Ischemia is a widespread and important clinical problem manifested in diverse conditions such as hemorrhagic shock, myocardial infarction, stroke, or local tissue trauma. After reperfusion, a period of microcirculatory no-reflow often ensues, leading to deterioration of microvascular and organ function. Leukocyte adhesion and cytotoxic release are important determinants of the reperfusion injury, but the role of leukocyte deformability and attendant capillary plugging in the injury remains controversial. This study is designed to test the central hypothesis that leukocyte deformability is a significant determinant of the microvascular resistance and tissue injury under pathological conditions and that alterations in cell deformability are primarily mediated by reorganization of the cell cytoskeleton. The long-range goal is to advance understanding of the cellular mechanisms responsible for deterioration of microvascular function in low-flow organ pathology.
The specific aims of the research are to measure the leukocyte deformability in vivo, the frequency and duration of leukocyte-capillary plugging, the microvascular resistance, and the extent of tissue injury during leukocyte activation by the chemoattractant peptide FMLP, and in a model of local ischemia and reperfusion in skeletal muscle, both with and without leukocyte cytoskeletal reorganization. Intravital microscopy of the rat spinotrapezius muscle will be employed to measure l) microvascular pressure gradients across single leukocyte-capillary entrance plugs using a dual micropuncture technique, 2) leukocyte plug dimensions and durations and vessel dimensions using video methods, and 3) propidium iodide uptake by non-viable skeletal muscle cells using fluorescence imaging. The measurements allow determination of the leukocyte deformability, the extent of leukocyte-capillary plugging, the microvascular resistance in arteriovenous capillary units, and the distribution of tissue injury in the same units. Using cytochalasin-D, colchicine, and pentoxifylline in conjunction with the FMLP and ischemia-reperfusion protocols, a test will be made of the hypothesis that leukocyte deformability is a significant determinant of microvascular resistance under pathological conditions, and that reorganization of cytoskeletal F-actin mediates the deformability changes. Histological reconstruction of serial sections will be used to determine the cellular composition of permanent plugs. Finally, using a hemodynamic computer network model to link the measured cell deformabilities to microvascular resistance changes, a test will made of the hypothesis that the macroscopic no-reflow response is quantitatively related to underlying changes in leukocyte cytoskeletal properties. The capability to link cellular or microcirculatory events to macroscopic organ hemodynamics provides a basis for understanding the relative roles of cell cytoskeletal reorganization and adhesion/cytotoxic release in ischemic injury, leading ultimately to improved therapeutic measures.
