Characterization of spinal HSP72 induction and development of ischemic tolerance after spinal ischemia in rats.
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Induction of heat shock protein (HSP72) has been implicated in the development of ischemic tolerance in several tissue organs including brain and spinal cord. In the present study, using an aortic balloon occlusion model in rats, we characterized the effect of transient noninjurious (3 or 6 min) or injurious intervals (10 min) of spinal ischemia followed by 4-72 h of reflow on spinal expression of HSP72 and GFAP protein. In a separate group of animals, the effect of ischemic preconditioning (3 or 6 min) on the recovery of function after injurious interval of spinal ischemia (10 min) was studied. After 3 min of ischemia, there was a modest increase in HSP72 protein immunoreactivity in the dorsal horn neurons at 12 h after reperfusion. After 6 min of ischemia, a more robust and wide spread HSP72 protein expression in both dorsal and ventral horn neurons was detected. The peak of the expression was seen at 24 h after ischemia. At the same time point, a significant increase in spinal tissue GFAP expression was measured with Western blots and corresponded morphologically with the presence of activated astrocytes in spinal segments that had been treated similarly. After 10 min of ischemia and 24 h of reflow, a significant increase in spinal neuronal HSP72 expression in perinecrotic regions was seen. Behaviorally, 3 min preconditioning ischemia led to the development of a biphasic ischemic tolerance (the first at 30 min and the second at 24 h after preconditioning) and was expressed as a significantly better recovery of motor function after exposure to a second 10-min interval of spinal ischemia. After 6 min ischemic preconditioning, a more robust ischemic tolerance at 24 h after preconditioning then seen after 3-min preconditioning was detected. These data indicate that 3 min of spinal ischemia represents a threshold for spinal neuronal HSP72 induction, however, a longer sublethal interval (6 min) of preconditioning ischemia is required for a potent neuronal HSP72 induction. More robust neurological protection, seen after 6 min of preconditioning ischemia, also indicates that HSP72 expression in spinal interneurons seen at 24 h after preconditioning may represent an important variable in modulating ischemic tolerance observed during this time frame.
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