The Study About The Mechanism Which Electroacupuncture At Jiaji Points（EX-B2） Protects Aganist Excitotoxicity Of Lumbar Spinal Neurons In ALS-SOD1^G93A Transgenic Mice

Objective:This present study was aimed to observe the effects of electroacupuncture at jiaji points (EX-B2) on disease progression and behavior in the transgenic mouse model of ALS(ALS-SOD1G93A), and to investigate the mechanism how electroacupuncture exerts anti-excitotoxic effects in lumbar spinal neurons.Methods:ALS-SOD1G93A transgenic mice were used for this study.Part one, We identified the ALS-SOD1G93A transgenic mice by PCR with the primer sequence and identification methods provided by Jackson Laboratory, and observed the phenotype of ALS-SOD1G93A transgenic mice.30 ALS-SOD1G93A transgenic mice were randomly divided into electroacupunct-ure group(group A), acupuncture group(group B) and control group(group C), 10 in each group. From the age of 60 days, ALS-SOD1G93A transgenic mice in group C were disposed by seizing and binding, ALS-SOD1G93A transgenic mice in group B were treated by acupuncture at jiaji points (EX-B2) and ALS-SOD1G93A transgenic mice in group A were treated by electroacupunctu-re at jiaji points (EX-B2) with the electrial stimulation at the frequency of 2 Hz,20min/time,2 times/week for 4 weeks. In this study, disease progression of each group were monitored 2 times/week from the age of 60 days to the end event occurrence, including suspending tail test, weight and rotarod test.Part two,33 ALS-SOD1G93A transgenic mice were randomly divided into electroacupuncture group(group A), acupuncture group(group B) and control group(group C),11 in each group.11 non-transgenic littermates were used as wild-type group. Group A, B and C were disposed with part one. Wild-type group wasn’t accepted any processing. HE staining and transmission electron microscopy were used to observe the anterior horn of the lumbar spinal cord pathological morphology, and detect the number of motor neurons. ELISA method was used to detect glutamate concentration in serum. The expression of EAAT2, GluR2 in the anterior horn of the lumbar spinal cord were detected by immunohistochemistry, and the level of EAAT2 and GluR2 protein of the lumbar spinal cord were investigated by immunoblotting.Results:1. ALS-SOD1G93A transgenic mice identification:PCR identification: Tested mice which appeared two bands in the lane were ALS-SOD1G93A transgenic mice, while only a band in the lane were wild-type littermates ones, and the positive rate was 40%±5%. Phenotypic characteristic:Symptoms typically occured at the age of 90 days (approximately) and extended to symmetrical paralysis of the hindlimbs at the age of 100-120 days followed by complete paralysis at the age of 140-150 days.2. Analysis of weight:Group A delayed the loss of mice weight compared with group C, the difference was statistically significant(P<0.05), though group B also delayed the loss of weight, there was no significant difference(P>0.05).3. Analysis of onset and survival:Group A delayed the onset of the disease by 8 days compared with group C, the difference was statistically significant (P<0.05). Interestingly, group B also slightly increased this value compared with group C, although there was no statistical significance (P>0.05). Group A and group B respectively prolonged the survival time by 10 days and 5 days compared with group C, the differences were statistically significant(P<0.01,P<0.05), and group A prolonged the survival time by 5 days compared with group B, the difference was statistically significant(P< 0.05). Kaplan-Meier survival analysis showed that the age of 147.6 days, the survival rate of group C was significantly lower than group B and group A.4. Analysis of rotarod test:From the age of 19 weeks to 21 weeks, group A and group B could significantly improve rotarod motor function compared with group C, the differences were statistically significant(P<0.05,P<0.01). and group A was more obvious than group B(P<0.05,P<0.01).5. Analysis of HE staining and transmission electron microscopy:Group A and group B could significantly reduce morphological lesions and increase the number of motor neurons compared with group C, Group A’effect was better than that of group B.6. Analysis of ELISA:Glutamate concentration in serum of group C was significantly increased(P<0.01), compared with group C, group A and group B were significantly decreased(P<0.01), and group A was more obvious than group B(P<0.05).7. Analysis of IHC:In the anterior horn of the lumbar spinal cord, EAAT2 and GluR2 protein expression of group C were significantly decreased (P<0.01), compared with group C, group A and group B were significantly increased(P<0.05,P<0.01), and group A obviously than group B(P<0.05, P< 0.01).8. Analysis of western-blot:EAAT2 and GluR2 protein in the lumbar spinal cord of group C were significantly decreased (P<0.01), compared with group C, group A and group B were significantly increased(P<0.01), and group A obviously than group B(P<0.01,P<0.05). Conclusion:1. Electroacupuncture at jiaji points (EX-B2) can improve motor functi-on, delay the onset of disease and prolong survival time in ALS-SOD1G93A transgenic mice, and the effect is better than acupuncture.2. Electroacupuncture at jiaji points(EX-B2) can improve morphological changes of motor neurons in the anterior horn of the lumbar spinal cord. Its effect is better than acupuncture.3. Electroacupuncture at jiaji points(EX-B2) can decrease glutamate con-centration in serum by upregulating the expression of EAAT2, and exert anti-excitotoxic effects by upregulating the expression of GluR2 protein. Its effect is better than acupuncture.