| Peripheral nerve injury(PNI)is a common clinical problem that often leads to different degrees of dysfunction and disability of the body.After PNI repair,up to 33%of patients exhibit a variety of signs and symptoms,such as loss of sensory and motor function,chronic pain,multiple functional disorders.In terms of physiological anatomy and functional behavior,the peripheral nerve plays a key role in relaying messages back and forth from the brain to different parts of the body.After PNI,on one hand,the nutrient supply to the target organ decreases,the long-term denervation causes atrophy of the target organ,and a decline in function,leading to irreversible changes.On the other hand,the neurons of the corresponding spinal cord segment are damaged,which further aggravate PNI.During peripheral nerve repair,many complex biological processes are involved,such as changes in various types of cells[12],the influence of various transcription factors[13],and the changes in the neuronal microenvironment.At present,advanced microsurgical repair is the first choice for the treatment of PNI,but the use of surgery alone is difficult to obtain satisfactory results,with low patient satisfaction.PNI repair has always been a difficult problem for clinicians.Appropriate electrical stimulation(ES)has been shown to accelerate the expression of neurotrophic factors and receptors,promote axon extension,and increase the specificity of axon docking,thus promoting PNI repair.The molecular mechanism by which ES promotes PNI repair remains poorly understood and needs to be further investigated.Based on the current situation of clinical application and basic research of PNI repair,in this study,we performed in vivo experiment to verify the promoting effect of ES on PNI repair,applied ES on Schwann cells(SCs)in vitro to observe changes in cellular biological behaviors,such as migration and proliferation of SCs,and predicted targeted genes for differentially expressed miRNAs that were involved in nerve repair.The study aimed to investigate the molecular mechanism by which ES promotes PNI repair,and verify the role of miRNA-target gene regulatory relationship in promoting PNI repair through both in vitro cell experiment and in vivo animal experiment.Methods:Experiment 1:Effect of ES on sciatic nerve repair after crush injury in ratsIn this part,rat model of sciatic nerve crush injury was established,and ES was applied.The effect of ES at certain intensity on the repair of sciatic nerve injury in rats was evaluated comprehensively by observation of general condition and behavior of rats,histopathological changes in sections of sciatic nerve,gastrocnemius and spinal cord,and changes in the expression of target proteins in sciatic nerve tissues.(1)Model establishment:Rats were randomly divided into ES,non-ES and sham operation groups.In the ES and the non-ES groups,rat model of grade IV sciatic nerve injury was established by using clamping method.After model establishment,continuous ES(intensity:5 mA,frequency:20 HZ,duration:1 h)was applied immediately for 1,7,and 14 days.(2)Observation of general condition and behavioral changes in rats:The general condition of rats in each was observed at each observation time point.The sciatic functional index(SFI)was calculated by walking track analysis in order to determine the degree of motor function recovery after sciatic nerve injury.Response latency to thermal pain was measured to detect the extent of sensory nerve recovery.(3)Histological changes in the sciatic nerves of rats:At each observation time point,the sciatic nerves of rats in each group were harvested.Hematoxylin-eosin(HE)staining was performed to observe histological changes in the sciatic nerves.Electron microscopy was used to observe myelin and axon regeneration.Immunofluorescence staining with S100β,myelin basic protein(MBP),and neurofilament(NF)antibodies was used to observe the dedifferentiation and proliferation of SCs,myelin sheath formation,and the extent of axon regeneration,respectively.(4)Histological changes in the gastrocnemius muscles of rats:At each observation time point,gastrocnemius muscles of rats in each group were harvested.Gastrocnemius muscle wet weight was measured and then photographed.HE staining was performed to observe the histological changes in muscle fibers.(5)Histological changes in the spinal cord of rats:At each observation time point,the L4-L6 segments of the spinal cord in each group were collected.HE staining was performed to observe the histological changes in the spinal cord,and toluidine blue staining was performed to further verify the changes in motor neuron in the anterior horn of the spinal cord.Experiment 2:Effect of ES on the biological behavior of rat SCsIn this part,the optimal parameters for ES applied on SCs were determined.The effects of ES on the specific biological behaviors of SCs were analyzed by morphological observation,cell counting assay,biochemical assays,flow cytometry and molecular biological assays.(1)Determination of the optimal stimulation parameters:SCs were inoculated on cell culture conductive slides.Cells in one group were treated with a single application of ES,and cells in the other group were treated with continuous applications of ES for a total of 3 days.Stimulation parameters consisted of a frequency of 20 Hz,current intensities of 30 mA,15 mA,5 mA,0 mA,a pulse duration of 100 μs,an on-off ratio of 1:2,and a stimulation duration of 60 min.The optimal stimulation parameters were determined by transwell migration assay.(2)Effects of ES on the biological behaviors of rat SCs:SCs were divided into two groups:a 0mA group(control group)and an ES group.Cells in the ES group received ES with a frequency of 20 HZ,intensity of 30 mA,pulse width of 100 μs,1:2 ratio,and stimulation duration of 60min.The migration of SCs was assessed using a transwell migration assay.The effect of ES on the proliferation of SCs was analyzed by using various methods,i.e.cell growth status was directly observed under a light microscope,cell viability and cell proliferation were evaluated by MTS assay,and the effects of ES on cell cycle were evaluated by flow cytometry.Meanwhile,RT-PCR was performed to detect the expression of key cell cycle-related proteins,thus clarifying the effect of ES on the proliferation of SCs.Experiment 3:Whole transcriptome analysis,bioinformatics analysis and validation in SCs treated with ESIn this part,based on the results regarding the effects of ES on the specific biological behavior of SCs obtained in Experiment 2,whole transcriptome analysis was performed to screen the miRNAs and their target genes that were involved in the changes in biological behaviors of SCs caused by ES at certain intensity.The miRNA-target gene regulatory relationships induced by ES were verified by molecular biology assays,such as RT-PCR,western blotting,plasmid transfection,and cellular assays.(1)Whole transcriptome sequencing and bioinformatics analysis in ES-treated SCs:Next-generation sequencing was implemented to identify circRNAs,lncRNAs,miRNAs and mRNAs in SCs treated with ES at 0 mA and 30 mA,respectively.The original image data of the sequencing results were analyzed using Bcl2fastq(v2.17.1.14)for image base calling and preliminary quality analysis,and the raw sequencing data generated were stored as FASTQ files to obtain sequence information of miRNA,lncRNA,mRNA,and circRNA.Based on the standard and combined analyses,the differentially expressed miRNAs were identified.Target genes regulated by these miRNAs were screened by clustering and enrichment analysis.(2)Validation of miRNA-target gene regulatory relationships:RT-qPCR and western blotting were used to verify the expression of differentially expressed miRNAs and their target genes in SCs treated with ES.Cell migration assay was used to detect the effect of differentially expressed miRNAs and their target genes on the migration ability of SCs.Then,miRNA mimic transfection,reversal assay by transfecting target gene-expressed plasmid and dual-luciferase reporter assay were performed to verify the regulatory relationships between miRNAs and their target genes.Experiment 4:Effects of the identified miRNAs on the repair of sciatic nerve crush injury in ratsIn this part,based on the regulatory relationships between the miRNAs and their target genes determined in vitro in Experiment 3,rat model of grade IV sciatic nerve injury was created,and rats were injected with corresponding miRNA mimics(agomir)and inhibitor(antagomir).The effects of the identified miRNAs on the repair of sciatic nerve injury in rats were assessed through observation of general condition of rats,histopathological changes in sections of sciatic nerve,and changes in the expression of target proteins in sciatic nerve tissues.Meanwhile,after creation of the rat model of grade IV sciatic nerve injury and application of ES,the miRNA-target gene regulatory relationships were verified again by fluorescence in situ hybridization(FISH)analysis combined with immunofluorescence.(1)Model preparation:Rat model of grade IV sciatic nerve injury model was established by crushing the sciatic nerve with a clamp.Rats were randomly divided into antagomiR,agomiR and control groups.Observation time points were set at 7 days,14 days postoperatively,and all rats in each group were subdivided into 7-day subgroup and 14-day subgroup.Rats in the 7-day subgroup received a single injection of antagomiR,agomiR and a mixture of PBS and matrigel,respectively,on the surgery day.In the 14-day subgroup,antagomiR,agomiR and a mixture of PBS and matrigel were injected into the rats on the surgery day and on days 7 after surgery,respectively.(2)Observation of the effects of miRNAs on the repair of sciatic nerve crush injury in rats:General condition of rats in each group were observed.HE staining was performed to observe histological changes in sciatic nerves.Immunofluorescence staining with S100β,MBP,and NF antibodies was used to observe the dedifferentiation and proliferation of SCs,myelin sheath formation,and extent of axon regeneration,respectively.(3)Validation of ES-miRNA-target gene:After application of ES in rat model of grade IV sciatic nerve injury,FISH combined with immunofluorescence was used to verify the miRNA-target gene regulatory relationships again.Results:Experiment 1:ES for the repair of sciatic nerve crush injury in rats(1)After establishment of rat model of sciatic nerve crush injury,sciatic nerves of rats were harvested.HE staining results of the crushed sciatic nerve revealed that the myelin sheath was damaged without normal fiber structure.Myelin sheath marker MBP wrapped around the axon and was in a missing and irregular state.The arrangement of SCs marker S100β was disordered.There were many broken fibers and the number of fibers decreased.These results indicated that most of the nerve fibers in the injured area were broken and their arrangement was disordered,suggesting that rat model of sciatic nerve crush injury was successfully established.(2)General condition and behavioral observation:There were no significant differences in general condition and response latency to thermal pain between ES and non-ES groups at each time point.At 14 days after surgery,complete footprints were observed in both groups,there was a significant difference in SFI between the ES and non-ES groups[(-74.65±1.79)vs.(-86.41±2.76),P<0.01].This suggests that ES can promote the recovery of sciatic nerve motor function earlier.(3)Histological changes of the sciatic nerve:HE staining of the sciatic nerve revealed that at 1 day after surgery,there was no significant difference in pathological results between the ES and non-ES groups.At 7 days after surgery,nerve fibers in the ES group were arranged more orderly and the number of infiltrated inflammatory cells was lower compared with the non-ES group.These differences were more marked at 14 days after surgery.Transmission electron microscope results of sciatic nerve ultrastructure showed that at 1 day after surgery,the sciatic nerve was injured in both the ES and non-ES groups.At 7 days after surgery,the myelin sheath and axon of the sciatic nerve tended to recover,and the myelin sheath was thick in each group,but the fibers in the non-ES group were sparse.At 14 days after surgery,the two groups both had thinner myelin sheath when compared to the observation at 7 days after surgery,however,the sciatic nerve was thicker,axon arrangement was better,and nerve fibers were denser in the ES group compared with the non-ES group.Immunofluorescence staining with S100β antibody showed that S100β protein exhibited a streamline trend in the longitudinal direction at 7 days after surgery,and the streamline trend was more continuous at 14 days after surgery.The results of immunofluorescence staining with MBP antibody were similar to those of immunofluorescence staining with S100-βantibody.The streamline trend of S100β and MBP expression in the ES group were not as continuous as those in the sham operation group.At 14 days after surgery,results of immunofluorescence staining with NF protein antibody showed that at the transverse level,most of the sciatic nerve axons in the ES group were surrounded by SCs,the diameters of the axons were uneven,but axon arrangement was regular.In the non-ES group,only a few sciatic nerve axons were surrounded by SCs,and axons were scattered and sparsely distributed.In the sham operation group,there were a larger number of sciatic nerve axons,and each axon was wrapped by SCs,axons were large in diameter,evenly distributed,and orderly arranged.These findings suggest that ES promotes the repair of the injured sciatic nerve by promoting myelin sheath formation and axon regeneration of the injured sciatic nerve.(4)Histological changes of the gastrocnemius muscle:At each time point,there was no significant difference in wet weight of the gastrocnemius muscle between the ES and non-ES groups.Wet weight of the gastrocnemius muscle in the ES group gradually decreased with time compared with the sham operation group.Gross observation results documented muscle atrophy after sciatic nerve injury.HE staining results showed that the cell viability in the gastrocnemius muscle was significantly stronger in the ES group than non-ES group at each time point.At 7 and 14 days after surgery,the number of nuclei was significantly higher,cells were smaller,but cell morphology was clear and uniform in size,and they were more densely arranged in the ES group when compared to the non-ES group.The non-ES group exhibited more obvious atrophy of muscle cells compared with the ES group.These findings suggest that ES can improve the morphology of the target tissues innervated by injured sciatic nerve.(5)Histological changes in the spinal cord:HE staining of the spinal cord revealed that at 1 day after surgery,spinal cord nerve cell bodies were slightly swollen,with decreased cell protrusions in the non-ES group.Spinal cord nerve cell bodies were slightly shrunk,with decreased cell protrusions in the ES group.At 7 days after surgery,spinal cord nerve cell bodies were obviously shrunk,with decreased cell protrusions,neuron arrangement was slightly irregular,and the numbers of neurons and Nissl bodies were decreased in the non-ES group.Spinal cord nerve cell bodies were slightly shrunk,with visible protrusions,some nuclei moved to the edge of the cells,and Nissl bodies were evenly distributed in the ES group.At 14 days after surgery,the pathological changes in the spinal cord tissues of rats in the non-ES group was similar with these observed at 7 days after surgery.The signs of neuronal injury repair were observed,some neurons exhibited regenerated nuclei,Nissl bodies were evenly distributed,and neurons with slightly large protrusions were seen in the ES group.Toluidine blue staining was employed to quantify the neuronal densities,the results showed that the densities of neurons in the anterior horn of the spinal cord in the ES and non-ES groups were 26.03±1.35 and 8.25±1.42,respectively,at 1 day after surgery,36.22±0.72 and 26.42±0.86,respectively,at 7days after surgery,43.72 ± 5.25 and 31.04 ± 2.17,respectively,at 14 days after surgery.The ES group showed significantly higher densities of neurons in the anterior horn of the spinal cord than the non-ES group at each time point.And the densities of neurons in the anterior horn of the spinal cord in the ES group increased with time,which was markedly lower than the sham operation group at 14 days after surgery(43.72 ± 5.25 vs.62.96±3.56).These findings suggest that ES can markedly improve the morphology and density of neurons in the corresponding upstream spinal cord segment of the injured sciatic nerve.Experiment 2:Effects of ES on the biological behaviors of rat SCs(1)The optimal parameters for ES applied on SCs in vitro:Transwell migration assay results showed that the optimal stimulation parameters included 20 Hz frequency,30 mA current intensity,100 μs pulse width,1:2 ratio,and 60 minutes duration.(2)In term of the effects of ES on the biological behaviors of SCs,the cell migration assay results showed that ES at 30 mA significantly increased the number of migrated SCs compared with ES at 0 mA(p<0.01).SCs treated with continuous application of ES showed significantly greater migration ability than those treated with a single application of ES.Cell proliferation MTS assay showed that ES at 0 mA and 30 mA both effectively maintained cell viability,but did not increase the proliferation ability of SCs.Experiment 3:Whole transcriptome sequencing,bioinformatics analysis,and validation of SCs treated with ES(1)Whole transcriptome sequencing and bioinformatics analysis of ES-treated SCs:The expression profiles of circRNA,lncRNA,miRNA,and mRNA were sorted out.The differentially expressed RANs were screened with FC≥2 and P<0.05 as the thresholds.The volcano plots of circRNA,lncRNA,miRNA,and mRNA expression were created using EdgeR.A total of 1467 differential expressed circRNAs,195 differential expressed lncRNAs,66 differential expressed miRNAs,and 86 differential expressed mRNAs were screened.The results of cluster analysis and enrichment analysis showed that the top three molecular functional items with high enrichment were "transcription co-repressor","fibronectin",and“mismatched DNA".The top three cell composition items with high enrichment were "nucleus","macromolecular complex",and "presynaptic active region".The top three biological process items with high enrichment were "positive regulation of transcription of RNA polymerase Ⅱ promoter","positive regulation of endocytosis" and"negative regulation of G protein-coupled receptor protein signal pathway".Among them,the items " nucleus" and "positive regulation of transcription of RNA polymerase Ⅱpromoter" enriched the greatest number of differentially expressed genes,i.e.,84 and 23 differentially expressed genes,with the-log 10(P-value)of 4 and 3 respectively.These findings suggest that after ES,the substance in the nucleus of SCs changes greatly,and the transcription activity changes.The specific functions of each differentially expressed gene were further analyzed in detail,and the heat map was drawn.The results showed that the up-and down-regulated genes were enriched in cell migration,oxidative stress,DNA damage repair,and cell proliferation and inhibition.Among these genes,MCC was most significantly down-regulated in the ES group.The miRNAs responsible for MCC regulation included miRNA-6328.miRNA-6328 was predicted to be a regulator of MCC expression.(2)Validation of miRNA-target gene regulatory relationships:the RT-PCR and western blotting validation results showed that MCC expression was down-regulated in response to ES.siRNA-specific knockdown of MCC expression in SCs promoted cell migration,conversely,overexpression of MCC decreased cell migration.RT-PCR revealed high expression of miRNA-6328 in the ES group.miRNA-6328 mimic assay results showed that the number of migrated cells in the miRNA-6328 mimic group was significantly higher than that of control group,whereas the number of migrated cells in the miRNA-6328 inhibitor group was significantly lower when compared to controls.RT-PCR and western blotting revealed low and high MCC expression in cells transfected with miRNA-6328 mimics and miRNA-6328 inhibitor,respectively.Reversal assay by transfecting plasmid expressing MCC,i.e.co-transfection of miRNA-6328 mimics with MCC plasmid and control mimics,showed that the enhancing effect of miRNA-6328 on the migration of SCs was reversed by MCC plasmid.All the above-mentioned results demonstrated that miRNA-6328 negatively regulated the expression of MCC,thus promoting the migration of SCs.The dual-luciferase reporter assay was performed to verify the regulatory relationships between miRNA and MCC,the results showed that cells co-transfected with luciferase reporter plasmid and miRNA-6328 mimics had low fluorescence value,miRNA-6328 regulated the expression of MCC by binding to the 3’-UTR region of MCC.The findings suggest that miRNA-6328 negatively regulates the expression of MCC by binding to its 3’-UTR region,which in turn promotes the migration of SCs.Experiment 4:Effects of miRNA-6328 on the repair of sciatic nerve crush injury in ratsIn order to observe the effect of miRNA-6328 on sciatic nerve repair,rat model of grade IV sciatic nerve injury model was established by crushing the sciatic nerve with a clamp.Rats were randomly divided into antagomiR-6328,agomiR-6328 and control groups,and were observed at two time points(7 and 14 days)after surgery.No significant change was observed in the general condition of rats in each group at each observation time point.At 14 days postoperatively,the hindpaw pads of rats in each group were completely suspended and were unable to touch the ground,with joint stiffness and muscle atrophy in the hind limbs.However,the HE staining results showed obvious improvement in arrangement of nerve fibers,neovascularization and myelin regeneration in the agomiR-6328 group and the control group compared with the antagomir-6328 group at 7 days postoperatively.New blood vessels were formed,myelin was regenerated,nerve fibers were arranged regularly,and inflammatory cells began to decrease in the agomiR-6328 group at 14 days postoperatively,these changes were significantly better than the antagomir-6328 group.Immunofluorescence staining with S100β antibody showed that at 14 days postoperatively,S100β protein expression exhibited a streamline trend in the longitudinal direction in the agomiR-6328 group and the control group,and the fluorescence density in the agomiR-6328 group was significantly higher than that in the control group,while the streamline trend of S100-βprotein expression was not apparent in the antagomiR-6328 group.Immunofluorescence staining with MBP antibody was performed to observe myelin formation,and similar results were obtained as those obtained from immunofluorescence staining with S100β.Immunofluorescence staining with NF protein antibody was performed to verify axonal regeneration,the results showed that in the cross section,many sciatic nerve axons were wrapped by SCs,axons exhibited varying diameters and were arranged more regularly in the agomiR-6328 and control groups at 14 days postoperatively.The control group had significantly reduced nerve fiber density when compared to the agomiR-6328 group.In the antagomiR-6328 group,only a few sciatic nerve axons were wrapped by SCs,and the axons were scattered and sparsely distributed.These results suggest that miRNA-6328 could promote myelin formation and axon regeneration in the injured sciatic nerve,thus facilitating the repair of sciatic nerve injury in rats.(2)Validation of ES-miRNA-target gene axis:after applying ES in rat model of grade Ⅳ sciatic nerve injury model,miRNA-target gene regulatory relationships was verified again by FISH combined with immunofluorescence,the results showed that the ES group exhibited high expression of miRNA-6328 and low expression of MCC,while non-ES group showed low expression of miRNA-6328 and high expression of MCC.The above results suggest that the ES-miRNA6328-MCC axis is an important mechanism of action of ES in the treatment of sciatic nerve injury.Conclusions:Our findings suggest that 1)ES can promote sciatic nerve repair after injury in rats;2)ES can promote the repair of sciatic nerve injury by enhancing the migration of SCs;3)miRNA-6328 down-regulates MCC expression by binding to 3’-UTR of mRNA,to inhibit its protein expression and enhance the migration ability of SC cells;4)ES can induce miR-6328 to regulate MCC,which in turn promote the repair of injured peripheral nerve.The ES-miRNA6328-MCC axis is an important mechanism of ES in the treatment of sciatic nerve injury. |
PDF Full Text Request