The pathogenesis and treatment of cerebral ischemia based on the mechanism of cytoskeletal changes

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IntroductionIncrease in intracellular calcium (Ca^<2+>) has been speculated to play a pivotal role in the progress of ischemic brain damage. Calpain, calcium-dependent neutral protease, and its endogenous inhibitor calpastatin, may be involved in the pathogenesis, by influencing the breakdown of fodrin, a major constituent of the membrane skeleton in the central nervous system (CNS). We examined the activity of calpain/calpastatin and the proteolysis of alpha-and beta-subunits of fodrin during ischemia in the rat.Methods and ResultsDecapitated heads of rats were incubated at 37ﾟC for 0-40 min. The proteolysis of fodrin in the homogenates were analyzed by Western blotting. The 100 000xg supernatant was applied to a DEAE-cellulose column, and the amount of m-calpain in the 0.4 M NaCl eluate was measured by spectrophotometry as the release of trichloroacetic acid-soluble peptides from azocasein in the presence of 5 mM Ca^<2+>. Calpastatin activity in the heat treated 100 000xg supernatant … More was determined by measuring the inhibition of bovine lung m-calpain.Degradation of alpha-and beta-fodrin were progressively increased with ischemia time. The amount of m-calpain showed no significant changes, and calpastatin activity decreased significantly in 40 minute-period of ischemia.When the heads of rats were incubated at 33ﾟC for 40 min, breakdown product (150-kD fagment) was decreased significantly comparing to that from the samples of normothermia (37ﾟC).In the brain subjected to 30 minute-forebrain ischemia, the amount of calpain was significatnly decreased, and the breakdown of alpha-fodrin was significantly increased during and 60 min after ischemia.Pretreatment with calpain inhibitor-1 (CAI) (iv.) showed the trend inhibition of calpain activation and significant attenuation of the breakdown of alpha-fodrin.Zymography, applicable to smaller samples, showed the regional variation of the m-calpain activation and breakdown of fodrin, which were marked in the hippocampus during (15min) and (60min) after ischemia.ConclusionsThe results suggest that ischemia induces the proteolysis of alpha-and beta-fodrin chiefly through an activation of m-calpain. Hypothermia and CAI provide substantial protective effect by modifying the calpain activity and hence the breakdown of the cytoskeleton of CNS. Less