Basic information concerning the mechanisms responsible for hypoxic cell death is lacking. The overall goal of the proposed project is to define the cellular mechanisms which are responsible for the onset of irreversible injury and cell death using isolated rat hepatocytes as a model system. Cell viability, cytosolic free calcium and pH, mitochondrial membrane potential, lysosomal distribution and integrity, cytoskeletal status, plasma membrane distribution and integrity, and cell surface morphology will be evaluated in single individual hepatocytes during hypoxia by quantitative digitized video microscopy (DVM). DVM will allow the direct observation of the dynamics of organelles and molecules in living cells, providing new information about the roles of these components in cellular function. The onset of irreversible cell injury and cell death and the progression or recovery from injury during reoxygenation will be determined with respect to these cellular functions. Since increased cytosolic free calcium, cell swelling, decreased energy supply, proteolysis and reactive oxygen species have all been hypothesized to play a role in cell death during hypoxia or following reoxygenation, pharmacologic stabilization regimes utilizing calcium antagonists, anaerobic substrates, osmotic agents, protease inhibitors, and scavengers of free oxygen radicals will be assessed with regard to their ability to delay or prevent the onset of irreversible injury and cell death. The role of the cytoskeleton in hypoxic injury will be examined using agents which modulate cytoskeletal structure and function. Additionally, cytoskeletal structure during hypoxia will be visualized using immunocytochemistry in combination with light and electron microscopy. Parallel experiments with suspensions of hepatocytes will also be carried out using conventional biochemical techniques. This project will provide fundamental, new information regarding mechanisms responsible for the onset of hypoxic cell death. This information will be important for the development of treatment modalities effective in the preservation of cells and tissue during hypoxic episodes.
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