The attachment of leukocytes to microvascular endothelium and migration into the interstitium is the hallmark of the inflammatory reaction that is especially deleterious in sepsis, shock, and episodes of ischemia/reperfusion. We have been able to demonstrate in previous studies that leukocyte entrapment in the microcirculation in the form of capillary plugging and post-capillary attachment to the endothelium constitutes a key facet of the disruption of tissue homeostasis. Our past studies, which were focused largely on intravascular events, will be expanded to explore the role of different leukocytes in tissue breakdown during the period of cell migration across the wall of microvessels and during amoeboid migration within the interstitium. Our proposed studies are designed to provide details of the key events during leukocyte mediated tissue injury, utilizing a newly developed digital fluorescent intravital microscopy approach in selected tissues of the rat. Our hypothesis proposes that activated leukocytes migrating in the tissue and activated endothelial cells serve as sources for free radical mediated tissue injury after stimulation. Fluorescent markers for peroxide formation, mitochondrial function and cell death will be utilized in conjunction with time-lapse microphotography to record sequential details of the events associated with leukocyte migration in the tissue. The membrane adhesion mechanisms (integrins and selectins) that are involved in interstitial migration will be investigated in the presence of selected stimulators. The mechanisms responsible for the production of oxygen free radicals and their consequences for parenchymal cell death will be quantified the first time in vivo at high optical resolution and identified. During the intravascular interaction between neutrophils and endothelial cells, peroxidation by iron catalyzed hydroxyl radical formation takes place in the region of close contact between the two cellular membranes. This mechanism will be explored in vivo under conditions where leukocytes are migrating into the interstitium in response to different proinflammatory stimulators. One observes regularly during migration of leukocytes across the microvascular wall, the development of micro-hemorrhagic erythrocyte diathesis and formation of interstitial pools of red cells. This phenomenon has been observed in many clinical situations but its underlying cellular and molecular mechanisms are unexplored. It is our hypothesis that formation of micro- hemorrhages requires an initial adhesion and diapedesis of leukocytes, and that red cell escape into the interstitium is associated with iron catalyzed hydroxyl radical formation and in turn enhanced parenchymal peroxidation and cell death. The mechanisms by which leukocytes in diapedesis form pores large enough for red cell escape will be investigated using intravital and electron microscopy in combination with modeling. The production of peroxides, cell death in the interstitium in the presence of micro-hemorrhages, and degeneration of connective tissue cells will be investigated utilizing digital fluorescent microscopy in vivo. A systematic program along these lines will serve to identify key mechanisms that lead to tissue damage in the presence of activated leukocytes and endothelial cells.
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