The principal objective of this proposal is to elucidate the mechanism of plasma membrane disruption during ischemia and to determine whether it is responsible for the onset of injury. Preliminary results indicate that ultrastructurally-inapparent plasma membrane injury occurs early during energy-deficient conditions and, although this injury, per se, does not rupture the plasma membrane, disruption can occur if cells are osmotically swollen, as might occur during ischemia and/or reperfusion. The specific goals are to evaluate the ultrastructurally inapparent membrane injury: its contribution to ischemic plasma membrane disruption, its pathogenesis, its relationship to irreversible injury, and its potential reversibility. In addition, the role and pathogenesis of cell swelling during ischemia will be investigated. The proposed studies will use in vitro preparations including total ischemia in dog heart, perfused rat heart, and perfused rat liver and substrate-free anoxia in incubated thin ventricular slices, perfused rat heart, and perfused rat liver. Plasma membrane damage will be assessed by electron microscopy, by enzyme release, and by plasma membrane permeability to normally-impermeant extracellular markers. The first experimental approach is to decrease intracellular volume prior to the onset of ischemia by perfusion with hypertonic media, which should prevent cell swelling during ishemia. It is expected that there will be no plasma membrane disruption when cell swelling is prevented, but exposure of the ischemia tissue to slightly hypotonic media to produce cell swelling should result in prompt rupture of the plasma membranes. The second aspect is to evaluate the temporal association between the development of the inapparent plasma membrane injury and the onset of irreversible injury. If the inapparent membrane injury is the cause of lethal injury when normal physiologic conditions are restored, then the experimental manipulations will affect equally the development of the inapparent membrane injury and the onset of irreversible injury. The potential reversibility of the inapparent membrane injury will be investigated by instituting hypertonic conditions prior to reoxygenation after a sufficient period of anoxia or ischemia to produce the inapparent membrane injury. Then it will be determined whether an eventual return to isotonic conditions can be accomplished without rupturing plasma membranes. The final aspect is to determine the factors responsible for cell swelling during ischemia. This will be accomplished by manipulating the composition of the extracellular fluid during anoxia and monitoring intracellular volume changes. The research could be of great therapeutic value if conditions are found where plasma membrane disruption can be prevented and the initial plasma membrane injury can be repaired.
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