| ObjectiveTo observe the effect of electroacupuncture combined with rich rehabilitation training on cerebral ischemic injury,in order to investigate the possible mechanism of RhoA and ROCK protein in Nogo signaling pathway intervention on nerve regeneration.MethodsHealthy rats(male SD rats of clean grade)90,of which 18 rats in sham group,SD rats were successfully modeled after randomly divided into model group,electroacupuncture group,rehabilitation group and electroacupuncture combined with rich rich rehabilitation training group(i.e.,Needle Kang(NK)group).The model of focal cerebral ischemia in rats was established by suture method.Model group,sham operation group were not given any intervention,electroacupuncture group only use electroacupuncture,rehabilitation group use rich enriched environment and the needle Kang group used electroacupuncture and Enriched Rehabilitative Training.The five groups were respectively sacrificed at 3,7days and 14 d.Intervene for 1,3,7,14 days,rats were observed by using the neurological deficit scores;Intervene for 1,3,7,14 days,The behavioral function of each group was observed by using the behavioral function scale;The levels of cerebral infarction were measured by TTC staining in different time;The changes of the histological changes were observed by HE staining on the cortex and hippocampus at 14 days of Intervene.The expression of RhoA and ROCK protein in the Nogo signaling pathway of the ischemic cortex and hippocampus in each group was detected by using three methods(Immunohistochemistry,Western blotting and RT-PCR).Results1.Neurological deficit score(NDS): Intervene for 1,3,7and 14 days,the five group of rats were evaluated by NDS,the results: Sham operation group,NDS was 0;NDS of model group were higher than the sham operation group at each time point,and the difference was statistically significant between groups(P<0.01);Intervene for 1,3days,three intervention group of NDS decreased compared with the model group,but there was no significant difference between groups(P>0.05);Intervene for 7,14 days,NDS of three intervention groups were significantly lower than the model group,the difference was statistically significant(P<0.01);the other intervention groups were higher than the needle kang group in NDS,there was statistically significant(P<0.01).2.Behavioral function score:(1)The Negative GeotaxisIntervene for 1,3,7 and 14 days rates were evaluated by negative geotaxis,the results: compared with the sham operation group after four time points of the negative geotaxis requiredtime,negative geotaxis time of model group was longer than that the sham operation group,the difference was statistically significant at each time point between the two groups(P<0.01);Intervene for 1,3days,compared with the model group,negative geotaxis required time of the three intervention groups were shorter than the model group,but no statistically significant(P>0.05);Intervene for 7?14days,negative geotaxis required time of the three intervention groups were shorter than the model group,the difference was statistically significant(P<0.05 or P<0.01);The enriched rehabilitative training group were compared with electroacupuncture group,the time for rehabilitation group were less,but there was no significant(P>0.05);the needle kang group was lower than the rich rehabilitation training group(P<0.05 or P<0.01).(2)Wire hang test:Intervene for 1,3,7,14 days four time points of each group of rats were evaluated by Wire hang test,the results: the time of forelimb stay online of model group rates lower than the sham group,there was statistically significant(P<0.01);Intervene for 1 days,compared with the model group,the three intervention groups of forelimb stays on line time longer than the model group,but was not statistically significant(P>0.05);Intervene for 3,7,14 days,the three intervention groups' time were shorter than that the model group,was statistically significant(P<0.01);the other two intervention group's time were longer than electroacupuncture group,was statistically significant(P<0.01);the time of the needle kang group was shorter than that of the rich rehabilitation training group(P<0.05).(3)Cylinder rearing test:Intervene for 1,3,7,14 days four time points of each group of rats were evaluated by Cylinder rearing test,the results: adhesive removal test time of the sham operation group were longer than the sham operation group(P<0.01);Intervene for 1,3days,compared with the model group,the three intervention group's adhesive removal time were shorter than the model group,but not statistically significant(P>0.05);Intervene for 7days or 14 days,the model group' s adhesive removal test required time were longer than the intervention group,have statistical significance(P<0.01);compared with needle kang group,the other two group's adhesive removal time were shorter than needle kang group,was statistically significant(P<0.05 or P<0.01);the needle kang group's time was shorter than that of the rich rehabilitation training group(P<0.05).3.The volume detection of cerebral infarction:After TTC staining,pale white was the ischemic area of rats.The boundary between ischemic area and normal tissue is not ambiguous,it can distinguish between normal and ischemic border,and the frontal,apical,temporal and basal ganglia can appear infarction.There was no pale white area in sham operation group;the model group had obvious pale area;Intervene for 3d,the three intervention groups compared with the model group,the model group pale region sizes were larger than the former,but there was no significant difference between pale zone volume(P>0.05);Intervene for 7,14 days,model group's pale area volume was larger than the the three intervention groups,and there was significant difference of the volume of pale white region(P<0.05 or P<0.01);compared with other two intervention groups,the pale white volume of the needle Kang groups decreased,there were statistically significant differences in the pale white area(P<0.05)4.Histological observation:HE staining,observed: cortex and hippocampus neurons of the sham operation group were few abnormal;brain edema in model group,the cells were arranged in disorder,some neurons showed the changing of vacuolar,neuronal nuclei had pyknosis,even the pathological changes of the nucleolus disappeared.After14 days,the pathological changes of neurons of the three intervention groups were reduced compered with the model group in the cortex and hippocampus.Intervene for 14 days,compared with other intervention groups,the inflammatory infiltration of the needle Kang groups was significantly reduced,and there were more neurons and fewer abnormal neurons in needle Kang groups.5.RhoA and ROCK Protein expression:(1)Cortical area:Immunohistochemistry: we found that the number of RhoA and ROCK2 positive cells was significantly increased after cerebral ischemia,and the number of RhoA and ROCK2 positive cells of sham operation group was less.The number of RhoA and ROCK2 positive cells of model group was significantly higher than that in sham operation group,there was statistical significance(P<0.01).Three the number of RhoA and ROCK2 positive cells in the intervention group was significantly lower than the model group,there was statistical significance(P<0.01).Compared with the other two groups,the number of RhoA and ROCK2 positive cells of the needle Kang groups were significantly lower than the other two groups,there was statistical significance(P<0.01).Westen blot: The RhoA and ROCK2 of the ischemic side were significantly enhanced,and the expression of RhoA and ROCK2 of the sham operation group was less.The RhoA and ROCK2 of model group were significantly higher than the sham operation group(P<0.01).RhoA and ROCK2 of the three intervention groups were significantly lower than the model group,there was statistical significance(P<0.01).the RhoA and ROCK2 of the needle Kang groups were significantly lower than those he other intervention groups,there was statistical significance(P<0.01).(2)Hippocampus:Immunohistochemistry: The results showed that the number of RhoA and ROCK2 positive cells in hippocampus of ischemic side was significantly higher.The expression of RhoA and ROCK2 of the sham operation group was less.The expression of RhoA and ROCK2 of the model group was significantly higher than the sham operation group(P<0.01).the expression of RhoA and ROCK2 of the three intervention groups was significantly lower than the model group,there was statistical significance(P<0.05 or P<0.01).The expression of RhoA and ROCK2 of the needle Kang group was lower than the other two groups,there was statistical significance(P<0.01).Westen blot: The expression of RhoA and ROCK2 in the hippocampus of the ischemic side was significantly enhanced,and the expression of RhoA and ROCK2 in the sham operation group was less.The expression of RhoA and ROCK2 of model group was significantly higher than the sham operation group(P<0.01).The expression of RhoA and ROCK of the three intervention groups was significantly lower than the model group,there was statistical significance(P<0.01).Compared with the other intervention groups,the expression of RhoA and ROCK2 of the needle Kang group were lower than other intervention groups,there was statistical significance(P<0.05 or P<0.01).6.RhoA and ROCK gene expression:(1)Cortical area:The RhoA and ROCK2 mRNA of the ischemic cortex were significantly increased after cerebral ischemia,and RhoA and ROCK2 mRNA expression of the sham operation group was less than that the sham operation group.The RhoA and ROCK2 mRNA of model group were significantly higher than the sham operation group(P<0.01).The RhoA and ROCK2 mRNA of the Three intervention group was significantly lower than the model group(P<0.01).Compared with other intervention groups,the expression of RhoA and ROCK2 mRNA of the needle Kang group were significantly lower,there was statistical significance(P<0.05).(2)Hippocampus:The expression of RhoA and ROCK2 mRNA of the ischemic side was significantly enhanced,and the expression of RhoA and ROCK2 mRNA of the sham operation group was less.The expression of RhoA and ROCK2 mRNA of model group was significantly higher than that the sham operation group(P<0.01).Three the expression of RhoA and ROCK2 mRNA of the three intervention groups was significantly lower than the model group(P<0.01).Compared with the other intervention groups,the expression RhoA and ROCK2 mRNA of the needle Kang group were significantly lower than the other intervention groups,there was statistical significance(P<0.05 or P<0.01).Conclusion1Compared with electroacupuncture or rehabilitation therapy,the combination of two methods is more effective in improving the neurological function of cerebral ischemia rats,and enhance the coordination and balance ability of limbs.2 Compared with electroacupuncture and rehabilitation therapy,the combination of two methods can reduce the infarct volume and pathological changes of focal cerebral ischemia rats.3 Electroacupuncture combined with enrich rehabilitation therapy can reduce the expression of RhoA,ROCK2 of ischemic area in cortex and hippocampus,which may be mechanism of the nerve remodeling by electroacupuncture combined with rehabilitation therapy. |
PDF Full Text Request