The Experimental Study On Expression Of Caspase-3, Bcl-2, Bax And Neuronal Apoptosis After Focal Cerebral Ischemia And Reperfusion In Rats

Objective: Stroke is the third largest cause of death in developedcountries. Within the last few years, there has been a growing interest in brain injury after cerebral ischemia. Neuron death following cerebral ischemia and reperfusion has apoptotic and a necrotic component. Apoptosis, programmed cell death (PCD) is an active and reversible process of cellular self-destruction with distinctive morphological and biochemical features. Neuronal apoptosis plays important role during brain injury after cerebral ischemia and the study of apoptic mechanism is signifying meaning to brain injury after cerebral ischemia. At present apoptic mechanism is not completely clear. Cumulative evidence indicates that apoptosis primarily occurs in the penumbra area, whereas rapid energy depletions inside the ischemic core causes acute necrosis. The penumbral area has been considered a therapeutic target in cerebral ischemia. Neuronal apoptosis occurs via activation of molecular signalling pathways resembling apoptosis in nonneuronal cells. Central mediators of apoptosis are two families of molecules: a group of cysteine aspartate proteases, called caspases, and molecules of the bcl-2 family, e.g. bcl-2, bax, and bid. Caspases initiate and execute cell death, while bcl-2 family members modulate death signalling and lead to release of pro-apoptotic molecules from the mitochondrial intermembranous space,e.g. cytochrome c and apoptosis inducing factor (AIF). It has not been reported in domestic and overseas literatures how to cure effectively cerebral ischmia disease. In the present study, rats focal cerebral ischemia-reperfusion (FCIR) injury model were successfully reproduced. To observe the expression of caspase-3 , bcl-2, bax protein and alteration of neuronal apoptosis after focal cerebral ischemia-reperfusion in rats. To study the relation of the expression of caspase-3, bcl-2, bax protein and the number of apoptic neuron and the relation among caspase-3, bcl-2 and bax. This study provide experimental foundation for clarifying apoptic mechanism and prevention and cure of cerebral ischemia-reperfusion damage.Methods: 70 adult Sprague-Dawley rats weighing 250 to 300g were usedand devided into sham operated group, cerebral ischemia-reperfusion groups ( 2, 6, 12, 24, 48 , 72h) at random. Each group has 10 rats. Rats focal cerebral ischemia-reperfusion injury model was established with suture embolimethod from Zea-longa' method improved. Occlusion of the left middle cerebral artery (MCA) was accomplished according to Longa et al. Briefly, rats were anesthetized and the left common carotid artery was exposed through a midline incision, and the internal carotid artery was isolated and carefully separated. A 4-0 nylon monofilament, whose tip was rounded by heating, was introduced up to 17 ± 0.5 mm from the bifurcation of the common carotid artery and the external carotid artery . The monofilament was introduced only up to 10mm in sham operated group. Rectal temperature was monitored and maintained between 36.5 ℃ and 37.5 ℃ with a heating pad. Reperfusion began 2h after middle cerebral artery occlusion (MCAO). In the operated group at prescriptive time point after FCIR and in the sham- operated group at 24h after FCIR , rats were anesthetized and intracardially perfused with nomal saline and then cold PBS containing 4% paraformaldehyde. The rats' brain tissue wasobtained from optic chiasma +7mm to bregma +11 mm . The dislodged brain tissues were fixed in 4% paraformaldehyde for 24h, paraffin sections were made. The slices were grouped into 6 sets and were used respectively for the staining of hematoxylin & eosin (H&E), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), immunohistochemic-al evaluation of caspase-3 and bcl-2 and bax, negative comparison. All data are expressed as mean ± SEM. Comparisons were performed by LSD (the least significant difference) t-test after ANOVA (analysis of variance). Values of P<0.05 were considered statistically significant.Results: The majority of caspase-3 - positive cells were distributedin the inner boundary zones (penumbra) of the cerebral cortex and caudate putamen of the occluded MCA area. On the other hand, only a few cells positively stained for caspase-3 were found in the ischemic core region in the ipsilateral cortex. Bcl-2-positive cells were distributed in the circumambience of ischemic core region. Bax-positive cells were distributed abroad in the ischemic core region and penumbra region. TUNEL-positive cells were distributed in the boundary zones (penumbra) and ischemic cores of the cerebral cortex and caudate putamen of the occluded MCA area. A negligible amount of caspase-3 , bcl-2 , bax , and TUNEL-positive cells were found in the cortical area of the contralateral side. In the sham-operated group, caspase-3, bcl-2, bax positive neurons and apoptotic neurone in cerebral cortex were negligible. Expression numbers of caspase-3 and bax were higher in 2h/rep6h highest in 2h/rep24h and 2h/rep48h. And they went down in 2h/rep72h. Bcl-2 was higher in 2h/rep2h and the highest in 2h/rep6h markedly. Apoptotic cells were higher in 2h/rep6h and were highest in 2h/rep24h and 2h/rep48h. Apoptotic cells went down in 2h/rep72h. The rate bcl-2 and bax is consistent with neuronal apoptosis.Conclusion: Expression of bcl-2 and activation of caspase-3 isnegative correlative. Expression of bax and activation of caspase-3 is positive correlative. The rate bcl-2 and bax is relative to neuronal apoptosis. Caspase-3 and bax protein play key role in ischemia brain injury, and they were involved in apoptosis of neuron after cerebral ischemia together. Bcl-2 play key role in antagonizing ischemia brain injury.