Process Of Neuronal Injury And Protect Effects Of Xinnaoshutong Capsule After Cerebral Ischemia And Reperfusion In Rats

Cerebrovascular disease is a clinical common, frequently encountered disease in the elderly. The rates of morbility, deformity and mortality are very high. It is one of the important diseases that lead significant harm to human health. Among them, the incidence of ischemic cerebrovascular disease is more accounted in the clinic. It is the focus and current research which is resume cerebral blood flow as soon as possible and, if possible, reverse the injury for nerve cells. But if we can not restore blood flow in time, it will lead brain ischemia and reperfusion to injury, and exacerbate the patient's conditions. Recently, it was found that cell injury is a dynamic, rapid development of the cascade after cerebral blood flow interruption and reperfusion of the brain. The injury cascade includes lots of pathological mechanisms of excitatory amino acid toxicity, energy metabolism obstruction, intracellular calcium overload, inflammatory response and so on.This study using two models: focal cerebral ischemia and reperfusion injury in rats and cortical neurons in primary culture induced by hypoxia/reoxygenation. From the integral and cellular levels, changes of neuronal apoptosis and necrosis during time-space as a focal point, to dynamic observe the time-space change during the process of neuronal injury after cerebral ischemia and reperfusion in rats. Using the clinical effective drug-Xinnaoshutong capsule to treat cerebral ischemia-reperfusion injury, to observe changes of oxygen free radicals, inflammatory cytokines, cytoskeleton and apoptosis. Combining with morphology to observe pathological changes in rat brains, to discuss the protective effects of Xinnaoshutong capsule and the possible mechanisms on cerebral ischemia and reperfusion injury in rats. At the same time, relating with the doctrine of"internal toxin hurting brain collaterals"in Chinese Medicine, from the pathophysiological process of neuron injury after cerebral ischemia and reperfusion and the protective mechanisms of Xinnaoshutong capsule, to provide modern scientific basis for the doctrine of internal toxin hurting brain collaterals, enrich and improve the basic theory of Chinese Medicine for transient cerebral ischemia and reperfusion injury.The experiment includes three parts:PartⅠ:Dynamic changes on morphology and apoptosis at time-space after cerebral ischemia and reperfusion injury in rats. (in vivo experiment)AIM:To discuss the dynamic changes on pathological morphology and apoptosis of the different region in the rat brain at different times of reperfusion after 1.5 hours of ischemia in rats.Methods: Animal model of focal cerebral ischemia and reperfusion is the thread occlusion technique induced middle cerebral artery occlusion in rats. Rats were randomly divided into seven groups: sham group, and 3h, 6h, 12h, 24h, 48h, 72h of reperfusion respectively after 1.5h of ischemia by transient occlusion of middle cerebral artery. Observing morphological changes of brain tissue and neuron by HE staining and Nissl staining. Observing ultrastructure changes of nerve cells by electron microscope. Analyzing apoptosis of cortical neuron by TUNEL staining.Results:Different degrees of neurological deficits and neuronal apoptosis were observed at different times of reperfusion after 1.5h of ischemia. The main injury of brain tissues were cerebral cortex and caudate putamen. Neurons were showed the phenomena of neuronal edema, gap widened between cells, and nuclear condensation by HE staining and Nissl staining. Neurons edma and similar apoptosis body were also showed by electron microscope. The trend of pathological injury of rat brain is the most serious between 12~48h of reperfusion after 1.5h of cerebral ischemia. Apoptosis appeared obviously between 12~48h of reperfusion, which peaked at 24h of reperfusion. Injury area of cerebral cortex was wider and apoptosis was much more than of caudate putamen.PartⅡ:Protective effects and mechanisms of Xinnaoshutong capsule after cerebral ischemia and reperfusion injury in rats. (in vivo experiment)AIM:From aspects of oxygen free radicals, inflammatory cytokines, cytoskeleton, apoptosis-regulating cytokines after cerebral ischemia-reperfusion injury, and combing with morphology to observe the cerebral changes, to study protective effects and mechanisms of Xinnaoshutong capsule on cerebral ischemia and reperfusion injury in rats,Methods: Animal model of focal cerebral ischemia and reperfusion is the thread occlusion technique induced middle cerebral artery occlusion in the rat. Rats were randomly divided into three groups: Sham group, 24h of reperfusion after 1.5h of transient cerebral ischemia model group, Xinnaoshutong capsule treatment group. Observing changes of cortical neurons by HE staining and Nissl staining, and ultrastructure changes of nerve cells by electron microscope. Detecting the activity or content of MDA, SOD, LD and LDH in ischemic cerebral brain tissues by spectrophotometry. Detecting the content of IL-1βand TNF-αin serum and ischemic cerebral brain tissue by RIA and ELISA respectively. Detecting changes of protein expression of MAP-2,β-tublin, Bax, Bcl-2, Fas, Fas-L and Caspase-3 by immunohisochemistry, and gene expression of Caspase-3 by RT-PCR.Results:(1) Obvious symptoms of neurological deficits and morphological changes, such as ischemic brain edema, nuclear condensation, were observed 24h of reperfusion after 1.5h of transient cerebral ischemia.(2) Xinnaoshutong capsule has the following effects on reperfusion after cerebral ischemia: improving the activity of SOD, whereas reducing activity or content of MDA, LD and LDH, reducing content of IL-1βand TNF-αin serum and ischemic cerebral brain tissue, increasing the protein expression of MAP-2 andβ-tublin, increasing the protein expression of Bcl-2 and the ratio of Bcl-2/Bax, and reducing the protein expression of Bax, Fas, Fas-L and caspase-3, then reducing the number of apoptotic cells, to alleviate cerebral injury and protect rat brain tissue after ischemia and reperfusion injury.PartⅢ:Protective effects and mechanisms of Tribulus terrestris L. Saponion(TTLS) on apoptosis in cultured rat cortical neurons. (in vito experiment)AIM:From the changes of intracellular calcium, mitochondria membrane potential and cytosolic Caspase-3/7 activity, to study the protective effects of Tribulus terrestris L. Saponion (TTLS) on apoptosis induced by hypoxia/reoxygenation in cultured rat cortical neurons, and to discuss the possible protective mechanisms.Methods: Rat cortical neurons in primary culture, and the apoptosis model was induced by hypoxia/reoxygenation. The condition of model neurons were made with mixture of 95% N2 and 5% CO2 for 3h induced hypoxia, and then with mixture of 95% O2 and 5% CO2 for 12h induced reoxygenation. Different concentration of TTLS is respectively treated from hypoxia until the end of reoxygenation. The activity of cortical neurons was observed by MTT, LDH releasing rate was detected by spectrophotometry, apoptosis neuron morphology and apoptosis percent of cortical neurons were analyzed by AO staining and flow cytometry with Annexin V-FITC and PI staining. Intracellular free Ca2+([Ca2+]i) with Fluo-3 fluometry and mitochondria membrane potential with JC-1 fluometry were observed with a confocal laser-scanning microscope and determined by mean fluorescent value, cytosolic Caspase-3/7 activity were measured by fluorescent plate reader. Bax protein expression were observed by immunohistochemical technique.Results:At different times of reoxygenation after 3h of hypoxia could be cause cortical neurons damage. In them, 3h of hypoxia and 12h of reoxygenation induced damage seriously, which was mainly with apoptosis. TTLS could enhance cortical neurons activity, reduce LDH releasing rate and neuronal [Ca2+]i, increase membrane potential, decrease cytosolic Caspase-3/7 activity, and reduce Bax protein expression, then to inhibit apoptosis of cortical neurons induced by hypoxia/ reoxygenation.Conclusion:Integrated analyzing the results of in vivo and in vito experiments, we drew a conclusion as follow:Different degrees of apoptosis and brain injury induced by focal cerebral ischemia and reperfusion. Apoptosis had positive correlation with damage of brain tissues. The main injury of brain tissues were cerebral cortex and caudate putamen, injury area of cerebral cortex was wider, pathological damage and apoptosis were focused on 24h of reperfusion. Therefore, this time of cerebral ischemia and reperfusion model could be used for the research of drug intervention. Xinnaoshutong capsule could decrease the damage of free radical and expression of inflammatory cytokines, inhibit intracellular calcium overload, keep mitochondria membrane potential stabilization, and adjust the expression of several apoptosis regulating cytokines, then to inhibit neuronal apoptosis. We speculated that occurrence and development and final outcome of ischemia and reperfusion injury accord with the doctrine of"toxin injury hurting brain collaterals"in Chinese Medicine. The redundant products of free radicals and its metabolites, excessive inflammatory cytokines and overload intracellular calcium correspond to the"internal toxin". The brain structure injury and neuronal apoptosis correspond to the final outcome of"internal toxin injury hurting brain collaterals". The internal toxin includes fire toxin, phlegm toxin and blood stasis toxin and so on, so must find and relieve the origin the toxin to eliminate the internal toxin. Xinnaoshutong capsule could activate blood circulation, resolve blood stasis and dredge brain collaterals to alleviate the injury of brain infarction induced by"blood stasis toxin", comprehensively improve damaged brain tissues, and get to the protective effect of neuron. It is suggest that Xinnaoshutong capsule has the effective prevention and treatment in cerebral ischemic cerebrovascular diseases.