Nerve Root Magnetic Stimulation Enhances Synaptic Plasticity Of Sensorimotor Cortex And Damaged Region Of Spinal Cord Via Ascending Pathway

PurposeAfter spinal cord injury（SCI）,neural circuit reconstruction is not limited to descending corticospinal tracts,but also includes the connection of proprioceptive afferents to local spinal circuits.Therefore,it is difficult to achieve neural circuit reconstruction and functional remodeling with only a single transcranial cortical target,and multi-target,multi-modal progressive and repeated interventions are required.Based on this principle,our research group innovatively designed a neural circuit-magnetic stimulation protocol（transcranial magnetic stimulation+nerve root magnetic stimulation）for patients with incomplete SCI,which has been confirmed in clinical and basic research.Based on the effectiveness of transcranial magnetic stimulation（TMS）in the treatment of SCI has been confirmed by many studies,this study focuses on nerve root magnetic stimulation（NRMS）.The research purpose includes:1)determine whether NRMS can improve motor function following SCI;2)explore whether NRMS can affect the nerve conduction function of sensorimotor conduction pathways;3)study whether NRMS can promote synaptic plasticity in sensorimotor cortex;4)explore whether NRMS can promote synaptic plasticity in the injured area of spinal cord.The purpose is to lay the foundation of behavior,neurophysiology,cytology and molecular science for the promotion and use of NRMS,and to provide technical support for the neuromodulation of the central nervous system and the promotion of neural repair.Method1.Construction of rat clamp-type T10 spinal cord injury modelT10 compressed SCI model of rats was constructed by using aneurysm clips,and whether the model was successfully constructed was determined by intraoperative performance,postoperative observation of hindlimb muscle tone and behavioral evaluation（Basso-Beattie-Bresnahan,BBB）.2.To explore the therapeutic effect and mechanism of NRMS on SCI ratsSD rats were divided into 3 groups:sham operation+sham stimulation（Sham+SS）group,spinal cord injury+sham stimulation（SCI+SS）group and spinal cord injury+NRMS（SCI+NRMS）group.The NRMS intervention was started 3 days after SCI,once a day,5 times a week,for a total of 3 weeks.The changes of hindlimb motor function of SCI rats were observed by BBB score,inclined plate test,modified Tarlov score and rotarod test before operation,1d,3d,7d,14d,and 21d after operation to evaluate the therapeutic effect of NRMS on SCI rats.SEP and MEP were detected before operation,3d,7d,14d,and 21d after operation,and their latency and amplitude were compared to evaluate the nerve conduction function of NRMS on sensory conduction pathway（spinothalamic tract）and motor conduction pathway（corticospinal tract）.H-reflex detection was performed before operation,7d and 21d after operation.The latency and amplitude of H wave and M wave were recorded,and the H/M wave amplitude ratio was calculated to evaluate the effect of NRMS on spinal cord presynaptic inhibition.On the 22nd day after the injury,the sensorimotor cortex and spinal cord injury area of the rats were taken,the synaptic structure of the sensorimotor cortex was observed by transmission electron microscope,and the synaptic ultrastructural parameters were calculated:the thickness of the postsynaptic dense（PSD）,the length of the synaptic active zone,the synaptic curvature and the synaptic cleft;the pathological changes of the spinal cord structure in the injured area were observed by Nissl staining;the morphology of the neuronal dendrites in the sensorimotor cortex and the injured area of the spinal cord was observed by Golgi staining,and the number of dendrites and dendritic spine density were compared;the differential genes and involved pathways in each group were analyzed by transcriptome sequencing and bioinformatics,and the differential genes Syp,Syn2,GAP43 and MAP2 were further verified by RT-PCR;the expression levels of synaptic plasticity-related proteins PSD95,Synapsin I,GAP43 and BDNF were quantitatively analyzed by Western blot;the expression of PSD95 in sensorimotor cortex was also detected by immunofluorescence assay;the release levels of neurotransmitters GABA,Glu,NE,Ach and DA were measured by liquid chromatography-multiplex mass spectrometry;the expression levels of neurotrophic factors BDNF,NGF,NT3 and NT4 were detected by ELISA.Result1.Successful establishment of T10 compressed spinal cord injury models of ratsDuring the operation,when the spinal cord of the rat was clamped,the rat showed severe hindlimb twitching and spastic tail flicking,and the spinal cord showed obvious hematoma after clamping.The evaluation BBB score was significantly lower than the sham group,and the recovery was slow and limited.2.NRMS promotes motor function recovery after spinal cord injuryOn day 7 after SCI,the BBB score,slope angle and rotarod time in the SCI+NRMS group were significantly higher than those in the SCI+SS group（P<0.01,P<0.01,and P<0.05,respectively）.At 14 days and 21 days after operation,BBB score,modified Tarlov score,the angle of inclined plane and rotarod time of the rats in the SCI+NRMS group were higher than those in the SCI+SS group,and statistically significant differences were observed（P<0.001）.3.The impact of NRMS on SEPOn postoperative day 3,compared with the Sham+SS group,the SEP latency of the two groups of SCI rats was significantly prolonged（P<0.001）,while no significant difference was observed between the two SCI groups.After the first week,the SEP latency of SCI rats was gradually shortened,and the latency of NRMS-treated SCI rats was significantly shorter than that of sham-stimulated SCI rats(P_7d=0.004,P_14d=0.0137,P_21d<0.001).However,there was no significant difference in SEP amplitude between the two SCI groups during the study period（P>0.05）.4.The impact of NRMS on MEPOn the 3rd day after the operation,compared with the rats in the Sham+SS group,the MEP amplitude of the SCI rats was significantly decreased（P<0.001）,and the latency was significantly prolonged（P<0.001）.There was no significant difference in MEP latency between the SCI+NRMS group and the SCI+SS group（P>0.05）.On postoperative days 7,14,and 21,NRMS treatment was found to significantly shorten the prolonged MEP latency caused by SCI(P_7d=0.0023,P_14d<0.001,P_21d<0.001).5.Influence of NRMS on H-reflexCompared with preoperative,the SCI+SS group had a shorter latency（P<0.001）and an increased amplitude（P<0.01）of the H wave in the SCI+SS group at one week after operation;The M-wave amplitude ratios were significantly decreased（P<0.05）.At 7d postoperatively,the latency of H waves in the SCI+NRMS group was significantly longer than that in the SCI+SS group（P<0.01）.For the amplitude of H waves,although there was a large difference between the two groups,this was only statistically significant at the end of the first week（P<0.01）.For the H/M amplitude ratio,at 7d and 21d after surgery,compared with the SCI+SS group,the SCI+NRMS group was significantly lower,and the difference was statistically significant（P<0.001）.6.The effect of NRMS on synaptic structure of the sensorimotor cortexCompared with the Sham+SS group,in the SCI+SS group,we observed significantly impaired synaptic ultrastructure,straight synaptic morphology,reduced curvature,fewer synaptic vesicles,and more vacuoles.The synaptic structure of the SCI+NRMS group was more regular than that of the SCI+SS group,suggesting that synaptic damage has a certain degree of recovery.Significant changes in synaptic ultrastructure after SCI included reduced thickness of the PSD,reduced length of the synaptic active band,and reduced curvature of the synaptic interface（P<0.001）,but not the width of the synaptic cleft（P>0.05）.The length of the synaptic active band was significantly increased after NRMS treatment（P<0.001）.7.The effect of NRMS on the pathological structure of injured area of spinal cordThe Nissl body in the Sham+SS group is blue-purple tabby-like,and the Nissl body is large and abundant.Most Nissl bodies in the SCI+SS group are small in size,some of which have been decomposed into sand grains and scattered,and a large number of voids appear.Compared with the SCI+SS group,the number of Nissl bodies in the SCI+NRMS group was significantly increased,indicating that the degree of neuronal damage was less and the functional state was better.8.The effect of NRMS on neuronal dendrite in the sensorimotor cortex and the injured area of spinal cordCompared with the rats in the Sham+SS group,the number of dendrites and the density of dendritic spines in the sensorimotor areas of the cerebral cortex of the rats in the SCI+SS group were reduced,which were improved after NRMS treatment.The density of dendritic spines showed that the density of dendritic spines in the SCI+SS group was remarkably lower than that in the Sham+SS group,and the density of the SCI+NRMS group was markedly higher than that in the SCI+SS group（P<0.001）.By Sholl analysis,the results demonstrated that the number of dendrites of neurons in the SCI+SS group was significantly reduced compared with the Sham+SS group at 30-130μm from the soma（P<0.001）.At 60-140μm from the cell body（except at 70μm）,the number of neuronal dendrites in the SCI+NRMS group was significantly increased in comparison with that in the SCI+SS group(P_60,90,140<0.05,P₈₀<0.01,P_100-130<0.001).Compared with the rats in the Sham+SS group,the rats in the SCI+SS group had glial scar formation in the spinal cord injury area,the number of neurons was reduced,and the number of dendrites was reduced,while the glial scar was not obvious in the SCI+NRMS group was compared with that in the SCI+SS group,and the numbers of neurons and dendrites were both increased.9.The effect of NRMS on the transcription level of genes related to synaptic plasticity in the sensorimotor cortex and the injured area of spinal cordIn the sensorimotor cortex,GO enrichment analysis showed that the functions of cell processes,neuronal axons,regulation of cell communication,and signal regulation were significantly enriched.Significantly enriched signaling pathways in KEGG included neuroactive ligand-receptor interaction pathway,calcium signaling pathway,glutamatergic synaptic pathway,chemokine signaling pathway,GABAergic synaptic signaling pathway,MAPK signaling pathway,c AMP signaling pathway,axon guidance pathway and other signaling pathways.PCR results showed that Syp,Syn2,GAP43 and MAP2 m RNA were significantly increased in the sensorimotor cortex of the SCI+NRMS group compared with the Sham+SS group（P<0.01）.Compared with rats in SCI+SS group,Syp,Syn2,GAP43 and MAP2 m RNA were significantly increased in sensorimotor cortex of rats in SCI+NRMS group(P_Syp,MAP2<0.05,PSyn2,GAP43<0.01).In the injured area of spinal cord,GO enrichment analysis showed significant enrichment for functions such as synapses,postsynaptic compacts,cell processes,neuronal processes,dendritic spines,and central nervous system development.Significantly enriched signaling pathways in KEGG included neuroactive ligand-receptor interaction pathway,glutamatergic synaptic pathway,calcium signaling pathway,GABAergic synaptic signaling pathway,dopaminergic synaptic signaling pathway,c AMP signaling pathway,synaptic vesicle recycling signaling pathway and other signaling pathways.The PCR results showed that,at three weeks after SCI,both Syp and Syn2 m RNA were significantly increased in the injured area of spinal cord of the rats in the SCI+NRMS group compared with the rats in the Sham+SS group（P<0.05）.Syp m RNA was significantly increased in the injured area of spinal cord of the rats in the SCI+NRMS group compared with the rats in the SCI+SS group（P<0.05）.10.The effect of NRMS on the expression of synaptic plasticity-related proteins in the sensorimotor cortex and the injured area of spinal cordWB results showed that three weeks after SCI,compared with rats in Sham+SS group,the expressions of PSD95,GAP43,Synapsin I and BDNF in the cortical sensorimotor areas of rats in SCI+SS group were significantly decreased(P_BDNF<0.05,P_{PSD95,GAP43,Synapsin I}<0.001).Compared with untreated rats,the SCI rats treated with NRMS had significantly increased expressions of PSD95,GAP43,and Synapsin I（P<0.001）.The results of immunofluorescence showed that the expression of PSD95 in SCI rats was significantly decreased compared with that in sham-operated group,and the percentage of PSD95-positive cells in the cortical sensorimotor area of rats in SCI+NRMS group was significantly increased compared with that in SCI+SS group,consistent with WB results.Compared with the rats in the Sham+SS group,the expressions of PSD95 and BDNF in the injured area of spinal cord of the rats in the SCI+SS group were significantly decreased（P<0.001）,and the expression of Synapsin I was significantly increased（P<0.001）;the expression of Synapsin I in the injured area of spinal cord was significantly increased in the rats of the SCI+NRMS group（P<0.01）.The expression of PSD95,GAP43 and BDNF were significantly increased in NRMS-treated SCI rats compared with untreated rats(P_PSD95<0.05,P_GAP43,BDNF<0.01).11.The effect of NRMS on neurotransmitter release in the sensorimotor cortex and the injured area of spinal cordCompared with the rats in the Sham+SS group,the release of Glu,Ach,NE and DA in the sensorimotor cortex of the rats in the SCI group was obviously increased（P<0.05）.Glu release was significantly increased in the sensorimotor cortex of the rats in the SCI+NRMS group compared with the rats in the SCI+SS group（P<0.01）.Compared with the rats in the Sham+SS group,the release of Glu and DA in the spinal cord injury area of the rats in the SCI group were significantly decreased(P_Glu<0.001,P_DA<0.01),and the release of Ach was significantly increased（P<0.05）.Compared with the rats in the SCI+SS group,the release of NE,Ach and DA in the spinal cord injury area of the rats in the SCI+NRMS group were significantly reduced(P_NE,_Ach<0.001,P_DA<0.05).12.The effect of NRMS on neurotrophic factors in the sensorimotor cortexCompared with rats in Sham+SS group,the expression levels of proteins BDNF,NGF,NT3 and NT4 in the sensorimotor cortex of rats in SCI+SS group were significantly increased(P_BDNF,NT4<0.01,P_NGF,NT3<0.001).In the SCI+NRMS group,only the expression of BDNF was up-regulated（P<0.05）.Compared with the rats in the SCI+SS group,the protein expressions of NGF,NT3 and NT4 were significantly increased in the sensorimotor cortex of the rats in the SCI+NRMS group（PNT4<0.01,PNGF,NT3<0.001）.13.The effect of NRMS on the expression of inflammatory factorsCompared with the rats in the Sham+SS group,the levels of IL-1β,IL-6 and TNF-αin the serum of the rats in the SCI+SS group were significantly increased（P<0.001）.The contents of IL-1βand IL-6 were significantly increased(P_IL-1β<0.05,P_IL-6<0.01).Compared with the rats in the SCI+SS group,the levels of IL-1β,IL-6and TNF-αin the serum of the rats in the SCI+NRMS group were significantly increased(P_IL-1β<0.05,P_IL-6<0.001,P_TNF-α<0.01).ConclusionBy activating the ascending sensory pathway,NRMS can effectively promote the motor function recovery of incomplete SCI rats due to increased corticospinal output;promote the nerve conduction function of the sensory conduction pathway and motor conduction pathway;enhance the presynaptic inhibition of the spinal cord by reducing the excitability of motor neurons in the anterior horn of the spinal cord;reduce the damage to the synaptic structure of the sensorimotor cortex by SCI and expand the length of the synaptic active band;reduce the pathological changes of the spinal cord in the injured area;and increase the number of dendrites and the density of dendritic spines of the neurons in the sensorimotor cortex and the damaged area of spinal cord to enhance the structural plasticity of the sensorimotor cortex and injured area of spinal cord in SCI rats.Moreover,NRMS can increase the expression levels of synaptic plasticity-related proteins and the expression of synaptic plasticity-related genes at the transcriptional level in the sensorimotor cortex and the injured area of spinal cord,promote the release of neurotransmitters in the sensorimotor cortex,inhibit the release of neurotransmitters in the injured area of spinal cord,and increase the expression of neurotrophic factors in the sensorimotor cortex to enhance the functional plasticity of the sensorimotor cortex and the injured area of spinal cord in SCI rats.In addition,NRMS was also able to suppress the inflammatory response following SCI.