Liver transplantation is the only effective, long term therapy for irreversible liver failure. An important impediment to the wider use of this surgery is the fact that human donor livers can be preserved for only about 8 hrs before irreversible damage occurs. The overall goal of this proposed project is to optimize conditions for liver preservation using the isolated rat liver as a model. Cell viability, metabolism, microcirculation, bile formation and structure will all be assessed ex vivo in isolated livers. Cell viability will be determined by trypan blue uptake and enzyme release, metabolism by miniature surface probes and by metabolite content and release, microcirculation by vascular casts and fluoroscein-dextran clearance, bile formation by bile release from the cannulated bile duct, and structure by light microscopy and by scanning, transmission and freeze-fracture electron microscopy. Storage conditions will be optimized with respect to ionic composition and osmolarity of the storage solution using Ca++-free Collins solution as a starting point. During cold, ischemic organic storage lack of oxygen and glycolytic substrates limits ATP generation and leads to depletion of adenine nucleotide pools. Substrates which support ATP formation (e.g., oxygen, fructose, adenosine) will be evaluated for their ability to extend viability. Cold, ischemic preservation will be compared to perfusion preservation during which ATP-generating substrates are infused continuously. Increased cytosolic Ca++ may serve as a final common pathway for irreversible cell injury. Pharmacologic stabilization regimes utilizing Ca++-entry blockers and calmodulin antagonists will be evaluated in extending the viability of stored livers. Nutritional status (e.g., fed vs. fasted vs. refed) of the donor animal will be assessed in extending viability of stored livers. Glycogen rich livers are expected to be more resistant to damage, and gluconeogenic substrates which replete glycogen may extend viability. Conditions for reoxygenating and reperfusing stored livers will be optimized with respect to ionic composition, osmolarity, ATP-generating substrates and Ca++ antagonists. Scavengers of activated oxygen and blockers of its formation including allopurinol will also be tested in reducing cell injury during reoxygenation. In the final phase of these studies, optimal storage protocols developed for isolated rat livers will be assessed in dogs following actual transplantation surgery. Optimized conditions for liver storage established in these studies will then be ready for testing in a clinical setting. This project will provide useful, clinically relevant new information on measures to prolong liver preservation for transplantation surgery.
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