εPKC May Contribute to the Protective Effect of Hypothermia in a Rat Focal Cerebral Ischemia Model

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Abstract

Background and Purpose—

Protein kinase C epsilon (εPKC) has been implicated as a neuroprotectant in vitro. We studied εPKC activation (by its localization and proteolysis) in a rodent stroke model and correlated the effects of hypothermia with εPKC activity after cerebral ischemia.

Methods—

Rats were subjected to permanent distal middle cerebral artery occlusion plus 1 hour of bilateral common carotid artery occlusion. Body temperatures were maintained at 37°C or 30°C during common carotid artery occlusion. Brains were harvested at 10 minutes, 4 hours, and 24 hours after common carotid artery release, and the cortex corresponding to the ischemic core and penumbra was dissected. εPKC localization after stroke was assessed by Western blot and immunofluorescence microscopy. A caspase-3 inhibitor was used to test whether εPKC cleavage is caspase dependent.

Results—

εPKC in the membrane fraction and whole-protein homogenates decreased moderately in the penumbra but decreased markedly in the ischemic core. Hypothermia blocked this decrease in both the ischemic core and penumbra. Confocal microscopy confirmed that neuronal εPKC expression decreased in the ischemic core at 4 hours after reperfusion, and this loss was prevented by hypothermia. Two carboxyl-terminal cleavage products of εPKC with molecular masses of 43 and 35 kDa were detected. Although the protein band of 43 kDa decreased after stroke, the 35-kDa band increased. Such changes were not dependent on caspase-3. However, hypothermia blocked changes in the cleavage form of 35 kDa but not 43 kDa after stroke.

Conclusions—

Moderate hypothermia preserves εPKC activity after stroke.

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