Distribution Alteration Of 5-hydroxytryptamine Neuron In Spinal Cord And Brain Stem Were Potentially Associated With Death Of Neural Cells In SOD1*G93A Mice

Objective:Recent research suggests that amyotrophic lateral sclerosis(ALS)is not only damaged by motor nerve cells,but also damaged by other cells.In this study,using G93A-SOD1 transgenic mice as the amyotrophic lateral sclerosis(ALS)animal model to observe and analyze the serotonin(5-TH)changes in the spinal cord and brainstem of the major damaged areas in ALS,as well as the relationship between5-TH changes and neuronal cell death.Methods:G93A-SOD1 transgenic mice were purchased,raised and propagated,and the offspring mice were tested and screened for transgenic mouse models by PCR.The mice were divided into groups(60-70 days in the non-onset group,90-100 days in the disease group,and 120-130 days in the progression group).The mice in each group were fixed with formaldehyde,and the brain and spinal cord of the mice were completely removed and fixed at 4 °C.Overnight,gradient dehydration,embedded in OCT,rapid freezing of liquid nitrogen,the tissue of spinal cord and brainstem was coronally cut on a Leica cryostatand collected on Superfrost Plus slides for fluorescent immunohistochemical staining.Using the dual fluorescent immunohistochemical staining to label 5-HT,astrocytes,NPC,neuronal cells and cell nuclei.Under the microscope,the positive cells of different staining were superimposed and imaged by image processing technique,and the distribution characteristics of positive cells were oberseved and the relationship between 5-TH changes and astrocytes,NPC,and neuronal cells were analyzed statistically.Results:1、The 5-TH mainly expressed in the grey matter of adult spinal cord,including the anterior horn(AH),the posterior horn(PH),the central lateral column(CLC)andthe central canal(CC)of cervical,thoracic and lumbar segment.AH and PH are the most abundant,followed by CC and CLC.2、There was no significant difference in the expression of Serotonin between the segments of the spinal cord in WT mouse group.The expression of Serotonin in the spinal cord AH,PH,CC decreased with age,and there was no significant difference in CLC.3、Compared TG with WT mice,the expression of 5-TH in AH,PH and CC at the pre-onset stage,and that in the PH at the onset stage significantly decreased,but in the cervical segment of spinal cord,The AH,PH and CC at the progression stage significantly increased In the thoracic and lumbar segment,the expression of 5-TH in the AH at the onset stage and that in the AH and PH at the progression stage significantly increased.Serotonin expression in PH was significantly reduced at onset,but AH was significantly increased in both WT and TG mice,which implied that the redistribution of 5-HT occurred between the AH and PH.4、The TPH2 mainly expressed in the white matter of spinal cord,especially in the FL.While compared between WT and TG mice,the TPH2 in the FL significantly decreased at the pre-onset stage,but that in the cervical segment significantly increased at the progression stage.The TPH2 distribution in the cervical segment was more than that in the thoracic and lumbar segment at the progression stage,that at the pre-onset and onset didn’t show significant difference between the cervical,thoracic and lumbar segment.5、The TPH2 is expressed in the cytoplasm of neurons,mainly distributed in the nuclei of brain stem,including the dorsal raphe nucleus(DR),the median raphe nucleus(MNR),the raphe magnus nucleus(RMG),the raphe obscurus nucleus(ROB),the raphe pallidus nucleus(RPA),the raphe pontine nucleus(RPN)and the lateral paragigantocellular nucleus(LPGI).In the WT mice,the TPH2 distribution in DR was the most abundant,the secondary abundance was in the MNR and RMG,the third was the ROB,RPA,RPN and LPGI.While compared between WT and TG mince,TPH2 in the ROB at the pre-onset stage significantly decreased,TPH2 in the RPN at the onset and progression stage significantly increased.Conclusion:This study suggests that the redistribution of Serotonin in the anatomical region of the spinal cord and the distribution and the changes of TPH2 in the spinal cord and brainstem anatomical regions in ALS and their close correlation with neuronal apoptosis may be related to the pathogenesis of ALS.