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KMID : 1005420050070040317
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Journal of Cerebrovascular and Endovascular Neurosurgery
2005 Volume.7 No. 4 p.317 ~ p.323
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Changes in HSP 70 Distribution to Diazepam After Transient Global Ischemia in Rat
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Kim Jin-Joo
Kim Jae-Kwang
Lim Yong-Su
Yang Hyuk-Jun
Park Cheol-Wan
Lee Keun
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Abstract
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Purpose: Cerebral ischemia leads to neuronal damage in specific area of the brain of both rodents and humans. After ischemia, the pyramidal cells in area CA1 of the hippocampus are particularly sensitive but death of these pyramidal cells in delayed at least 3-4 days after a transient cerebral ischemia. Induction of Heat shock proteins 70 (HSP70) has been mainly observed in resistant area, and thus a protective effect of HSP70 has been proposed. The presence of HSP70 is a good indicator of the general pathophysiologic stress response to ischemia, but not necessarily a marker for neuronal survival. The present study investigated the change of HSP70 in ischemic and diazepam treated ischemic rat, and evaluated the distribution of HSP70 in the rat brain.
Methods: Fisher344 rats were used in the present experiments. Mild transient ischemia was induced by two-common carotid artery occlusion and reperfusion. Ischemia-treated and sham-operated rats were injected i.p. with 10 mg/kg diazepam or vehicle at 30 minutes, and again 90 minutes following the onset of reperfusion. After 1, 2 and 7 days following the reperfusion, brains were removed and immunohistochemistry and Western blotting for HSP70 were performed. For immunostaining, brain tissues were prepared following intracranial perfusion and frozen sections cut on a cryostat. Sections were incubated with a mouse monoclonal antibody by free-float method. Hippocampal tissues at the indicated time were prepared for Western Blot analysis. For the determination of protein levels of HSP70, tissue extracts were subjected to ECL Western Blot analysis using primary antibody. The result from Western Blotting was expressed numerically through the image analysis.
Results: HSP70 expression is markedly increased in 2 day groups of mild transient cerebral ischemia after reperfusion. HSP70-positive cells were observed in the cerebral cortex, striata terminalis, hippocampus after 2 days following the onset of ischemia. HSP70 expression increased in ischemic damaged rat brain at 1 day after reperfusion and immunostaining became particularly intense in the cerebral cortex and hippocampus. At 7 days HSP70 expression were increased in ischemic rat and decreased in the diazepam treated rat brain. In preconditioning ischemia, there is no ischemic cell death due to necrosis and apoptosis.
Conclusion: HSP 70 expression increased in diazepam-treated group after 2 days following the onset of the ischemia. But, after 7 days following the onset of the ischemia, HSP 70 expression of diazepam-treated group decrease more than diazepam-untreated group. In the morphologic findings, HSP 70-positive cells were observed in the cerebral cortex (S1 cortex, insular cortex, auditory cortex, visual cortex), stria terminalis, hippocampus (CA1 And CA3 region) after 2days following the onset of ischemia.
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KEYWORD
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HSP 70, Cerebral Ischemia, Diazepam
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