| Objective:Stroke, also known as cerebrovascular accident, is an emergent cerebral blood circulation disorder. Stroke can be classified into two major categories:ischemic and hemorrhagic, about85%of strokes are caused by ischemia. Due to its the high incidence and poor prognosis, stroke has emerged as the leading cause of disability and one of the top three causes of mortality in the elderly, which is regarded as serious threat to human health. Chinese medicine believes that the pathogenesis of ischemic stroke might be attributed to virtual six factors, including weak, fire, wind, phlegm, qi and blood. The interaction of these six factors under certain conditions might lead to the sudden onset of stroke. Acupuncture, the representative non-drug treatments of traditional Chinese medicine, is easy to be operated with high safety, meeting the healthy demands of modern society. The therapeutic benefits of acupuncture in the treatment of ischemic cerebrovascular disease have been extensively confirmed in clinical practice. Given the clinical benefits of acupuncture and the important roles of angiogenesis in the recovery of cerebral ischemia, we planed to investigate whether angiogenesis is involved in the therapeutic effects of electroacupuncture (EA) in cerebral ischemia and the possible mechanism. The present researches would provide experimental basis for the modulatory effects on angiogensis and the protective effects on ischemic brain injury of EA.Methods:Male SD rats with clean grade were randomly divided into EA group, model group, sham operation group (n=10). Middle cerebral artery occlusion was induced by intraluminal filament in EA group and model group for60min, and then the blood flow was restored. After reperfusion for2h, rats in EA group received EA at Baihui, Zusanl i and Baihui with density wave (frequency:2Hz/15Hz, intensity:1mA) for30min and this treatment was repeated every12h. Tweenty-four hours later, brain tissue was harvested and the level of VEGF mRNA was determined by real time PCR. Immunoblot was performed to detect the expression of VEGF and immunohistochemistry was used to examine the expression of angiogenesis-related factors in the ischemic area after24hours’reperfusion. At72h after reperfusion, the behavior impairment of the experimental animals and the microvessel density of the ischemic area were evaluated. In addition, the infarct volume was determined by TTC stain method, the apoptosis was detected with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and the expression of apoptosis-related protein was determined by immunoblot.Results:(1) The neurological injury score gradually decreased with the prolongation of reperfusion time for24h,48h and72h in model group and EA group, and at each time point EA group compared with model rats the neurological injury score of EA group was significantly lower than that of model group(P<0.05). Histomorphological examination showed normal tissue structure without edema or infiltration of inflammatory cells in sham-operated rats. In model group, the abnormal histomorphology manifested as loose tissue structure, interstitial edema, a large number of cell necrosis with nucleoli disappear and disorganized structure. The histological alterations were obviously alleivated in EA group.(2) The TTC stain showed that the rat brain tissue were stained bright red without white ischemic infarction in the sham operated group. In model group, most regions of the middle cerebral artery-distributed area was stained white, whereas there was only a smaller region of the brain tissue was white in EA-treated rats (P <0.01). In addition, there are few CD34-positive cells in the brain tissue of sham operation group. The brain tissue from model group showed visible CD34expression. There was significantly increased microvessel density in ischemic areas in the EA group when compared with that of model group (P<0.05).(3) Data analysis showed that there was very low level of VEGF mRNA in the sham operation group while the expression of VEGF mRNA obviously increased in the model group. The expression of VEGF mRNA further increased in the EA group(P<0.01). To further investigate the effects of EA on for angiogenesis in rat brain tissue with ischemia-reperfusion, immunoblot was performed to detect VEGF protein levels in brain tissue. The results showed that EA treatment significantly increased brain VEGF protein levels as compared with model group (P<0.05).(4) In sham operation group, there was no obvious G-CSF expression in brain tissue. There was visible G-CSF expression in cerebral tissue of model group, while the EA treatmen significantly increased G-CSF expression in ischemic areas as compared with model group (P<0.05). The expression of Ang-1was also very low in sham operation group. There was visible Ang-1expression in ischemic areas of model group. The expression of Ang-1was further increased after EA treatment (P<0.05).(5) In sham operation group, there were few apoptotic cells, whereas there were massive apoptotic cells in ischemic areas of the model group. The number of apoptotic cells decreased significantly in the EA group, as compared with model group (P<0.01). To further analyze the anti-apoptotic mechanisms of EA, the anti-apoptotic protein Bcl-2level was determined using immunoblot method. The results showed that the expression of Bcl-2was significantly higher than that of model group (P<0.01). We also examined the pro-apoptotic protein Bax levels in brain tissue, and we found that the level of Bax in the EA group was significantly lower than that in model group (P<0.05).Conclusion:EA may upregulate VEGF mRNA level and increase VEGF protein level in the ischemic area, which might promote angiogenessi and increase microvessel density of the ischemic area. Other angiogenic factors such as G-CSF and Ang-1also play important roles in the angiogenesis processes stimulated by EA. In addition, EA treatment might reduce the number of apoptotic cells of the ischemic area through regulating Bax/Bcl-2, thereby attenuating the pathological morphological damage of the ischemic area and reducing the infarct volume. These effects of EA might corporately reduce neurobehavioral disorders in rats with cerebral ischemia and reperfusion, providing effective cerebral protective effect. |
PDF Full Text Request