| [Objective]In this study,we tried to find out whether "Three Methods and Three Points"could regulate the expression of NRG 1 and ErbB2 in sciatic nerve injury part and L4-6 spinal cord of SNI model rats,leading to the repair and regeneration of injured nerve.NRG1 and ErbB2 were important factors affecting the regeneration of myelin sheath.Combined with behavioral,morphological and mechanism research,this experiment further clarified the mechanism of Tuina intervention to promote the repair and regeneration of myelin sheath after peripheral nerve injury,improved the possible mechanism of Tuina intervention to promote the repair and regeneration of injured nerve,and provided the relevant theoretical basis for clinical treatment.[Method]Animals(102 SD rats)were divided into groups by random number method,that is 21 normal rats,27 sham operated rats,27 model rats and 27 the "Three Methods and Three Points" group rats.We used sciatic nerve crush injury to establish rat models.The control method was grasping and binding,and the treatment method was "Three Methods and Three Points" Tuina intervention.The behavioral changes were evaluated by inclined plane test and threshold of photothermal tolerance.The changes of myelinated fibers were observed by Transmission electron microscope and immunofluorescence.Western-blot was used to detect the expression of NRG1 and ErbB2 in sciatic nerve injury part and L4-6 spinal cord.In a word,the study was done centering on the effect of Tuina intervention on the myelin sheath of injured sciatic nerve fiber."Three Methods and Three Points" Tuina intervention was timing,quantitative and qualitative stimulation.The Tuina manipulation simulator was used."Three Methods" were pointing-manipulation,dailing-manipulation and kneading-manipulation."Three Points" were BL37,GB34 and BL57."Three Methods" were successively applied to "Three Points" on the operation side of SNI rats,with one method manipulating on one point lasting for 1 min.The intervention force is 4N.There were totally 20 times interventions during the experiment.[Result]1.Tuina could promote the reco’very of motor function in SNI rats.Results of inclined plane experiment:28 days after the operation(after 20 times of intervention),the results of the "Three Methods and Three Points" group were higher than those of model group(P<0.05),closing to the level of normal group(P<0.05)and sham operation group(P<0.05).Tuina could significantly promote the recovery of muscle strength and motor function of SNI rats.2.Tuina could promote the recovery of sensory function in SNI ratsThreshold of photothermal tolerance results:28 days after operation(after 20 times of intervention),the results in model group were significantly higher than those in normal group(P<0.05)and sham operation group(P<0.05).The results of the "Three Methods and Three Points" group were significantly lower than those of model group(P<0.05),and reached the level of normal group(P>0.05)and sham operation group(P>0.05).Tuina could significantly promote the recovery of pain and sensory function in SNI rats.3.Tuina had a positive effect on the myelinated nerve fiber in the injured sciatic nerve part of SNI rats3.1 Transmission electron microscope observation resultsTwenty-eight days after operation,The injured area of sciatic nerve was observed under the transmission electron microscope:in the model group,some myelinated fibers were found to have severe myelin sheath collapse;the myelin sheath lamellae were scattered and the gap was enlarged;the axons were severely atrophied or even disappeared.After 20 times of"Three Methods and Three Points" Tuina intervention,it could be seen that compared with the model group,the structure of myelinated fibers was improved.The phenomenon of lamellar separation was reduced.The degree of axon atrophy was significantly reduced.The myelin sheath occasionally collapsed.The separation of lamellae occurred occasionally.The axons were slightly atrophic.Tuina could significantly improve the structure of myelinated fibers in the injured area of sciatic nerve.3.2 G-ratio statistical resultsG-ratio is the ratio of axon diameter to myelinated fiber diameter,which could reflect the thickness of myelin sheath.G-ratio is 0.6 in the peripheral nervous system of normal adult rats.Twenty-eight days after operation,the G-ratio of the model group(0.38±0.15)was significantly lower than that of the sham operation group(0.56±0.06)(P<0.05);after 20 times of Tuina intervention,the G-ratio of the "Three Methods and Three Points" group(0.54±0.10)was significantly higher than that of the model group(P<0.05),reaching the level of the sham operation group(P>0.05).Tuina could significantly improve the thickness of myelin sheath and promote its repair and regeneration.3.3 Statistical results of diameter of myelinated fiberThe thickness of myelin sheath and function vary with the diameter of myelinated fiber.Under the transmission electron microscope,myelinated fibers with small and medium diameter(1.5-5μm)were found mostly in the injured area of sciatic nerve.Twenty-eight days after operation,the diameter of myelinated fiber in model group was significantly lower than that in sham operation group(P<0.05);after 20 times of intervention,the diameter of myelinated fiber in the "Three Methods and Three Points" group was significantly higher than that in model group(P<0.05),closing to the level of sham operation group(P<0.05).3.4 Immunofluorescence of sciatic nerveUnder normal circumstances,Schwann cells and myelin sheath in the injured area of sciatic nerve had strong fluorescence signal,and a large number of myelinated fibers were arranged compactly and had good continuity.Twenty-eight days after the operation,myelinated fibers were absent,and some myelin sheath disintegrated.There was a great deal of apoptosis of Schwann cells.After 20 times of Tuina intervention,the continuity of myelinated fibers and the survival of Schwann cells were better than those in the model group.Tuina could improve the continuity of myelinated fibers in the injured area of sciatic nerve to some extent and protect the survival of Schwann cells.4.Tuina could regulate the expression of NRGl-ErbB2 in L4-6 spinal cord and injured area of sciatic nerve in SNI rats.4.1 Changes of NRG1 expression in L4-6 spinal cord of SNI rats before and after Tuina intervention.Three days and 7 days after operation(before intervention),the expression of NRG 1 in L4-6 spinal cord of model group and the "Three Methods and Three Points" group increased significantly(P<0.05).Twenty-eight days after the operation(after 20 times of intervention),the expression of NRG 1 in L4-6 spinal cord of model group and "Three Methods and Three Points" group reached normal level(P>0.05).The results of the "Three Methods and Three Points",group were significantly higher than those of model group(P<0.05).4.2 Changes of ErbB2 expression in L4-6 spinal cord of SNI rats before and after Tuina interventionThree days and 7 days after operation(before intervention),the expression of ErbB2 in L4-6 spinal cord of model group and the "Three Methods and Three Points" group increased significantly(P<0.05).Twenty-eight days after the operation(after 20 times of intervention),the expression of ErbB2 in L4-6 spinal cord of model group and the "Three Methods and Three Points" group reached the normal level(P>0.05).In conclusion,the NRG1-ErbB2 signal in the L4-6 spinal cord increased significantly after 3 days and 7 days after operation,and then decreased to the normal level,suggesting that the NRG1-ErbB2 signal in the spinal cord was closely related to the process of nerve injury,repair and regeneration.The expression of NRG1 in L4-6 spinal cord was increased by 20 times of intervention of Tuina.4.3 Changes of NRG1 expression in injured area of sciatic nerve in SNI rats before and after Tuina interventionThree days and 7 days after operation(before intervention),the expression of NRG 1 in the injured area of sciatic nerve in the model group and the "Three Methods and Three Points"group increased significantly(P<0.05).Twenty-eight days after the operation(after 20 times of intervention),the expression of NRG1 in the injured area of sciatic nerve in the model group and the "Three Methods and Three Points" group reached the normal level(P>0.05).4.4 Changes of ErbB2 expression in the injured area of sciatic nerve in SNI rats before and after Tuina interventionThree days and 7 days after operation(before intervention),the expression of ErbB2 in the injured area of sciatic nerve in model group and "Three Methods and Three Points" group increased significantly(P<0.05).Twenty-eight days after the operation(after 20 times of intervention),the expression of ErbB2 in the injured area of sciatic nerve in model group and the "Three Methods and Three Points" group reached normal level(P>0.05).The results of the "Three Methods and Three Points" group were significantly higher than those of model group(P<0.05).In a word,after operation,NRG1-ErbB2 was highly expressed in the acute stage(3 days after operation)in the injured sciatic nerve,and then gradually decreased to the normal level.The changes of NRG1 and ErbB2 were not completely synchronous:The expression of NRG 1 in the injured sciatic nerve of the model group and the "Three Methods and Three Points"group reached the normal level as early as 7 days after operation,while the expression of ErbB2 in the model group and the "Three Methods and Three Points" group reached the normal level at 28 days after operation.Twenty times of Tuina intervention could improve the expression of ErbB2 at the level of the injured sciatic nerve.[Conclusion]1."Three Methods and Three Points" Tuina intervention could improve the muscle strength of hind limbs of SNI rats,improve the pain sensory function of SNI rats,promote the recovery of motor and sensory function after sciatic nerve injury.It was proved that Tuina intervention on peripheral nerve injury had good treatment effect of in science research.2."Three Methods and Three Points" Tuina intervention could significantly improve the ultrastructure of injured sciatic nerve,reduce the degree of myelin degeneration,promote the repair and regeneration of myelinated fibers.Morphological evidence for Tuina treatment of peripheral nerve injury was provided.3."Three Methods and Three Points" Tuina intervention could improve the expression of NRG1 in L4-6 spinal cord and ErbB2 in injured area of sciatic nerve in SNI rats.The mechanism of Tuina in the treatment of peripheral nerve injury was improved."Three Methods and Three Points" Tuina intervention could promote the repair and regeneration of injured sciatic nerve.The behavioral performance(motor and sensory function)was the improved of SNI rats.The mechanism was to improve the expression of NRG1 in L4-6 spinal cord and ErbB2 in the injured area of sciatic nerve in SNI rats,protect the survival of Schwann cells,and have a benign effect on the injured myelinated fibers.That is,it could improve the continuity of nerve fiber and the degeneration degree of axon and myelin sheath,and promote its regeneration. |
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