Effect Of High Frequency Repetitive Transcranial Magnetic Stimulation On Axonal Regeneration After Spinal Cord Injury In Mice And Its Underlying Mechanism

The incidence of traumatic spinal cord injury due to car accidents is continuing to rise.Although advances in medical technology have effectively reduced the mortality rate of patients with traumatic spinal cord injury,how to facilitate the recovery of neurological function after spinal cord injury remains a worldwide challenge.After traumatic spinal cord injury,many patients are left with paraplegia or quadriplegia,which can burden individuals,families,and society.The main reason for the difficulty in restoring neurological function is that the growth capacity of most neurons in the adult central nervous system is inhibited,resulting in the inability to regenerate axons,and the altered local microenvironment after spinal cord injury also creates a huge obstacle to axon regeneration.A variety of neurostimulation tools,including epidural electrical stimulation and brain stimulation,have been progressively used in clinical trials for the treatment of SCI patients in recent years.Epidural electrical stimulation has shown promising results in several clinical trials.Brain stimulation includes transcranial direct current stimulation and magnetic stimulation,which is based on the principle of stimulating neurons in the brain and spinal cord by electricity or magnetically generated electricity.Although there are high level of evidences supporting the efficacy of r TMS(repetitive transcranial magnetic stimulation)in depression,pain,and recovery of motor function after strok,there is still a lack of high-quality clinical studies to support its efficacy in patients with spinal cord injury,and the specific mechanisms are still not clearly understood.However,even so,as a non-invasive,safe,and costeffective treatment,the clinical application of r TMS for spinal cord injury patients remains attractive.Therefore,the primary goal of this study is to investigate the efficacy and potential molecular mechanisms of HF-r TMS(high-frequency repetitive transcranial magnetic stimulation).In this project,we successfully constructed a HF-r TMS device for mice and used mouse T8 dorsal hemisection as a spinal cord injury model.From histological and behavioral experiments,we found that HF-r TMS could promote axonal regeneration and facilitate motor function recovery after SCI in mice.And by extracting the mouse cerebral cortex after HF-r TMS for Western Blot,we found that HF-r TMS significantly upregulated the level of p MEK in the mouse cerebral cortex.Further,by raising transgenic mice with key genes of the B-Raf-MAPK signaling pathway and using the Cre-Lox P conditional gene expression system,we found that the promotion of axonal regeneration after SCI by HF-r TMS was inhibited after selective knock-out MEK genes,suggesting that the MEK genes are essential for the HF-r TMS-promoted axon regeneration.Meanwhile,we got similar results when we selectively overexpressed B-RAF in the neurons,both axonal regeneration and motor function recovery were observed.Therefore,we conclude that the B-Raf-MAPK signaling pathway is one of the key mechanisms of HF-r TMS in treating SCI,and activation of neuronal endogenous B-Raf-MAPK signaling pathway also contributes to axon regeneration and functional recovery after SCI.Finally,we explored how the B-RafMAPK signaling pathway promotes axonal regeneration and functional recovery after SCI in mice by translational ribosome affinity purification technology and highthroughput sequencing.Genomic enrichment analysis was performed for genes with statistical differences in the differential expression analysis.We found that axon guidance,regulation of actin cytoskeleton,calcium signaling pathway,glutamatergic synapse,and neurotrophin signaling pathway,which related to axon regeneration were significantly enriched.Previous studies have shown that TMS can affect neuronal Ca2+ influx,while in vitro studies have also shown that calcium influx can activate the RAS and thus the downstream B-RAF-MAPK.In summary,we have concluded a possible mechanism by which HF-r TMS promotes axonal regeneration and motor function recovery.HF-r TMS affected neuronal Ca2+ activity and thus activated the RAS-B-Raf-MAPK signaling pathway,and the activation of the RAS-B-Raf-MAPK signaling pathway in turn mobilized the activation of several signaling pathways related to axonal regeneration,thereby maintaining the growth capacity of axonal growth cones and increasing synaptic plasticity,promoting axonal regeneration and the formation of new synapses,and resulting in the facilitation of functional recovery after SCI.