Ischemia-reperfusion (I-R) injury of skeletal muscle remains a significant limiting factor in the successful outcome of major limb traumatic injuries associated with prolonged periods of ischemia. Research-to-date by others and in our own laboratory indicates that hyperbaric oxygen (HBO) has significant potential as a treatment for I-R injury. The long-term objectives of this project are to determine the basic mechanisms and optimal dosing schedules of HBO treatment for l-R in skeletal muscle. I-R injury in the microcirculation is characterized by an early significant increase in neutrophils adhering to the endothelium of postcapillary venules that is associated with a severe and progressive vasoconstriction in adjacent microarterioles. Administration of HBO ameliorates these deleterious microcirculatory events when given up to l hour after reperfusion. Although these findings are exciting and have potential therapeutic effects, the mechanisms by which HBO antagonizes neutrophil- endothelial adhesion and prevents microarteriolar vasoconstriction remain unknown. The specific aim of this proposal is to investigate these mechanisms by experiments in 4 related areas. First, to determine the effect of HBO on pulmonary leukosequestration. HBO has the potential to induce sequestration of neutrophils in the lungs which may decrease delivery of these cells to the ischemic muscle during reperfusion. This will be tested using a rat gracilis muscle I-R injury model evaluating neutrophil-to-muscle delivery and lung PMN accumulation by quantitative histology and myeloperoxidase activity. Second, to determine the effect of HBO on PMN-endothelial interaction focusing on the neutrophil CD18 adhesion molecule. A monoclonal antibody (MAB) against CD18 will be used in the in vivo rat gracilis muscle microcirculation preparation to quantitate neutrophil endothelial adhesion and determine if adhesion is CD18 dependent. Neutrophils will then be collected from the reperfused effluent of the ischemic gracilis muscle to see if HBO is altering expression of CD18 by use of labeled MAB. Third, to determine the role of thromboxane (Tx) in the arteriolar vasoconstriction associated with I-R and the effect of HBO. A TxA2 antagonist will be used in the microcirculation model to determine the effect on arteriole vasoactivity by measuring vessel diameters during reperfusion. Thromboxane concentration will then be measured from the gracilis venous effluent during reperfusion to determine if HBO is decreasing production of this vasoactive substance. Finally, the optimal dosing and functional outcome of HBO will be tested in a clinically relevant rabbit hindlimb model of I- R. This research should provide insight into the mechanisms of HBO in I-R injury which ultimately could significantly impact limb salvage after traumatic injury and could have even broader implications in the treatment of I-R injury in other organ systems.