Organ transplant therapy has been increasingly utilized in recent years for the clinical treatment of diseased organs. Expanded use of this mode of treatment will come only when mechanisms for the long term storage of donor organs are found. Cryopreservation is an obvious answer yet, to date, freezing of whole organs has been largely unsuccessful; the technology that permits cryopreservation of isolated research will continue using mammalian organs, the problems and solutions to organ cryopreservation can be attacked directly using a vertebrate model animal with a natural tolerance of freezing. The wood frog, Rana sylvatica, readily survives for weeks in a frozen state with up to 65 % of total body water as extracellular ice; freeze tolerance is the key to winter survival for this terrestrially- hibernating species. Studies in my laboratory investigate the molecular mechanisms and biochemical regulation supporting natural freeze tolerance in R. sylvatica. The proposed research provides an organ-specific analysis of the stresses of freezing, the actions of cryoprotectants, and metabolic responses to the frozen state in the wood frog. Isolated cell systems (hepatocytes, synaptosomes) from frogs and laboratory rats are employed for model studies to determine of the effects of freezing and the actions of cryoprotectants (both natural and the agents commonly used in medical cryopreservation) in preserving both structural and metabolic integrity during freezing. Particular emphasis is placed on metabolic actions of cryoprotectants; these have received little prior attention but appear to be key to the choice of glucose as the natural cryoprotectant in the wood frog. The involvement of metabolic depression as a mechanism of ischemia tolerance for long term freezing survival is evaluated and the metabolic regulation controlling cryoprotectant synthesis in liver and permitting the extreme hyperglycemia of the frozen state is assessed.
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