The Alteration On The Peripheral Nerve Of Amyotrophic Lateral Sclerosis And Effects Of Fibroblast Growth Factor 9 In Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis?ALS?is a neurodegenerative disease characterized by progressive loss of motor neurons.The peripheral nerve is a bridge connecting neurons and muscles,and a healthy peripheral nerve can ensure the body's normal material and information exchange.When the peripheral nerve appears to damage that may cause motor dysfunction.During development of the ALS disease,motor neuron selective death in the central nervous system,limbs are also progressive muscular atrophy.Peripheral nerve as a bridge between neurons and muscle,it plays an important role in ALS disease.In the study of amyotrophic lateral sclerosis,most of the research focuses on the changes of the central nerve system,and the changes and effects of the peripheral nervous system are rarely noticed.Therefore,we investigated the changes of peripheral nerves in the amyotrophic lateral sclerosis by observation and experiment on the peripheral nerves of the SOD1-G93A transgenic mice.Part?:The effect of mutant SOD1 on the peripheral nervesObjective:To investigate morphology of peripheral nerve on SOD1-G93A transgenic mice in different periods,and detect the expression of myelin sheath and axon related gene and protein in peripheral nerve of the SOD1-G93A transgenic mice,to explore the effect of mutant SOD1 on the peripheral nerves.Methods:The genotypes of transgenic mice were identified by PCR amplificationmethod,theSOD1-G93Atransgenicmiceandthe non-transgenic control mice were divided into two groups.Firstly,SOD1-G93A transgenic mice and the non-transgenic control mice were chosen at P15 and P30,the ultrastructure of sciatic nerve were observed,and real-time quantitative PCR assay were taken to explore the expression of mRNA levels of myelin related genes between SOD1-G93A transgenic mice and the non-transgenic control mice at P30.Secondly,SOD1-G93A transgenic mice and the non-transgenic control mice were chosen to be observed at the early of postnatal symptoms?60 days?,the early symptoms?90 days?and late symptoms?120 days?.Thirdly,the changes of axon and myelin sheath in the sciatic nerve were observed in the late stage?120 days?by using the protein imprinting method and immunofluorescence method.Fourth,the behavioral changes of SOD1-G93A mice were observed in the late stage of disease?120 days?.Results:1.Electron microscopy and toluidine blue staining were used to observe the morphology of SOD1-G93A transgenic mice and the non-transgenic control mice at P15 and P30.Moreover,during the development of peripheral nerve,the formation of the sciatic nerve sheath was observed,and the percentage of the myelinated fibers between SOD1-G93A and the control mice was not significant.In addition,thickness of myelin sheath and axon of the SOD1-G93A transgenic mice and the non-transgenic control mice at P30were counted,thickness of myelin sheath and axon between the SOD1-G93A transgenic mice and the non-transgenic control mice showed no difference.2.The methods of real-time quantitative PCR were used to observe the mRNA expression of myelin associated genes in the SOD1-G93A transgenic mice and the non-transgenic control mice at P30.The data showed that there were two groups of SOD1-G93A transgenic mice and the non-transgenic control mice without statistical significance.The results also suggest that the mutant SOD1 gene does not affect the development of peripheral nerves.3.Electron microscopy and toluidine blue staining were used to observe the morphology of the SOD1-G93A transgenic mice and the non-transgenic control mice at P60 and P90.According to the statistics,the sciatic nerve of the SOD1-G93A transgenic mice showed a significant proportion of the myelosheath and axonal degeneration at P90,while the peripheral nerve of the non-transgenic control mice was relatively intact.4.Electron microscopy and toluidine blue staining were used to observe the morphology of the SOD1-G93A transgenic mice and the non-transgenic control mice at P120.The sciatic nerve of SOD1-G93A transgenic severely damaged at P120.5.Fluorescence staining was used to detect protein level of the myelin and axonal associated proteins in the sciatic nerve of the SOD1-G93A transgenic mice and the non-transgenic control mice.The expression of axonal associated protein decrease in the sciatic nerve of the transgenic mice of SOD1-G93A at P120,compared with the sciatic nerve of the control mice.6.It was found that the motor function of the SOD1-G93A mice at the late stage of disease decreased,and the length of step decreased by the experiment of the rotatory rod and the step test,.Conclusion:During the process of sciatic nerve development,the mutated SOD1 gene does not affect the formation of the sciatic nerve myelin sheath.In the SOD1-G93A mice,the sciatic nerve showed progressive damage.Especially in SOD1-G93A mice,he myelin sheath and axon were significantly denaturated at P120.Part?:The changes of peripheral nerves were observed in theperipheral nerves of SOD1-G93A mice?120 days?and the effect of the mutant SOD1 gene on regeneration of peripheral nerve.Objective:The purpose of this study is to explore the effect of mutanted SOD1 gene on the peripheral nerve,including the influence of inflammation and blood nerve barrier.The survive of SCs were detected to investigate the change of peripheral nerve in the SOD1-G93A mice.In addition,the peripheral nerve of the SOD1-G93A transgenic mice and the non-transgenic control mice were crushed to observe effect of the mutanted SOD1 gene on peripheral nerve regeneration.Methods:fluorescence staining was used to observe the expression of inflammatory correlative factors in the late stage of SOD1-G93A transgenic mice.To observe the damage of the blood nerve barrier in the peripheral nerves of the transgenic mice,the mice were injected with the Evan's blue dye of the SOD1-G93A transgenic mice and the control mice.The apoptosis of the sciatic nerve in the SOD1-G93A transgenic mice and the non-transgenic control mice was detected by Tunel kit.In addition,the peripheral nerve of the SOD1-G93A transgenic mice and the non-transgenic control mice were crushed at P30.Electron microscopy was applied to explore the ultrastructure of peripheral nerve between two groups 7 days and 28 days after injury.At the same time,BrdU staining was applied to observe the proliferation of Schwann cells between two groups of mice 7 days after the crush.The mRNA expression of inflammation related genes and myelin related genes were detected by real-time quantitative PCR 7 days after the crush..In addition,the recruitment of inflammatory cells in two groups were observed by fluorescence staining.Results:1.The method of fluorescent staining were used,the expression of CD68MCP-1,CD86 and TGF-?were found increased in theperipheral nerve of the SOD1-G93A transgenic mice,compared with sciatic nerve of the control mice.2.Evan's blue were injected to the mice,we found that the blood nerve barrier in the peripheral nerve of the SOD1-G93A mouse was damaged.The apoptosis of Schwann cells in the peripheral nerve was observed.Compared with the control mice,the death of SCs were obviously observed in the sciatic nerve of SOD1-G93A mice.3.The peripheral nerve of the SOD1-G93A transgenic mice and the non-transgenic control mice were crushed.7 days after crush,real-time quantitative PCR were used,the non-transgenic control mice were found recruit more inflammatory cells.At the same time,real-time quantitative PCR results also showed that the correlation gene of myelin was significantly increased in the peripheral nerve of the non-transgenic control mice.4.The peripheral nerve of the SOD1-G93A transgenic mice and the non-transgenic control mice were crushed.7 days after crush,BrdU stainings were used,compared with the SOD1-G93A transgenic mice,the proliferation of Schwann cell in the non-transgenic control mice increased significantly.5.The ultrastructure of sciatic nerve in SOD1-G93A transgenic mice and the non-transgenic control mice were observed by electron microscopy in the28 days after crush.The results showed that there were more myelinated fibers in the non-transgenic control mice,compared with the SOD1-G93A mice.Conclusion:In the sciatic nerve of the SOD1-G93A mice,severe inflammatory reaction occurred in the late stage of disease,and the blood nerve barrier was damaged.The apoptosis of the sciatic nerve in the SOD1-G93A mice increased significantly.After acute injury intervention in the peripheral nerves of SOD1-G93A mice,the mutant SOD1 gene was found to affect regeneration of the peripheral nerve.Part?:The effects of FGF9 Schwann cells knockout on the peripheralnerve of the SOD1-G93A miceObjective:To observe the effects of FGF9 Schwann cells knockout on the peripheral nerve of the SOD1-G93A mice.Methods:Fluorescence staining was used to observe the expression of FGF9inperipheralnerveofFgf9^fl/flmice.Themiceof SOD1-G93A;Fgf9^fl/fl;P0-cre and SOD1-G93A;Fgf9^fl/fll/fl were obtained by multiple hybridization.Using real-time quantitative PCR method to observe the knockout efficiency of FGF9 in peripheral nerves of Fgf9^fl/fl;P0-cre mice.Fluorescence staining and real-time quantitative PCR were used to detect the secretion of inflammatory related factors in the peripheral nerves of SOD1-G93A;Fgf9^fl/fl;P0-cre and SOD1-G93A;Fgf9^fl/fll/fl mice.The ultrastructure ofperipheralnervebetweenSOD1-G93A;Fgf9^fl/fl;P0-creand SOD1-G93A;Fgf9^fl/fll/fl mice were observed by electron microscopy.Tunel staining were used to detect the apoptosis of peripheral nerves in SOD1-G93A;Fgf9^fl/fl;P0-cre and SOD1-G93A;Fgf9^fl/fll/fl mice.Results:1.Fluorescence staining revealed a strong expression of FGF9 in the peripheral nerves of Fgf9^fl/fll/fl mice.And the results of mRNA showed that the expression of FGF9 decreased in the sciatic nerve after FGF9 SCs knockout.2.It was found that the peripheral nerves of SOD1-G93A;Fgf9^fl/fl;P0-cre and SOD1-G93A;Fgf9fl/fl mice a large number of inflammatory factors were secreted at P120 by fluorescence staining and real-time quantitative PCR.The peripheral nerves of SOD1-G93A;Fgf9^fl/fll/fl mice were found recruit more inflammatory cells and secrete more inflammatory cytokines.3.Afterobservationtheultrastructureofsciaticnervein SOD1-G93A;Fgf9^fl/fl;P0-cre and SOD1-G93A;Fgf9^fl/fll/fl mice,the statistical resultsshowedthatthedamageofperipheralnerve SOD1-G93A;Fgf9^fl/fl;P0-cre mice injury of a mouse was relatively slight,and more axons were retained.4.Using Tunel staining,it was found that peripheral nerve apoptosis was more significant in SOD1-G93A;Fgf9^fl/fll/fl mice,compared with the peripheral nerves of SOD1-G93A;Fgf9^fl/fl;P0-cre mice.Conclusion:We found FGF9 may recruit more inflammatory cells during peripheral nerve injury,FGF9 exacerbated the peripheral nerve degeneration.