| Objective:1.To investigate the neuroprotective effect of transcranial intermittent thetaburst stimulation(iTBS)for the acute and subacute stage of transverse SCI in rats.2.To investigate the mechanism of axon regeneration and motor function recovery for the chronic stage of transverse SCI in rats treated with transcranial iTBS.Methods:80 SD female rats weighing 180-200g were selected.All rats were randomly divided into the Normal group(n=20),the Control group(n=20),the sham iTBS group(n=20),and iTBS group(n=20).In addition to the Normal group,the other groups of rats underwent total spinal cord transection at T10.After the SCI model was successfully established,the rats in the Control group were not treated with transcranial magnetic stimulation.The rats in the iTBS group were treated 72 hours after SCI with iTBS.Once a day,5 times a week,until sampling.The rats in the sham iTBS group were treated with ineffective magnetic stimulation 72 hours after SCI,that is,the effective stimulation surface of the circular coil was perpendicular to the skull of the rats,and the treatment parameters were the same as those in the iTBS group.The behavioral scores of rats in each group were scored weekly.One week after treatment,tissues of the sensorimotor cortex(SMC)were collected for inflammatory cell detection,including immunofluorescence expression of CD68 and quantitative detection of CD68,CCR7 and CD206 by western blot.At 2 weeks after treatment,microglia expression and neuronal apoptosis in SMC tissues were detected,including immunofluorescence analysis of IBA-1,Cleaved caspase3 and NeuN,and quantitative western blot detection of Cleaved caspase3 and Pro-caspase3.At 4 weeks after treatment,the SMC tissues and the spinal cord tissues in the rostral injury region of rats in each group were collected for mRNA sequencing.The inflammation,neuronal injury and apoptosis,astrocytes,synaptic plasticity and axon regeneration were detected on the spinal cord tissue at the injured area,including immunofluorescence analysis of CD68,ATF3,Cleaved caspase3/NeuN,GFAP,GAP43,NF and quantitative western blot detection of Cleaved caspase3,Pro-caspase3,PSD95,SYN,NF and GAP43.At 8 weeks after treatment,motor evoked potential was detected on 5 rats in each group.After that,we performed BDA tracing in the SMC of 3 rats and the long descending propriospinal tract(LDPT)of cervical spinal cord of 2 rats in each group.At 2 weeks after nerve tracing,the spinal cord tissues of the injured area were collected from rats in each group for immunofluorescence analysis of 5-hydroxytryptamine and BDA.What’s more,the SMC,raphe nucleus and LDPT tissues were also collected for immunofluorescence analysis of c-Fos.Results:1.The results of iTBS on the treatment of acute and subacute stages of transverse SCI in rats showed that(1)Expression of inflammatory cells and apoptosis in neurons of SMC after 1 or 2 weeks of iTBS treatment:The number of M1-type macrophages(CCR7 marker),microglia cells(CD68,IBA-1 marker)and neuronal apoptosis(Cleaved caspase3/NeuN co-marker)in the SMC of the iTBS group was significantly lower than in the Control group and the sham iTBS group.The protein expression level of M2-type macrophages(CD206 marker)was significantly higher than that of the Control group and the sham iTBS group,the difference was statistically significant,but there was no statistically significant difference between the Control group and the sham iTBS group.The results of mRNA sequencing in the SMC showed that transcranial iTBS treatment significantly down-regulated the expressions of pro-inflammatory and pro-apoptotic genes and more up-regulated the expressions of anti-inflammatory and anti-apoptotic genes compared with the sham iTBS group.(2)Expression of inflammatory cells and apoptosis in neurons of the injured spinal cord after 4 weeks of iTBS treatment:In the iTBS group,the number of inflammatory cells(CD68 marker)in the regions rostral and caudal to/in the injury site of the spinal cord was significantly lower than that in the Control group and the sham iTBS group,and the number of apoptotic neurons(Cleaved caspase3/NeuN comarker)in the regions rostral and caudal to the injury site of the spinal cord was also lower than that in the Control group and the sham iTBS group,with statistically significant differences.This result was also confirmed by western blot analysis in the rostral tissues of the injured area,but there was no statistical significance between the Control group and the sham iTBS group.The results of mRNA sequencing in the spinal cord tissues showed that transcranial iTBS significantly down-regulated the expressions of pro-inflammatory and pro-apoptotic genes and more up-regulated the expressions of anti-inflammatory and anti-apoptotic genes compared with the sham iTBS group.(3)Neuroprotective effect of iTBS on the injured spinal cord:Compared with the Control group and the sham iTBS group,the number of damaged neurons labeled by ATF3 in the regions rostral and caudal to the injury site of the spinal cord in the iTBS group was significantly decreased.What’s more,the number of astrocytes labeled by GFAP in the regions rostral and caudal to/in the injury site of the spinal cord in the iTBS group was significantly decreased compared with the Control group and the sham iTBS group.However,there was no significant difference between the Control group and the sham iTBS group.The results of mRNA sequencing in the spinal cord tissues showed that transcranial iTBS down-regulated neuronal damage factor genes and up-regulated excitatory neuron genes and neurotrophic factor and receptor genes,thus promoting neuron survival.2.The results of iTBS on the treatment of chronic stage of transverse SCI in rats showed that(1)Recovery of motor function after iTBS treatment:After 8 weeks of treatment,the amplitude of motor evoked potentials,the average score of BBB test and the number of hind limb grid steps in iTBS group was significantly higher than those in the Control group and the sham iTBS group,while there was no statistical significance between the Control group and the sham iTBS group.(2)Results of synaptic plasticity in injured spinal cord after iTBS treatment:Compared with the Normal group,the synaptic formation related proteins SYN and PSD95 were decreased after SCI by western blot analysis.After 4 weeks of treatment,the expression levels of SYN and PSD95 in the iTBS group were significantly higher than those in the Control group and the sham iTBS group.Moreover,the results of mRNA sequencing in the spinal cord tissues showed that compared with the sham iTBS group,the iTBS group had higher levels of mRNA enrichment involved in neuronal activation,neurotransmitter,neuronal metabolism,axon regeneration and synaptic plasticity.(3)Results of axonal regeneration in the injured area of spinal cord after iTBS treatment:After 4 weeks of treatment,compared with the Control group and the sham iTBS group,the area of GAP43 positive and NF positive nerve fibers in the regions rostral and caudal to the injury site of the spinal cord in the iTBS group was significantly increased.In addition,it was also significantly increased compared with the sham iTBS group in the central of the injury site.However,there was no significant difference between the Control group and the sham iTBS group.At the same time,the results of western blot analysis in the rostral tissues of the injured area were consistent with these results.After nerve tracing,the number of BDA positive and 5-HT positive nerve fibers in the regions rostral to the injury site of the spinal cord in the iTBS group was significantly greater than that in the Control group and the sham iTBS group.At the same time,the number of 5-HT positive nerve fibers in the central and caudal of the injured area was also significantly increased,but there was no significant difference between the Control group and the sham iTBS group.(4)Results of activation of motor circuits by iTBS treatment:After transcranial iTBS treatment,neurons expressing c-Fos can be detected in the sensorimotor cortex,raphe nucleus,LDPT,and thoracic spinal cord in the injured area.What’s more,synaptic contact was formed between BDA labeled nerve fibers and c-Fos labeled neurons in the raphe nucleus and LDPT.Conclusion:1.Transcranial iTBS therapy could regulate the homeostasis of brain and spinal cord immune microenvironment in acute and subacute stages by regulating the genes of brain and spinal cord neurons and the microenvironment,so as to prevent the infiltration of inflammatory cells,neuronal damage and apoptosis and excessive proliferation of astrocytes as much as possible.It provided a good neuroprotective basis for promoting axon regeneration and motor function recovery in chronic period.2.Transcranial iTBS initiated nerve regeneration and regulated neuroplasticity by activating brain and spinal cord neurons.3.Transcranial iTBS promoted the recovery of autonomic motor function by remodeling motor circuits,possibly including 1)corticospinal tract(CST)—5-HT pathway,2)CST—LDPT pathway,3)CST—5-HT—LDPT pathway.4.This study discovered that after activation of neural pathways through transcranial iTBS,key genes initiated axon regeneration and neuroplasticity,which is expected to be used as regulatory targets in subsequent studies. |
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