The general focus of this laboratory has been the study of acute pulmonary insufficiency. Currently, our aims are to examine the mechanisms of injury following 2h of hindlimb ischemia, to define the mediators released and their role in the induction of hindlimb and pulmonary microvascular permeability. It is believed that reperfusion following ischemia will lead to synthesis of the chemoattractants and chemoativators leukotriene (LT)r and oxygen free radicals. These agents, directly or indirectly by stimulating polymorphonuclear leukocyte (PMN) entrapment and thromboxane (Tx) A2 synthesis are thought to moderate local endothelial cell (EC) permeability by causing disassembly of actin filaments. This alteration in the cellular cytoskeleton permits instability and enlargement of interendothelial junctions. If the ischemic stimulus to the hindlimb is sufficient to cause release of chemoattractants and chemoactivators into the circulation, pulmonary damage may be the consequence of the direct action of these agents ont he microvasculature as well as the secondary consequence of PMN entrapment with release of vasotoxic agents including TxA2 in the lungs. Hindlimb ischemia in the anesthetized rat or sheep will be produced by tourniquet occlusion for 2h. Plasma titers of the cicosanoids LTB4 and TxB2 will be measured by high pressure liquid chromatography and radioimmunoassay. In sheep with a hindlimb venous tourniquet or a left atrial balloon the degree of microvascular permeability in the hindlimb or lung will be assessed by measure of lymph flow and lymph/plasma protein concentration in lymph collected from the popliteal lymphatic or caudal mediastinal lymph node respectively. In order to test the location of eicosanoid synthesis, titer in plasma and lymph will be assayed and experiments conducted in animals depleted of circulating PMN using chemotherapy, anti-PMN antibodies or crystalloid perfusion of the hindlimb. Isolated PMN as well as the rat model will be used to assay PMN activation by flow cytometry and to quantitate pulmonary leukosequetration and edema following hindlimb ischemia. In order to document the importance of a mediator, experiments will be designed as often as possible to: (1)assay levels of the putative mediator and relate its appearance to pathophysiologic alterations (2) assess the ability of antagonists to prevent the pathophysiologic effect and (3) infuse a putative mediator such as LTB 4, oxygen free radical or Tx mimic and demonstrate the occurrence of the pathophysiologic effect. In vitro methods using pulmonary artery and pulmonary microvessel endothelial cells (EC) grown on an amnion membrane or microcarrier beads will be used to study mechanisms of PMN diapedesis and EC permeability. The emphasis will be on quantitating the ability of the eicosanoids by means of membrane cytoskletal modifications to mediate both diapedeses and macromolecular permeability. Taken as a whole these data should define a role of eicosanoids, oxygen free radicals and PMN in the local and pulmonary response to ischemia as well as demonstrate a role for actin filaments in modifying the microvascular barrier.
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