The Experiment Exploration About The Effect Of COX-2 Expression In Lumbar Spinal Of ALS-SOD1^G93A Transgenic Mice By Electroacupuncture At Jiaji Ponts

Purpose:Influencing on survival,behavior,histomorphology and COX-2 protein expression in ALS-SOD1^G93A transgenic mice by Jiaji electroacupuncture,which is aimed at observing neuroprotective effect of electroacupuncture at Jiaji point on ALS-SOD1^G93A transgenic mice and its possible mechanism and then laying a solid theoretical basis for the clinical treatment of amyotrophic lateral sclerosis.Method:Part one Effect of Jiaji electroacupuncture on Survival and Behavior of ALS-SOD1^G93A transgenic mice:1.Laboratory animals and identification:transgenic mice of the SPF grade ALS-SOD1^G93A were introduced in American Jackson laboratories,according to the identification program proposed by Jackson laboratories,the gene identification of PCR amplification was proceed by interleukin2（IL-2）regarded as the internal reference,and then doing a phenotype identification with the mouse disease.2.Grouping and disposal:taking 18 ALS-SOD1^G93A positive mice and dividing the mice into electroacupuncture group,acupuncture group and model control group.Every group has 6 mice.We started to intervene when mice is 60 days.Electroacupuncture group and acupuncture group both do an intervention by acupuncturing both sides of L1～2、L5～6 Jiaji points.The mice of the acupuncture group were treated with 2Hz current on the same side of trunk.The frequency of electroacupuncture group and acupuncture group both are 20min/time,2times/week.The model group and the wild control group were given the same time and same duration as the acupuncture group to grap and bind,20min/time,2times/week.3.Neurological function score:suspension test was used to score the lower limb function of the suspended mice.Incidence period was decided when neurological score equals to 1 with consequent two days.Terminal event and survival period were decided when the neurological score equals to 4.4.Behavior test:the muscle strength and limb coordination of mice were determined by swivel test.Part Two Effect of Jiaji electroacupuncture on morphology of the anterior horn of the lumbar and COX-2 protein expression of ALS-SOD1^G93A transgenic mice:1.Grouping and disposal:taking 36 ALS-SOD1^G93A positive mice and randomly dividing into electroacupuncture group,acupuncture group and model control group.Every group has 12 mice.12 wild-negative brood mice were regarded as Control group.Positive mice were treated with experimental one and the negative control mice were were given the same time and same duration as the acupuncture group to grap and bind.2.HE staining,transmission electron microscopy:observing the morphological changes of motor neurons in the anterior horn of the lumbar spinal cord of mice.3.Immunohistochemistry was utilized to detect an expression situation of neurons COX-2 of anterior horn of the lumbar spinal cord with 125-days mice.Result:1.Offspring ALS-SOD1^G93A mice determination:after the gene identification of PCR（?）plification,a part of the mice carry positive ALS-SOD1^G93A mutant gene,which were propagated by positive mice.Positive rate is 40%±5%.Phenotype can be seen that suspension test is positive about the 94-days mice,appearing slow action and hind legs drag about 110-120 days,reaching the end of the period about 130-140 days.2.The analysis of incidence period and survival period:The onset of acupuncture group was delayed by 5 days compared with the model group.The incidence of electroacupuncture group was delayed by 9 days compared with the model group.There is a significant difference between the above two results,and is a statistical significance（P<0.05）.The survival period of acupuncture group was delayed by 6 days compared with the model group.The survival period of electroacupuncture group was delayed by 11 days compared with the model group.There is a significant difference between the above two results,and has a statistical significance（p<0.05）.3.Weight:starting from the 19th week,the weight of acupuncture group and electroacupuncture group was significantly higher than the model control group,and the electroacupuncture group is superior to the hand group.Having significant difference and statistical significance（p<0.05）.4.Analysis of Behavioral Experiment Results:starting from the 17th week,the incubation period of the model control group began to decrease,the latency of the chiropractic and electroacupuncture groups decreased from 18th weeks.From the preclinical comparison results of each stage of two groups of mice we can found that the latency of electroacupuncture group was significantly higher than that of model control group at the beginning of 17th week.Having significant difference and statistical significance（p<0.05）。5.Morphology:Anterior horn of the lumbar spinal cord of mice of HE staining and electron microscopy control group obviously put up loose edema,vacuolization,motor neurons degeneration,nerve fibers dissolved and disappeared.Spinal anterior horn motor neurons of mice of chiropractic group and electroacupuncture group has a lower Degree of denaturation.electroacupuncture group is superior to the hand group,and has a small number of visible Nissl body.6.Expression of COX-2 Protein Content:immunohistochemical staining results found that comparing with the model group,the expression of COX-2 in the chiropractic group and electroacupuncture group was significantly down-regulated.Electroacupuncture group is superior to the hand group,and has significant difference and statistical significance（p<0.05）。Conclusion:1.Electroacupuncture at Jiaji Point improves the behavior of ALS-SOD1^G93A transgenic mice,delays onset time and extends the survival period.2.Electroacupuncture at Jiaji Point can improve the morphological changes of motor neurons of the spinal cord anterior horn of ALS-SOD1^G93A transgenic mice,and the effect is better than acupuncture treatment.3.Electroacupuncture at Jiaji can alleviate oxidative stress injury in nerve cells,reduce the toxic effects of glutamate on lumbar medulla neurons and play a neuroprotective effect by the down-regulation of COX-2 expression of motor neurons of the spinal cord anterior horn of ALS-SOD1^G93A transgenic mice.