NEUTROPHILS IN ISCHEMIA REPERFUSION INJURY IN SHOCK

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A significant portion of the injury that occurs following ischemia and reperfusion has been established to occur during the reperfusion period. This reperfusion injury is leukocyte mediated and blocking leukocyte adherence with monoclonal antibodies provides significant protection from injury. Likewise, mild hypotheremia (1-4 degrees C) during ischemia and reperfusion results in a significant reduction in injury. Ischemia-reperfusion is seen in a variety of clinical disorders including myocardial infarction, stroke, organ transplantation, hemorrhagic shock and traumatic injuries. Clearly, this broad spectrum of clinical disorders that may have a component of reperfusion injury places it as a major cause of mortality and morbidity in the United States. Prolonged ischemia in these disorders results in significant injury due to tissue hypoxia and rapid return of perfusion will reduce this type of injury. However, reperfusion of previously ischemic tissue can initiate an inflammatory response that results in significant additional tissue injury. Blocking leukocytes adhesion has been shown experimentally to significantly reduce portion of this reperfusion injury. Likewise, mild hypothermia has been shown to reduce the injury that occurs in ischemia and reperfusion. The hypotheses of this project are: 1) The expression and/or activation of leukocyte adhesion molecules on endothelial cells or on neutrophils (pMNs) is dependent on small changes in temperature (1 to 4 degrees C). Adhesion molecule function is reduced during mild hypothermia, thus providing protection from ischemia and reperfusion injury. and 2) Significant tissue protection results from mild hypothermia during the immediate post reperfusion period (4-8 hours). Therefore, maintaining patients slightly hypothermic during this period may provide protection from injury and be of significant therapeutic benefit. The specific aims are: Aim 1 A: To determine the stimulated expression of adhesion molecules CD62P, CD62E, CD106, and CD54 as well as expression of mRNA by cultured HUVECs using ELISA and Northern blot analysis at 33, 35, 37, and 39 degrees C. Aim 1 B: By understanding the mechanism of hypothermia-mediated inhibition of gene expression in endothelial cells, it will be possible to reverse or activate the effects of hypothermia in vivo - Aim 2: To measure indicators of PMN function and activation following stimulation of leukocytes with PMA, heat killed bacteria, and C5a. Temperature will be set at 33 degrees C, 35 degrees C, 37 degrees C and 39 degrees C. Aim 3: To determine the effect of mild hypothermia on leukocyte rolling and adherence under flow conditions at temperatures of 33, 35, 37, and 39 degrees C. Aim 4: To determine the quantitative changes in expression of CD62P, CD62E and CD54 and their mRNA in rabbit ear ischemia- reperfusion at ambient temperatures of 22 degrees C and 24 degrees C. Aim 5: To determine the timing of hypothermia that is effective in preventing tissue injury following ischemia and reperfusion. Aim 6: To determine the protective effects of reduced temperature following hemorrhagic shock and resuscitation. The experiments of this project will use cell and molecular biology techniques and will be completed by using in vivo and in vitro experiments.