Repetitive Nerve Stimulation In Patients Of Amyotrophic Lateral Sclerosis And Study Of ETV1-D38G Transgenic Mouse Model Of Amyotrophic Lateral Sclerosis

Objective:The goal of this study is to illuminate the features of repetitive nerve stimulation(RNS)in Chinese patients with amyotrophic lateral sclerosis(ALS),in hoping of new discoveries to understand more about mechanism of the disease.Methods:1.Clinical and electrophysiological data of 149 cases diagnosed with ALS(clinical possible cases excluded)who underwent RNS were enrolled and analyzed retrospectively.2.Needle electromyography(EMG)and RNS were performed in all cases.LF RNS(low frequency RNS,3Hz)were performed in accessory nerve,axillary nerve,ulnar nerve,femoral nerve,common peroneal nerve,facial nerve and median nerve.HF RNS(high frequency RNS,20Hz)were only performed in ulnar nerve in a part of cases.3.SPSS22.0 software package was used for T-test and chi-square test.Results:1.The age at diagnosis of these ALS patients ranged from 22-78 years old(54.5± 10.8).There is a male to female ratio of 1.6:1.2.Decremental responses to LF RNS(?10%)in at least one nerve were detected in 84(56.4%)cases,most commonly seen in nerves supplying proximal muscles:accessory nerve and axillary nerve.There was no decremental response seen in facial nerve.3.Incremental responses to HF RNS(?60%)in ulnar nerve were observed in 7(6.5%)cases who underwent the 20Hz stimulation.5 of these cases have decremental response to LF RNS in accessory nerve at the same time.4.In 109 nerves with decremental response,62(56.9%)nerves had continuous decremental pattern,more than U-shape recovery(38,34.9%)and atypical ones(9,8.2%).5-19 cases showed definite decremental responses in LF RNS in accessory nerve while no abnormality were found in EMG and neurological examination of sternocleidomastoid muscle,supplied by accessory nerve.3 of these cases showed incremental response in HF RNS in ulnar nerve.Conclusions:1.Decremental responses in RNS can be commonly observed in ALS patients,especially in accessory nerve and axillary nerve2.Normal EMG,no weakness in physical examination of sternocleidomastoid muscle and Decremental response to LF RNS in accessory nerve indicates a "dying-back" process,with destruction of NMJs prior to motor neurons in the anterior horn.3.Incremental responses in ulnar nerve and dominant continuous decreased decremental pattern implies the damage of NMJs includes both post and pre synaptic membrane,which leads to the understanding that NMJ destruction may be an overall involvement of the synapse,not restricted to a certain region.Objective:Genetic study of amyotrophic lateral sclerosis(ALS)is hot nowadays.This study is based on a Chinese ALS pedigree we discovered which may has a new mutation gene ETV1.Therefore,we constructed a new ALS transgenic mice model with the new mutation ETV1-D38G,to study the relationship between ETV1 gene and ALS.Methods:1.The offspring generated by mating of hemizygous were identified by DNA extraction of tails,PCR and agarose gel electrophoresis.Male offsprings were further divided into three groups:homozygous,hemizygous and wide type control.Existence of mutation site were confirmed DNA sequencing.All mice were kept in situation met the standard of SPF(Specific pathogen Free).2.General condition,weight and motor function were evaluated after 60 days postnatal of the mice.Electromyography in gastrocnemius muscle was performed in 1 homozygous and 1 control after anesthesia,to see if any spontaneous potential appeared3.1 homozygous and control were put to death by breaking the neck.Lumbar enlargement and gastrocnemius muscle was removed.All tissues were embedded in paraffin and sliced.Number of neurons in anterior spinal horn and morphology of muscle were observed by light microscope after HE and Nissl staining.Meanwhile,immunohistochemical stain with antibody to ubiquitin and immunohistochemical stain with anti-glial fibrillary acidic protein(GFAP)antibody of the spinal cord were performed to see if there were ub positive inclusions and gliosis.4.3 homozygous and controls were put to death by breaking the neck.Whole spinal cord were removed for RNA extraction.RNA sequencing were further performed.Final data was analyzed by DAVID Bioinformatics Resources 6.8 system.5.SPSS 22.0 software package was used for Normality test and T-test.Results:1.19 homozygous,27 hemizygous,15 wide type controls were identified for experiment at last.2.During evaluation of general condition,weight,motor dysfunction and life span,there were no obvious difference observed between transgenic mice and wide type controls.There were no spontaneous potential and neurogenic MUPs observed in transgenic homozygous during EMG.3.No abnormality were found in HE,Nissl staining,immunohistochemical stain with ubiquitin and immunofluorescent stain with GFAP.4.Result of RNA sequence revealed a small amount of genes with expressing change:24 genes(FDR?0.5),including 12 with significant change(FDR?0.1).There were no clear correlation between the result and ALS.Conclusion:1.Through the observation of mice's behavior,detection of electrophysiology,the neuropathological study of neural system and muscle and RNA sequence,transgenic mouse model didn't present with a phenotype,the relationship between gene mutation ETV1-D38G and ALS remain unclear.2.The possible reason underlying includes:environmental interference;weak consistency between animal model and human disease;the pedigree also had(CAG)n abnormal amplification in gene ATXN3,ALS in this pedigree might be a coefficient by the two gene mutations.3.In further research,construction of "double hit" offspring by mating of ETV1-D38G transgenic mouse and SCA3 mouse and observation with SCA3 mouse and wide type controls may give more information on how the gene ETV1 correlates with ALS.