Study Of The Effect Of MTOR Activation Together With Task-based Rehabilitative Training On Axon Regeneration After Spinal Cord Injury In Mice

Objective:To investigate the effects of combining mTOR activation and task-based rehabilitative training on axon regeneration and upper limb function recovery after cervical 5 spinal cord injury in mice.Methods:1.Building PTEN inhibition pyramidal neurons model in mice:A total of 10 C57/BL mice were randomly and equally divided into PTEN group(n=5)and Control group(n=5),and Lenti-Pten-RNAi?Lenti-scrambled-eGFP(virus titers were all 8E+8 TU/mL)were injected into right motor cortex layer?area of them respectively.After injection14d,animals were sacrificed.The virus transfection efficiency and the level of PTEN in motor cortex layer?area were tested by immunofluorescence assay.The level of PTEN in two groups of pyramidal neurons was compared by nonparametric multiple t test.2.Observing the tracing effect of BDA in corticospinal tract:BDA tracer was injected into right motor cortex layer?area,animals were sacrificed after injection 14d.The corticospinal tract was dectected at cerebral cortex?medulary pyramid and cervical cord area.3.A total of 40 C57/BL mice were randomly and equally divided into PTEN RNAi+Exercise group(n=5)?PTEN RNAi group(n=5)and Control+Exercise group(n=5)?Control group(n=5),and Lentiviral particles(Lenti-PTEN-RNAi or Lenti-Scrambled-EGFP)were injected into the right sensorimotor mouse cortex in four experimental groups respectively.Two weeks after injection,all mouse groups received a left C5 crush injury.We performed task-based rehabilitative training for 4 weeks on the PTEN RNAi+Exercise and Control+Exercise groups.At 8 weeks after injury,all animals were sacrificed.The Endogenous regenerative capacity of pyramidal neurons were assessed by immunofluorescence double-labeling of PTEN/p-S6;the axon regeneration were observed by immunofluorescence double-labeling of BDA/GFAP;the formation of synapse were observed by immunofluorescence double-labeling of BDA/Synapsin?;the axon sprouting and syntagmatic relation of neuron in the anterior horn of gray matter above lesion were observed by immunofluorescence double-labeling of BDA/NeuN.The horizontal ladder and rearing test were used to assess motor function for forelimb at3 days,2 weeks,4weeks,6 weeks and 8 weeks after injury.Non-parametric multiple t test and one-way ANOVA were used to compare the corresponding indicators among groups.Results:1.Lenti-Pten-RNAi and Lenti-scrambled-eGFP were injected into the right motor cortex the first?layer accurately.The pyramidal neurons were infected by virus.In addition,the expression level of PTEN in PTEN group was significantly lower than that in Control group(P<0.05).2.14 days after BDA injection,BDA labeled pyramidal neurons were visible in the motor cortex,and BDA labeled corticospinal tracts were clearly visible in medulla pyramid and cervical spinal cord.3.At 8 weeks after injury,compared with Control+Ex group and Control group,PTEN RNAi+Ex group and PTEN RNAi group showed higher level of p-S6 in layer?of motor cortex(P<0.05).The number of regenerated axons in PTEN RNAi+Ex group and PTEN RNAi group was significantly higher than that in Control+Ex group and Control group(P<0.05),and longer distance in the PTEN RNAi+Ex group(P<0.05).Compared with the PTEN RNAi group,more synaptic structures on regenerated axons were observed in the PTEN RNAi+Ex group(P<0.05).Above lesion,more neurons that were surrounded by BDA~+axons were observed in PTEN RNAi+Ex group and the Control+Ex group than that in PTEN RNAi group and Control group.The error rate of horizontal ladder in PTEN RNAi+Ex group was lower than that in other groups at 6and 8 weeks after injury(P<0.05);the usage rate of left forelimb in rearing test in PTEN RNAi+Ex group was higher than that in other groups at 8 weeks after injury(P<0.05).Conclusion:1.The technology of RNA interference can suppress the expression of PTEN successfully in layer?of motor cortex in mice.2.By BDA tracing,the morphology of CST in the spinal cord can be observed clearly.3.Combining mTOR activation with task-based rehabilitative training can further promote axon regeneration and synaptic remodeling,and play a positive role in the formation of new neural circuits above the lesion and the recovery of upper limb motor function.