This project will examine the mechanisms of lung ischemia/reperfusion (I/R) injury with the ultimate goal to discover therapy for I/R an important clinical problem. We will use a new model of lung I/R in awake sheep; after 12 hours of unilateral lung ischemia, reperfusion is associated with a prolonged increase in flow of protein-rich lung lymph, compatible with increased microvascular surface area, abnormal permeability or a combination of both. We propose to apply methods we developed in other models to distinguish increased permeability from surface area during I/R. Because we find no changes in hemodynamics or regional distribution of pulmonary blood flow during reperfusion, we propose that the """"""""no-flow"""""""" phenomenon does not occur in lung reperfusion and will test this hypothesis further. Severe hypoxemia occurs in this model and is associated with increased wet/dry weights and inflammation in both lungs, despite only unilateral ischemia. Neutrophils are increased in both lungs when measured in lung biopsy or in bronchoalveolar lavage at 4 hours of reperfusion. We proposed that the neutrophil is critical to lung I/R, and will investigate this by depletion of circulating neutrophils before I/R. Our data indicated an increase in concentrations of metabolic products of arachidonate, including PGE2, 6-ketoPGF1 alpha, and TxB2; we propose to determine if these and other humoral mediators, including PGD2, LTB4, LTC4, platelet activating factor and tumor necrosis factor increase significanly during I/R. Lipid peroxidation products, including conjugated dienes and malondialdehyde, increase during reperfusion and suggest that free radical oxidant injury is contributory to the lung dysfunction. We will test the hypothesis that the oxidative component of the lung I/R is independent of xanthine oxidase (XO) in our model; we will administer inhibitors of XO before reperfusion and make measurements of XO in alveolar macrophages during both ischemia and reperfusion. Further we propose that reduced glutathione is an important protector against I/R. We will increase lung glutathione by n- acetylcysteinse administration before reperfusion, and will measure reduced and oxidized glutathione as a marker of oxidant stress as well as a marker of antioxidant reserve, in cells of lung lymph and bronchoalveolar lavage before and during I/R injury. Because adenosine provides effective prevention of I/R in heart and intestine, perhaps by inhibition of neutrophil superoxide production, we will infuse adenosine before lung reperfusion to investigate protection. Similarly, we will peform pilot investigation of agents reported to protect in other reperfusion models and which are appropriate for use in whole animals, for instance antiproteases. Finally, we propose to develop a model of lung transplantation in sheep, to complement our active clinical lung transplant program, utilizing information gained herein and applied to lung preservation.
