Study Of Hegu Electroacupuncture Improving The Survival In Mice With Endotoxemia

ObjectiveThere is little knowledge about Hegu acupoint's central nervous conduction.In this study,we are about to inject neurotropic retrograde tracer virus into Hegu acupoint to understand the nerve conduction pathway from the spinal cord to the brain after acupuncture at Hegu acupoint.Observe the activation of neurons in the brain area after electroacupuncture at Hegu acupoint with different current intensities through the expression of c-Fos protein.The endotoxemia model by LPS will be applied to study the protective effect of electroacupuncture at Hegu acupoint on the survival of mice.The ?₂ adrenergic receptor agonist clonidine and the ?₂ adrenergic receptor antagonist Yohimbine will be injected through the catheter into the locus coeruleus cannula,together with Hegu electroacupuncture,and observe its effect on the survival rate of mice with endotoxemia and discuss the possible mechanism of Hegu electroacupuncture improving the survival.MethodsPart 1: The mice were divided into 3 groups: the virus expression group for 3 days,the virus expression group for 4 days,and the virus expression group for 5 days,each group with 4 mice.Pseudorabies virus(PRV)was injected into the right Hegu acupoint,and the brain and spine were perfused at different time points,and the expression of PRV was observed by immunofluorescence.Part 2: The mice were divided into 4 groups: 3mA group,0.5mA group,0mA group,and blank group,each group with 3 mice.Electroacupuncture at Hegu acupoint or without electroacupuncture for 15 mins.After 1.5h,the brain was perfused,and the activation of brain area(c-Fos)was observed by immunofluorescence.Part 3: The mice were divided into 2 groups: 3mA pretreatment group and 0mA pretreatment group,each group with 10-11 mice.Electroacupuncture at Hegu acupoint for 15 minutes in advance,and then intraperitoneal injection of the pre-experimented LPS lethal dose(LD80).Sera were collected 1.5 hours later,and the mortality within 7 days was counted.Part 4: The mice were divided into 4 groups: yohimbine pretreatment group,5%DMSO pretreatment group,yohimbine plus 3mA electroacupuncture pretreatment group and clonidine pretreatment group,each group with 10-11 mice.All agents were pre-administered via cannula into locus coeruleus for 15 minutes,and yohimbine plus 3mA electroacupuncture pretreatment group was administered with electroacupuncture for 15 minutes later,and then intraperitoneal injection of the pre-experimented LPS lethal dose(LD80).Sera were collected after 1.5 hours and the mortality within 7 days was counted.Took all the sera from the third part and the fourth part to detect the TNF-? level by a mouse TNF-? ELISA kit.Results1.Results of part 1: Pseudorabies virus tracer neurons were expressed in the posterior horn of the cervical spinal cord after 3 days,expressed in the bilateral reticular nucleus and locus coeruleus after 4 days,and expressed in the unilateral basolateral amygdala and primary/secondary motor cortex after 5 days.2.Results of part 2: The co-expressed rate of c-Fos/TH neurons in the locus coeruleus in the Hegu acupoint 3mA group was significantly higher than that in the 0.5mA group,0mA group and the blank group.(ANOVA,F=223,***P<0.001)3.Results of part 3: LPS can reach 80% mortality(LD80)when injected into the abdominal cavity with 16mg/kg(2/10).Pretreatment with 3mA electroacupuncture at Hegu acupoint can significantly improve the survival rate of mice with endotoxemia induced by LPS(7/9),while pretreatment with 0mA electroacupuncture at Hegu acupoint cannot improve the survival rate of model mice(2/10).(3mA,n=9;0mA,n=10,log rank test,X²=7.182,**P=0.007)4.Results of part 4:(1)Direct administration of yohimbine into locus coeruleus accelerated the death of LPS-induced endotoxemia mice but affected little on long-term survival rate(1/9).(Yohimbine pretreatment,n=9;blank,n=10,log rank test,X²=2.856,P=0.091,Wilcoxon test,X²=5.231,*P=0.022)(2)5%DMSO does not affect the short-term and long-term survival rate of LPS-induced endotoxemia mice(3/10).(5%DMSO,n=10;blank,n=10,log rank test,X²=0.045,P=0.832,Wilcoxon test,X²=0.814,P=0.367)(3)Yohimbine plus electroacupuncture 3mA pretreatment cannot improve the survival rate of LPS-induced endotoxemia mice(3/10).(Yohimbine pretreatment,n=9;yohimbine plus electroacupuncture pretreatment,n=10,log rank test,X²=2.148,P=0.143)(4)Direct administration of clonidine into locus coeruleus can significantly increase survival in LPS-induced endotoxemia mice(7/10).(Yohimbine pretreatment,n=9;clonidine pretreatment,n=10;blank,n=10;5%DMSO,n=10,Mantel Cox test,X²=9.780,*P=0.021)(5)Hegu acupoint 3mA electroacupuncture pretreatment and clonidine pretreatment can significantly reduce the serum TNF-? level in LPS-induced endotoxemia mice.Yohimbine pretreatment group,5%DMSO pretreatment group,yohimbine plus 3mA electroacupuncture pretreatment group cannot significantly reduce the serum TNF-?level in LPS-induced endotoxemia mice.(n=7 per group,two-side Student's unpaired t test,t=2.289,*P=0.041;t=3.837,**P=0.002;t=3.897,##P=0.002)ConclusionAccording to the above results,it is proved that the ascending nerve pathway after acupuncture at Hegu acupoint is from the posterior horn of the spinal cord to the reticular nucleus to the locus coeruleus to the basolateral amygdala and the primary/secondary motor areas of the cerebral cortex.3mA electroacupuncture at Hegu acupoint can obviously activate the noradrenergic neurons of locus coeruleus while 0mA cannot.Pretreatment with 3mA electroacupuncture at Hegu acupoint or direct administration of ?₂ adrenergic receptor agonist clonidine into locus coeruleus can significantly increase the survival rate of mice with endotoxemia induced by LPS and reduce serum TNF-? levels.This effect is achieved by activating the ?₂ adrenergic receptor of locus coeruleus noradrenergic neurons.If the ?₂ adrenergic receptor antagonist yohimbine is directly administered into the locus coeruleus earlier,the protective effect of electroacupuncture disappeared.