| [Objective]In this study,sciatic nerve injury(SNI)was used as an example to establish a model of sciatic nerve crush injury to study peripheral nerve injury.Based on the comprehensive evaluation of behavior,morphology and molecular biology,the SNI model rats were intervened by three methods and three points,and the effects of tuina on the survival of spinal motor neurons and the activation state of microglia in SNI model rats were investigated.From the perspective of spinal cord nerve regeneration inhibition pathway,whether the tuina can inhibit the growth of cone-retraction and promote axonal regeneration by inhibiting the expression of key proteins Nogo-A and NgR in the Nogo-NgR signaling pathway.Through the study of three methods and three points tuina intervention to promote neuronal axon regeneration,promote the mechanism of motor function recovery,explore the scientific connotation of tuina treatment of peripheral nerve injury,and then provide a theoretical basis for the treatment of clinical peripheral nerve injury diseases[Methods]This study established a rat model by using sciatic nerve crush injury.Rats received Chinese tuina in accordance with the principle of Three Methods and Three Points,which three points of Yinmen,Chengshan and Yanglingquan for qualitative and quantitative,once daily for 20 days by using the tuina manipulation simulators.The sciatic nerve function index was used to evaluate the recovery of fine movements in rats,and the incline plane test was used to evaluate the recovery of hindlimb muscle strength in rats.The survival of spinal motor neurons was observed by immunofluorescence.The activation status of microglia marker CD11b was observed by fluorescence staining to explore the effect of tuina on the morphological changes of spinal cord.The molecular phylogenetic technique was used to detect the expression of Nogo-A and NgR in the spinal ventral horn.Through the comprehensive analysis of behavioral,morphological and molecular biological results,the effects of tuina on the recovery of sciatic nerve function in SNI rats were evaluated,and the mechanism of action of tuina was discussed from the perspective of spinal neuroactive substance morphological changes and nerve regeneration inhibition pathways.[Results]1.Tuina promoted the recovery of motor function related behavior in SNI rats1.1.Sciatic nerve function index(SFI)After 7 days of model establishment,the SFI results of the model group were significantly lower than those of the sham-operation group(P<0.01).On the 5th day of intervention,the SFI results of the model group were significantly lower than that of the sham operation group(P<0.01).On the 10th day of intervention,compared with the sham operation group,the rats in the model group were still significantly decreased(P<0.01);the tuina group had an increasing trend compared with the model group(P<0.01),but still lower than the sham operation group.After 15 days of intervention,the rats in the tuina group were significantly higher than the model group(P<0.01),but still lower than the sham operation group.After 20 days of intervention,the model group was significantly lower than the sham operation group(P<0.01);while the tuina group was significantly higher than the model group(P<0.01),and it was close to the sham operation group.1.2.Incline plate testAfter 7 days of model establishment,the results of the incline plate test of the model group were significantly lower than those of the sham-operation group(P<0.01).On the 5th day of intervention,the model group was still significantly lower than the sham operation group(P<0.05),and there was no significant difference between the tuina group and the model group(P>0.05).On the 10th day of intervention,compared with the model group,the results in the tuina group increased(P>0.05).And the results in the model group were significantly lower than those in the sham operation group(P<0.05).The tuina group was significantly higher than the model group(P<0.05),and was close to the sham operation group.2.Tuina improved the number of neurons in the ventral horn of the SNI rat and the activation status of microglia2.1.Spinal motor neurons immunofluorescence tracer resultsAt 7 days after model establishment,compared with the sham operation group,the number of jfluorescent gold retrograde tracing neurons in the L4-L6 segm ent of the injured side of the model group was decreased(P<0.05).After 20 days of intervention,compared with the sham operation group.the number of model group was significantly reduced(P<0.01),and neuronal number were significantly increased in the tuina group compared with the model group(P<0.05).2.2.Morphological changes of microglia marker CD11b in the ventral horn of the spinal cordAfter 7 days of modeling,the sham-operation groups of CD11b-labeled microglia were unactivated and scattered in the L4-L6 segment of the ventral horn of the spinal cord.Compared the sham-operation group,the model group had L4-L6 segments of the ventral side of the injured spinal cord.The CD11b-labeled microglia were activated and clustered in the vicinity of motor neurons,which were in close contact with neurons.After 20 days of intervention,CD11b-labeled microglia in the L4-L6 segment of the spinal cord of the model group showed significant high activation.In the state,compared with the model group,the degree of activation of microglia in the tuina group was weak.3.Tuina promoted axonal regeneration by reducing the expression of NgR in the ventral horn of the spinal cordThe results of western-blotting and immunohistochemistry of Nogo-A and NgR proteins in the L4-L6 segment of the ventral horn of the spinal cord:The results of western-blotting test showed that there was no significant difference in the expression level of NogoA between the model group and the sham-operation group(P>0.05).After 20 days of intervention,the expression level of NogoA in the model group was increased compared with the sham operation group(P>0.05),there was no significant difference in the expression level of NogoA between the model group and the tuina group(P>0.05).The results of immunohistochemistry showed that there was no significant difference in the expression level of NogoA between the model group and the sham-operation group(P>0.05).After 20 days of intervention,There was no significant difference in the expression level of NogoA between the model group and the tuina group(P>0.05).The results of western-blotting test showed that the expression of NgR in the model group was higher than that in the sham-operation group(P<0.05).After 20 days of intervention,the expression level of NgR in the tuina group was lower than that in the model group(P<0.05).The results of immunohistochemistry showed that the expression of NgR in the model group was higher than that in the sham operation group(P<0.05).After 20 days of intervention,the expression level of NgR in the tuina group was lower than that in the model group(P<0.05).[Conclusion]1.Three-method three-point tuina manipulation promoted the fine movement of SNI rats and the recovery of hind limb muscle strength,increased the number of ventral horn motoneurons in SNI rats,and reduced the activation of microglia in spinal ventral horn.It was proved that tuina had a promoting effect on the recovery of nerve fibers,which was related to the activation of microglia,and provided behavioral and morphological basis for the treatment of nerve injury diseases by tuina.2.Three-method and three-point tuina reduced the expression of NgR in the ventral horn of the spinal cord,suggesting that tuina may reduce the activation of NgR in the spinal cord,prevent the activation of the NogoA-NgR inhibitory signal,and thus promote the regeneration of axons.One of the mechanisms that illuminate the treatment of peripheral nerve injury by tuina is to inhibit the activation of NogoA-NgR signaling. |
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