| PrfaceStroke is a major cause of death and disability in the elderly. A phenomenon of " ischemic tolerance" in animals previously subjected to short ischemic episodes, rendering the more tolerant to subsequent occurrences of persistent cerebral ischemica. " Preconditioning " is a phenomenon in which brief exposure to ischemia makes brain less vulnerable to damage after longer ischemia, ischemic tolerance has drawn considerable attention as it provides a way to study the mechanisms of endogenous neuroprotection. The mechanism of " ischemic tolerance" is not well understood. Evidence supporting apoptotic modulation in cerebral ischemia includes the slow progressive nature of selective neuronal cell death in the hippocampus following global ischemia. The upregulation of apoptosis - promoter genes may be indicative of a tendency toward cell death. The overexpression of apoptosis - suppressor genes in brain may reflect a mechanism compatible with endogenous neuroprotection.To further investigate the potential role of Bel - 2 and Bax as an effecter of ischemic tolerance in brain. We used the intraluminal suture technique to precondition the striatum against subsequent focal ischemia and studied the expression of Bel - 2 and Bax protein in the region of tolerant stratum.Experimental Data1. subjectsMale Wistar rats weighing 240 to 270g were, used in all experiments and were allowed free access to food and water before and after all procedures, animals were randomly divided four group: the sham -operated group ( SO group), the ischemic preconditioning group (IP group),the ischemic preconditioning-cerebral ischemic group(IPCI group) and cerebral ischemic group (CI group) .2. ReagentTTC,Bcl -2,Bax and POD kits were supplied by WuHan Bostor experimental Co.Experimental Methods1. Animal methodThe rats were anesthetized with. 10% Chloral hydrate (350mg/ kg). The bifurcation of the common carotid artery (CCA) was exposed and external carotid artery ligated with suture. The internal carotid artery (ICA) was isolated and separated from the vagus nerve. The origin of the middle cerebral artery (MCA) was occluded by introducing a 3 -0 monofilament nylon suture (its tip rounded by heating) into the ICA lumen, through the CCA, the suture was gently advanced 17 to 20mm past CCA bifurcation, then the CCA was ligated and the wound closed.The sham - operated group underwent the same surgical procedure,anesthesia, the suture was inserted and advanced for a distanceof 5mm beyond the ICA/ECA bifurcation. The CI group underwent 60 minutes MCA occlusion. Two - 5 - minutes MCA occlusion separated by 5 minutes reperfusion was used to induce precondition. 3 days later, the IPCI group were again challenged with 60 minutes of focal ischemia.2.TTCBrain were rapidly removed and sectioned coronally at 2 - mm intervals. The sections were immersed in 2% 2,3,5 - triphenyltetrazoli-um hydrochloride (TTC) in saline for 30 minutes at 37 C and then transferred to 4% paraformaldehyde. Six sections were analyzed for infarction size by using a computerized imaging analysis system. Infarction volume was calculated by summing the infarction areas of all sections and multiplying by the slice thickness.3. Perfusion Fixation EmbedmentAfter animals were anesthetized, they were perfused transcardial-ly with saline (100ml) and 4% paraformaldehyde (100ml). Brain were removed and postfixed in the same fixative for 24 hour, then were embedded in pafaffin. And 6-m-thick coronal sections were taken at - 0. 3mm from the bragma. Some section were stained with cresyl violet, and some section were for immunocytochemistry4. immunocytochemistrythe sections were deparafinized , then heated and boiled by microwave. Nonspecific activity was blocked with normal goat serum. Brain sections then were incubated at 37 for 2 hour with rabbit monoclonal antibody against mouse Bel - 2 or Bax, and then incubated 20 minutes with goat anti-rabbit immunconjugate. The section for Bcl-2 or Bax staining were counterstained wit... |
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