| Objective: To observe the effects of electroacupuncture combined with rich rehabilitation training on angiopoietin-1(Ang-1)and endothelium-specific receptor tyrosine kinase 2,To study the effect of Tie-2)expression on the recovery of neurological function and angiogenesis in the ischemic side of rats with focal cerebral ischemia at different time and different intervention methods,and to explore the possible mechanism.In order to combine the traditional acupuncture method with the modern rehabilitation technology,and to provide experimental basis,in order to be effective and reasonable in clinical use.Method: 1.170 clean male Sprague Dawley(SD)rats were purchased from the experimental animal center.27 of them were divided into sham operated groups by random number table method.The remaining 143 rats were used to establish MCAO model by Zea longa modified thread bolt method.Then 108 rats were randomly divided into 4 groups: model group(27 rats),electroacupuncture group(27 rats),rehabilitation group(27 rats)and acupuncture combined rehabilitation training group(27 rats).According to the different course of treatment,the five groups were subdivided into 3 days,7 days and 14 days.2.The rats in the treatment group can be intervened when they are awake after anesthesia(about 2-3 hours after operation).The electroacupuncture group and the electroacupuncture combined rehabilitation group(the acupuncture group)selected Baihui,Fengfu,supplemented by Neiguan and Xinshu as the stimulation points;the rehabilitation group and the acupuncture group also received rehabilitation therapy intervention 2-3 hours after the operation,providing rich rehabilitation environment and rehabilitation exercise training;the sham operation group and the model group received routine feeding without any treatment.The rats in the five groups were treated or fed continuously for 3,7 and 14 days.3.Two types of neurological deficit physical signs(Zea longa's 5-point scoring standard and modified mnss16 scoring system)were used to evaluate the neurological injury and recovery of the five groups in four time periods(4 hours,3 days,7 days and 14 days)after operation;the five groups were observed in four time periods(5 minutes after inserting the line,intervention 3)by laser Doppler blood flow instrument Regional cerebral blood flow on days,7 days and 14 days;the damaged area of brain tissue was observed by TTC staining;the pathological changes of cerebral cortex in ischemic area of five groups were observed by HE staining at three different time periods(intervention for 3 days,7 days and 14 days);the pathological changes of cerebral cortex in three different time periods(3 days,7 days and 14 days)were observed by Western blot and RT-q PCR at three different time periods(3 days,7 days and 14 days))The expression of Ang-1 and Tie-2 protein and gene in different intervention methods(electroacupuncture,rehabilitation training,electroacupuncture combined rehabilitation training);the microvessel density was observed by immunohistochemistry and Weidner method.Result: 1.Neurological deficit score 1.1 Zea longa's 5-point score Compared with the sham operation group,the rats in the model group scored 0 in four time periods(4 hours,3 days,7 days and 14 days)after operation.Compared with the sham operation group,the rats in the model group scored higher in four time periods(P < 0.01),and gradually decreased with the increase of time periods.Compared with the model group,the rats in the electroacupuncture group and the rehabilitation group were in the intervention There was no significant difference between 4 hours and 3 days(P > 0.05)The scores of 7 days and 14 days were significantly lower than those of the model group(P < 0.01);compared with the model group,the scores of 3 days,7 days and 14 days in the four time periods(4 hours after operation,3 days after intervention,7 days and 14 days)were lower than those of the model group(P < 0.01);compared with the electroacupuncture group and the rehabilitation group,there was no significant difference at 4 hours after operation(P >0.05),the score of acupuncture group was lower than that of electroacupuncture group(P < 0.05),there was no significant difference between acupuncture group and rehabilitation group(P > 0.05),and the score of houshikang group was lower than that of electroacupuncture group and rehabilitation group on 7 and 14 days(P < 0.05 or P < 0.01);there was no significant difference between electroacupuncture group and rehabilitation group in 4 hours,3 days,7 days and 14 days(P > 0.05).And from the general trend,with the prolongation of the expectation of electroacupuncture combined with rich rehabilitation training,the better the recovery of neural function in rats.1.2 Modified m NSS16 scoring system After the operation,the scores of rats in the sham operation group at four time periods(4 hours,3 days,7 days,14 days)were all 0 Compared with the sham operation group,the scores of four time periods in the model group were higher than those in the sham operation group(P < 0.01)and gradually decreased with the increase of time periods;compared with the model group,there was no significant difference in the electroacupuncture group,the rehabilitation group and the electroacupuncture group(P > 0.05),and the scores of three,seven and 14 days after intervention were lower than those in the model group,with a significant difference(P < 0.01);Compared with the model group,the scores of the three groups were significantly lower(P < 0.01)in the four time periods(4 hours after operation,3 days after intervention,7 days after intervention,14 days after intervention);compared with the electroacupuncture group and the rehabilitation group,there was no significant difference(P > 0.05)in the four hours and 3 days after intervention,and the scores of the seven days and 14 days after intervention were significantly lower(P < 0.05)There was no significant difference between the electroacupuncture group and the rehabilitation group(P > 0.05).2.Regional cerebral blood flow(r CBF)After the operation,the cerebral blood flow of rats in the sham operation group was stable at about 200 pu in four time periods(5 minutes,3 days,7 days and 14 days).Compared with the sham operation group,the cerebral blood flow of rats in the model group was lower than that in the sham operation group(P < 0.01);compared with the model group,there was no significant difference between the electroacupuncture group and the rehabilitation group at 5 minutes and 3 days(P > 0.05)The blood flow at 7 and 14 days was significantly higher than that of the model group(P < 0.01);compared with the model group,the cerebral blood flow at 3,7 and 14 days was significantly higher than that of the model group(P < 0.01);compared with the electroacupuncture group and the rehabilitation group,there was no significant difference at 5 minutes(P > 0.05)The cerebral blood flow of rats in the three,seven and 14 day intervention group was significantly higher than that in the electroacupuncture group and the rehabilitation group(P < 0.01);there was no significant difference between the electroacupuncture group and the rehabilitation group(P > 0.05).3.Histopathological observation Sham operation group: there was no obvious loss of cell structure of brain tissue in the ischemic area of rats,vascular endothelial cells and glial cells were arranged in order,the structure was compact and hierarchical,the cytoplasm of nerve cells in brain tissue was rich,the nucleolus was clear and there was no obvious hyperchromatic condition,neurons;3 days after operation group: the change was most obvious,four groups(model group,electroacupuncture group,rehabilitation group,acupuncture group)were presented Interstitial edema,swelling and uneven density of nerve cells,deep staining of nerve cells,no significant difference was found in the four groups;7 days after the operation: the gap between the electroacupuncture group and the rehabilitation group decreased,tending to be slightly ordered.Compared with the model group,the interstitial edema after the brain tissue injury was significantly reduced,and the degree of repair was better.The edema of the cells in the acupuncture group was significantly reduced compared with the other groups.14 days after the operation,the edema of the cells in the ischemic brain tissue in the three groups was significantly improved,and the cell structure and quantity were increasingly normal There were clear nuclei in the treatment group,with clear layers,orderly arrangement,normal distribution of morphology and quantity,uniform cytoplasm,and the degree of injury was significantly reduced compared with the treatment group at other time points.Among them,the improvement of acupuncture group was the most obvious under the same treatment time.4.Western blot to detect the expression of Ang-1 and Tie-2 After operation,the sham operation group was divided into three periods(3 days,7 days and 14 days))The expression of Ang-1 and Tie-2 in model group was higher than that in sham operation group(P < 0.01);the expression of Ang-1 and Tie-2 in electroacupuncture group,rehabilitation group and model group were intervened for 3 days,7 days and 14 days,respectively The expression of Ang-1 and Tie-2 in acupuncture group was significantly higher than that in rehabilitation group(P < 0.01);the expression of Ang-1 and Tie-2 in acupuncture group was significantly higher than that in model group(P < 0.01);the expression of Ang-1 and Tie-2 in acupuncture group was significantly higher than that in electroacupuncture group(P < 0.01);the expression of Ang-1 and Tie-2 in acupuncture group was significantly higher than that in electroacupuncture group(P < 0.01)There was significant difference between the two groups(P < 0.05 or P < 0.01);there was no significant difference in the three courses of treatment(3 days,7 days and 14 days)between the electroacupuncture group and the rehabilitation group(P > 0.05).5.Detection of Ang-1 m RNA and Tie-2 m RNA expression by RT-q PCR The expression of Ang-1 m RNA and Tie-2 m RNA in the ischemic side of rats in the sham operation group was higher than that in the sham operation group(P < 0.01 or P < 0.01)Compared with the model group,the expression of Ang-1 m RNA and Tie-2 m RNA in the treatment group was significantly higher than that in the model group on the 3rd,7th and 14 th day of intervention(P < 0.01);compared with the model group,in the acupuncture group,the expression of Ang-1 m RNA and Tie-2 was significantly higher on the 3rd,7th and 14 th day of intervention(P < 0.01)The expression of Ang-1 m RNA and Tie-2 m RNA in the acupuncture group were significantly higher than those in the electroacupuncture group(P < 0.05 or P < 0.01)and the electroacupuncture group(P < 0.05 or P < 0.01)On the 3rd and 14 th day,the level of Tie-2 m RNA in EA group was lower than that in rehabilitation group(P < 0.05),and there was no significant difference between EA group and rehabilitation group(P > 0.05).6.Immunohistochemistry(IHC)was used to detect the expression of Ang-1 and Tie-2 positive cells After the operation,the positive cells of Ang-1 and Tie-2 in the ischemic side of rats in the sham operation group did not change significantly in three time periods(3 days,7 days and 14 days),but only expressed in trace;compared with the sham operation group,the positive cells of Ang-1 and Tie-2 in the model group were higher than those in the sham operation group(P < 0.01 or P < 0.01)Compared with the model group,the number of Ang-1 and Tie-2 positive cells in the electroacupuncture group and the rehabilitation group was significantly higher than that in the model group(P < 0.01 or P Compared with the model group,the number of Ang-1 and Tie-2 positive cells in the three groups was significantly higher(P < 0.01),and the number of Ang-1 and Tie-2 positive cells in the three groups was significantly higher(P < 0.05)(There was no significant difference between the electroacupuncture group and the rehabilitation group(P > 0.05).7.Microvessel density(MVD)After the operation,there was no significant change of microvascular density(MVD)on the ischemic side of rats in three time periods(3 days,7 days,14 days)in the sham operation group,but only micro expression;compared with the sham operation group,the microvascular density(MVD)in the three time periods in the model group was higher than that in the sham operation group(P < 0.01 or P < 0.01)Compared with the model group,the microvascular density of the acupuncture group and the rehabilitation group was significantly higher(P < 0.01)than that of the model group on the 3rd,7th and 14 th day of intervention(P < 0.05);compared with the model group,the microvascular density of the acupuncture group and the rehabilitation group was significantly higher(P < 0.01);compared with the electroacupuncture group and the rehabilitation group,the microvascular density of the acupuncture group was significantly higher(P < 0.01)The microvascular density was higher in the electroacupuncture group than that in the rehabilitation group(P < 0.05 or P < 0.01).There was no significant difference between the electroacupuncture group and the rehabilitation group at 3 days(P > 0.05).There was a difference between the rehabilitation group and the electroacupuncture group at 7 and 14 days after intervention(P < 0.05 or P < 0.01).Conclusion: 1.Three treatment methods(electroacupuncture,rehabilitation training,electroacupuncture combined rehabilitation training)can promote the repair of neurological damage,increase the blood supply of the injured side,restore the function of brain tissue,and significantly improve the pathology of the injured side of brain tissue.Combined therapy is better than single therapy,and the combination of electroacupuncture and rehabilitation can play the best effect of mutual assistance and mutual supplement 2.Electroacupuncture combined with rehabilitation training can up regulate the expression of Ang-1 and Tie-2 on the ang / Tie-2 signal pathway,increase the microvessel density in the ischemic area,and promote the neovascularization of the injured brain area.The combined therapy is superior to the simple electroacupuncture and rehabilitation therapy. |
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