Efforts to develop effective measures for the treatment of stroke have generally been based on the implicit assumption that one, or at the most several, factors control the progressive brain injury that occurs during the early hours of focal brain ischemia. Postischemic progression of brain damage appears to be extremely multifactorial. There is a finite probability that the assumption underlying most therapeutic stroke trials that seek to identify a dominant or controlling factor that determines postischemic progression of brain damage is incompatible with the fundamental nature of the problem. Postischemic progression of brain damage may be the result of a constellation of minor causes and the quest for a dominant or controlling cause would then be ultimately futile. Unconventional approaches may be required to arrest cellular destruction in brain ischemia. This project continues to investigate mammalian hibernation, a state of natural tolerance to severely reduced blood flow and oxygen delivery. Efforts to isolate and identify the factor or factors that regulate the controlled metabolic depression and tolerance of profound brain ischemia that forms the essence of natural hibernation are in progress. In hippocampal slices, hibernation confers robust resistance to hypoxia and glucose deprivation as compared to slices from non- hibernating ground squirrels and rats at 37 degrees C, 20 degrees C and 7 degrees C. This indicates that hibernation involves tolerance to an in vitro form of ischemic stress that is not strictly dependent on temperature. Protein synthesis in the hippocampal slices was found to be greatly depressed at the same incubation temperatures. Protein synthesis (PS), in vivo, was below the limit of autoradiographic detection in brain sections, and in brain extracts was determined to be 0.04% of the average rate from active squirrels. Further, it was threefold reduced in cell-free extracts from hibernating brain at 37 degrees C, eliminating hypothermia as the only cause for protein synthesis inhibition (active 0.47 plus/minus 0.08 pmol/mg protein/min; hibernator 0.16 plus/minus 0.05 pmol/mg protein/min, p<0.001). PS suppression involved blocks of initiation and elongation and its onset coincided with the early transition phase into hibernation. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2a are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a threefold increase in ribosomal mean transit times in cell-free extracts from hibernators (active 2.4 plus/minus 0.7 min; hibernator 7.1 plus/minus 1.4 min,p<0.001). Phosphorylation of eEF2 is increased,eEF2 kinase activity is increased and protein phosphatase 2A activity is decreased during hibernation which contribute to the elongation block. No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate trickle blood flow and reduced substrate and oxygen availability. Brain levels of growth arrest and DNA damage-inducible protein-34 (GADD-34) have been found to increase during hibernation. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state. - hibernation, cerebral ischemia, stroke, homeostasis, protein synthesis,metabolism, blood flow, hippocampal slice
