| Objective:To explore whether electro-acupuncture at the Qiaomai acupoints "Shenmai" and "Zhaohai" may improve the limb spasm in rats after stroke by adjusting the glycinergic neurotransmitter transmission system,so as to provide theoretical basis for clinical use of electroacupuncture in the treatment of limb spasm after stroke.Methods:From 60 Sprague-Dawley male rats,50 rats were randomly selected for right middle cerebral artery ischemia-reperfusion modeling,and the remaining 10 rats were divided into sham operation group.After 3 days of model building,the rats that failed to be modeled were screened out according to the evaluation results of Zealonga method and modified Ashworth scale.The remaining rats were randomly divided into electroacupuncture(EA)group,model group,electroacupuncture +strychnine.Strychnine,a glycine receptor blocker,was injected intraperitoneally before electroacupuncture.After the grouping was completed,electroacupuncture started.The electroacupuncture points were "Shenmai" and "Zhaohai" points of the affected limb.The electroacupuncture parameters were 100 HZ continuous wave.The stimulation intensity was based on the rhythmic contraction of the limbs.The electroacupuncture dose was once a day for 30 minutes each time for 6 consecutive days.Before and after the intervention,the m NSS neurological function score was used to assess neurological function,the Feeney balance test was used to assess exercise capacity and balance and coordination ability,and the modified Ashworth scale was used to assess changes in muscle tension.After sampling,Elisa was used to detect the content of glycine(Gly)in rat brainstem and spinal cord lumbar enlargement tissue,western-blot(WB)and real-time PCR were used to detect the expression of glycine receptor(Gly R),N-methyl-D-aspartate receptor(NMDAR),glycine transporter 1(Gly T1)and glycine transporter 2(Gly T2)in stem and spinal lumbar enlargement tissues.Results:1.Nerve function score and Feeney balance test results: The rats in the sham-operated group had no symptoms of neurological damage before and after treatment.Before treatment,compared with the sham operation group,the m NSS score and Feeney score of the model group,EA group,and EA + strychnine group increased(P<0.01),and there was no statistical difference between the groups(P>0.05);After treatment,compared with the model group,the m NSS score and Feeney score of rats in the EA group and EA + strychnine group were reduced,but only the difference with the EA group was statistically different(P<0.01).Compared with the EA group,the EA + strychnine group score increased and it was statistically significant(P<0.05).2.Muscle tone score results: Rats in the sham operation group did not show symptoms of increased muscle tone before and after treatment.Before treatment,compared with the sham operation group,the modified Ashworth scale scores of the model group,EA group and EA + strychnine group increased significantly(P<0.01),and there was no statistical difference between the groups;After treatment,the modified Ashworth scale score in the EA group was significantly lower than that of the model group,and there was a statistical difference(P<0.01).Compared with the EA group,the modified Ashworth scale score of rats in the EA + strychnine group increased,and there was a statistical difference(P<0.05).3.TTC staining results: Four rats were randomly selected from the sham operation group,model group,EA group and EA + strychnine group.Although after9 days of recovery period,it was still observed that there were obvious cerebral infarction areas in the right cerebral hemisphere,and the infarct size was model group > EA + strychnine group > EA group.4.Elisa results: Compared with the sham operation group,the glycine content of the model group decreased,and the difference was statistically significant(P<0.05),indicating that after MCAO,the glycine content in the brainstem and spinal lumbar enlargement of SD rats decreased;Compared with the model group,the levels of glycine in the brainstem and spinal lumbar enlargement of the EA group increased,and the difference was statistically significant(P<0.01,P<0.05),indicating that EA could increase the contents of glycine in the brain stem and spinal lumbar enlargement.5.Western blot results: In lumbar enlargement tissues,compared with the sham operation group,the expression of Gly Rα1 and Gly Rβ in model group were decreased(P<0.05,P<0.01),the expression of Gly T1 and NMDAR in the model group were increased(P<0.05).Although the expression of Gly T2 protein increased,there was no significant difference(P<0.05).Compared with the model group,the expressions of Gly Rα1 and Gly Rβ in EA group were increased,while the expressions of Gly T1,Gly T2 and NMDAR in EA group were decreased(P<0.05);In the brain stem tissue,compared with the sham operation group,the expressions of Gly Rα1 and Gly Rβ in the model group were decreased(P<0.05,P<0.01),and the expressions of Gly T1 and NMDAR were increased(P<0.05,P<0.01),with statistical significance,while the expression of Gly T2 was not significant(P>0.05).Compared with the model group,the expressions of Gly Rα1 and Gly Rβ in EA group were increased(P<0.05,P<0.01),and the expressions of Gly T1,Gly T2 and NMDAR were decreased(P<0.05,P<0.05,P<0.01),with statistical difference.6.RT-PCR results: In the spinal lumbar enlargement,compared with the sham operation group,the Gly R m RNA content in the model group was lower(P<0.01),and the content of Gly T1 m RNA,Gly T2 m RNA and NMDAR m RNA increased(P<0.01,P<0.05,P<0.01),and the difference was statistically significant.Also compared with the sham operation group,the content of Gly R m RNA in the brainstem of the model group decreased(P<0.05),and the content of Gly T1 m RNA,Gly T2 m RNA and NMDAR m RNA increased(P<0.05,P<0.01,P<0.05);Compared with the model group,the levels of Gly R m RNA in brain stem and spinal cord lumbar enlargement tissues of EA group were increased,while the levels of Gly T1 m RNA,Gly T2 m RNA and NMDAR m RNA were decreased(P<0.01).Conclusions:1.Electroacupuncture at the Qiaomai acupoints can effectively promote the recovery of neurological function and improve the motor function and balance ability of rats with stroke and muscle spasm.2.Electroacupuncture at Qiaomai acupoints can relieve the degree of muscle spasm in stroke rats.3.Electroacupuncture at qiaomai acupoint can increase the expression of Gly and Gly R in brain stem and lumbar enlargement,reduce the expression of Gly T1,Gly T2 and NMDAR,further improve the efficiency of glycine neurotransmitter transmission system,produce stronger inhibitory nerve signals,and reduce the excitability of lower motor neurons,which may be one of the mechanisms of alleviating limb spasm in rats after stroke. |
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