying the presence of these components individually. In addition, to test the hypothesis that dropping the """"""""energy charge"""""""" during ischemia leade to Ca++ influx which in turn converts xanthine dehydrogenase into xanthine oxidase (XO) by activating Ca-dependent protease, the energy charge, Ca-dependent proteases, xanthine dehydrogenase and oxidase, and calcium influx will be measured. Calcium slow channel blocking agents will be used in an attempt to inhibit the calcium influx into the myocardium. The role of molecular 02 during reperfusion will be evaluated by a) using an anoxic perfusion medium and progressively evaluating an increased 02 content in different groups of animals, and b) perfusing at a reduced flow rate, gradually increasing the rate in additional studies. The results of these experiments will be correlated with measurements of myocardial perservation: the high-energy phosphate levels (ATP, CP), CPK, coronary blood flow, 02 consumption, lactate extraction, infarct size, myocardial contractility, etc. The potential sources of the oxygen-derived free radicals during ischemic (neutrophil-mediated) and post-ischemic reperfusion (XO + hypoxanthine + 02 mediated) periods may be quite different, and the above experiments will lead us to derive a conclusion as to the relative significance of such mechanisms. This research will focus attentiion on the clinical events surrounding reperfusion after acute myocardial infarction and cardioplegic preservation during open-heart surgery. The interventions proposed (e.g. use of calcium channel blocking drugs or anti-inflammatory agents and cardioplegic alterations) could have direct clinical relevance to improve myocardial preservation.
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