| The research on prostheses at home and abroad is moving towards the direction of active and intelligent prostheses,requiring the prostheses to have the information feedback capabilities for accepting movement awareness to simulate normal walking,which means that the amputee should meet certain requirements on the state of the remaining muscles and nerves.Therefore,research on how to deal with residual limb muscles and nerves is of great significance to the application of prosthetics.Targeted muscle reinnervation(TMR)is a novel neural machine interface technology,there are many examples at home and abroad that have been reported to apply this technique clinically in the patients with high amputation.When an amputee with a TMR interface imagines doing a specific limb movement,the nerve signals generated by the brain are transmitted to the "target muscles" through nerves,and the electromyogram(EMG)generated by muscle contraction can be detected non-invasively with surface electrodes.The intelligent prostheses could predict the physical movements that the amputee wants to perform via measuring the EMG decoding.The influence of TMR on the survival of the residual tibial nerve and its cell bodies,as well as the re-neuralization of the specific targeted muscle NMJ,is closely related to the utilize of intelligent prostheses after TMR.Therefore,this study selected gastrocnemius muscle,tibial nerve and corresponding spinal lumbosacral enlargement as the research objects to analyze the changes in tissue morphology and related protein expression after treadmill training on the postoperative in the TMR rats.Explore the effect of TMR on the survival of the residual tibial nerve and its cell bodies,and the regeneration of the targeted muscle NMJ,and evaluate the promoting effect and possible mechanism of treadmill training on the recovery of residual motor neuron function after TMR.Objection: 1 Compare the effect of several simulation models of peripheral nerve function reconstruction after paraplegia,and find suitable animal models of targeted muscle nerve function reconstruction.2 Explore the effect of TMR on the survival of the residual tibial nerve and its cell bodies,and the reneuralization of the specific targeted muscle NMJ.3 Evaluate the promoting effect and possible mechanism of treadmill training on the recovery of residual motor neuron function after TMR.Method: 1 Establish a tibial nerve dissection model,a TMR model,a TNR model,and a NR model.Footprint analysis was used to calculate the SFI index of rats in each group,and the effects of several models of peripheral nerve function reconstruction after simulated paraplegia were evaluated through the maintenance rate of muscle wet weight,skeletal muscle HE staining and MASSON staining assays.2 Establish a TMR model and behavioral testing was performed: calculate the SFI index of rats in each group by footprint analysis;weigh bilateral muscle wet weight,calculating the maintenance rate of muscle wet weight,evaluate the effect of TMR on skeletal muscle fibers via skeletal muscle HE staining and MASSON staining,measure the number of NMJ and expression of ACHE,expression of tibial nerve S100 B,spinal cord neurons Ch AT,SYN and PSD95 by immunohistochemical detection,detecte the number of tibial nerve axons and myelin thickness through LFB staining.3 Establish a TMR+RUN model and behavioral testing was performed: calculate the SFI index of rats in each group by footprint analysis;weigh bilateral muscle wet weight,calculate the maintenance rate of muscle wet weight,and evaluate the effect of TMR on skeletal muscle fibers by HE staining and MASSON staining of skeletal muscle;measure the number of NMJ and expression of ACHE,tibial nerve S100 B and tibial nerve MBP,the number of axon and nyelin thickness by immunohistochemical detection,investigate the expression of spinal neuron SYN m RNA and PSD95 m RNA via PCR detection,detect the expression of skeletal muscle NRG1/ Erb B2,BDNF and trk B in the spinal cord through WB analysis.Result:1.SFI index of the TMR group was significantly higher than the TNT group but was lower than the control group,TNR group and NR group,(P<0.01).The gastrocnemius muscle wet weight was significantly higher in the TMR-6W group than in the TNT group,but was lower than the control group,TNR group and NR group(P<0.01).The muscle cells in the TMR group still showed atrophy and the gap widened significantly,however,compared with the TNT and NR group,TMR group was significantly improved,and the muscle cells were arranged regularly(P <0.05).Compared with the TNT and NR groups,the content of collagen fibrin in the muscle space of the TMR group was increased,the collagen fibrin content was significantly reduced in the TNT and NR groups,and the results showed significant difference(P <0.05).2.There was no significant difference in SFI index between TMR-6W and TMR-12 W groups,but both were significantly higher than the TNT group,and the difference was statistically significant(P<0.01).There was also no significant difference in the wet weight maintenance rate of the gastrocnemius muscle between the TMR-6W and TMR-12 W groups,but both were significantly higher than the TNT group(P <0.01).The muscle cells in the TMR group still showed atrophy and the gap widened significantly,however,TMR group was significantly improved,and the muscle cells were arranged regularly compared with the TNT group(P <0.05).The content of collagen fibrin in the intermuscular space of rats in the TMR group increased.However,it was significantly reduced in the TNT group,and the difference was significant(P<0.05).Compared with the TNT group,the number of Ach E-positive NMJ in the gastrocnemius muscle of the TMR-6W group and the TMR-12 W group increased significantly,and the Ach E immunofluorescence intensity was significantly enhanced(P<0.05).Moreover,compared with the TNT group,the number of axons in TMR-6W and TMR-12 W both increased,and there was a statistical difference(P<0.05).However,there was no statistical difference between TMR-6W group and TMR-12 W group.The myelin sheath thickness of TMR-6W group has no obviously increased compared with TNT group,but notably increased in TMR-12 W group(P <0.05).The expression of tibial nerve S100 B was much lower in the TMR-6W group and TMR-12 W group than in the TNT group(P<0.01),however,there was no significant difference between TMR-6W group and TMR-12 W group.The nerve cells in TMR-6W group are densely arranged,and the mainly large-sized neurons were clearly observed.The cytoplasm was uniformly stained,and the number was significantly higher than that of the TNT group(P<0.01).Compared with TMR-6W group,the nerve cells further increased in TMR-12 W group(P <0.01).The number of cholinergic neurons in the spinal cord and the Ch AT expression level in the TMR-6W and TMR-12 W groups were significantly increased compared with the TNT group(P<0.05),however,the difference between TMR-6W and TMR-12 W groups was not significant.Immunofluorescence staining also revealed the number of cholinergic neurons in the spinal cord were significantly increased and the immunofluorescence intensity of SYN was significantly enhanced in the TMR-6W and the TMR-12 W groups compared with TNT group.3.SFI index of the TMR+RUN group was significantly lower than the control group but was obviously higher than the TMR and TNT groups,and the difference was statistically significant(P<0.01).Moreover,the gastrocnemius muscle wet weight was significantly higher in the TMR+RUN group than in the TNT and TMR groups(P<0.05).HE staining results showed that the cross-sectional area of myocytes in the TMR+RUN group reached the normal level,which was significantly higher than that in the TNT and TMR groups.The content of collagen fibrin in the intermuscular space of rats in the TMR+RUN group increased,but the difference is not significant compared with the control group(P>0.05),however,it was significantly reduced compared with the TNT and TMR groups(P<0.05).Compared with the TNT and TMR groups,the number of Ach E-positive NMJ in the gastrocnemius muscle of the TMR+RUN group increased significantly,and the Ach E immunofluorescence intensity was significantly enhanced(P<0.05).Further,the expression of NRG1 and Erb B2 were significantly higher in TMR+RUN group than in TNT and TMR groups(P<0.05).Compared with TMR and TNT groups,the expression of MBP in TMR+RUN group was notably increased,the number of axons increased(P<0.05),and the thickness of myelin sheath also increased significantly(P <0.05).The expression of tibial nerve S100 B was much lower in the TMR+RUN group than in the TNT and TMR groups(P<0.01).The nerve cells in TMR+RUN group are densely arranged,and the cytoplasm was uniformly stained,and the number was significantly higher than that of the TNT and TMR groups(P<0.01).Compared with the TNT group,the levels of SYNm RNA and PSD-95 m RNA in the anterior horn of the spinal cord of the TMR group were up-regulated,but there was not significantly difference(P>0.05).However,the level of PSD-95 m RNA in the TMR+RUN group(0.95±0.02)was significantly up-regulated(P<0.05).Compared with TNT and TMR groups,the protein expression of BDNF and Trk B in TMR+RUN group increased significantly(P<0.05).Conclusion:1.To a certain extent,the TMR group model can restore the exercise ability of rats,protecting the remaining muscles,and the model exhibited a high survival rate.Therefore,it was the most suitable animal model for studying the reconstruction of muscle nerve function after peripheral nerve interruption.2.TMR technique enables the reconnection of residual nerve fibers and the target muscle,restores motor function in the hind limb at the injured side,and to a certain extent,it could improve the microenvironment for the survival of residual nerve fibers,neural cell bodies and NMJ regeneration,therefore,it is more conducive to reinnervation of hind limb muscles.3.Rehabilitation training after TMR enables the reconnection of residual nerve fibers and the target muscle,restores motor function in the hind limb at the injured side.The mechanism may be related to the improvement of the NMJ regeneration microenvironment,promotion of the regeneration of axons and myelin,and protection of the survival of neurons after the rehabilitation training. |
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