| Objective:In this experiment,a rat model of sciatic nerve clamping injury was established,and the rats with sciatic nerve injury were treated by electro-acupuncture at "Huantiao" point.The functional recovery,changes in the morphology of nerve fibers,changes in myelin sheath and changes in MIF expression of rats in each group at different treatment time were observed to measure the degree of sciatic nerve injury repair,and compare the difference between treatment group and model group.To further explore the effect of electroacupuncture at "Huantiao" point in the repair of sciatic nerve injury and its influence on MIF.Materials and Methods:Eighty-one healthy female Sprague-Dawley rats(SPF grade),aged about 6 months,weighted 250±20 g were selected.The 81 rats were evenly divided into three groups: sham-operated group,model group,and huantiaoxue group according to the experimental design scheme and random number table,with 27 rats in each group.Each group was divided into 3 subgroups: 3d subgroup,7d subgroup,and 14 d subgroup according to 3 time points,with 9 rats in each subgroup.Third degree sciatic nerve injury was established by referring to clamping method.After the rats were anesthetized,they were fixed on the experimental bench and placed in the prone position to expose the right lower limb.The rats were first shaved and disinfected at the junction of buttock and thigh.The incision was made along with the direction of sciatic nerve to find the sciatic nerve.The sciatic nerve was clamped with hemostatic forceps and detained at the 3rd detent position of hemostatic forceps.After clamping under pressure about 5kg for 10 s,the hemostatic forceps buckle was opened for 10 s,the hemostatic forceps clamped for 10 s again according to the above method and opened for 10 s.The procedure was repeated for three times until the width of the nerve injury was about 2 mm,the sterilization was carried out,and then the incisions were sutured layer by layer..After the model was established completely,all rats were fed regularly.The rats in the sham-operated group and the model group were not treated with acupuncture.However,in order to eliminate the human interference factors,the rats in the sham-operated group and the model group were given daily fixation management according to the fixation method of treatment group.After successful modeling,the rats in the Huantiao treatment group began to be treat by electro-acupuncture of the point "Huantiao" after the rats fully awakened,the needle was retained when the muscle tissue of lower extremity at the affected side of rat's twitched quickly and the toe quiver phenomenon appeared.At this point,the acupuncture needle handle was connected to the electro-acupuncture instrument,one pole of which was connected to the Huantiao point,and the other pole was connected to the rats' tail to make an indifferent electrode.According to the specified time points,the treatment lasted for 3d,7d or 14 days respectively using dilatational wave under frequency of 2Hz and current of 1mA,15 min/once,once/day.The sciatic nerve function index was measured,the motor function values of rats were calculated,and the recovery was compared;the neuroelectrophysiological test was conducted in rats to determine their nerve conduction velocity to more accurately estimate the degree of neurological recovery in rats of each group.After the end of the measurement,the rats were sacrificed by overdose anesthesia and the damaged sciatic nerve segments were extracted and fixed for future detection.The SFI measurement was performed to calculate the functional recovery of the sciatic nerve.The extent of recovery of sciatic nerve was investigated by MNCV measurement.The pathological morphology of the damaged sciatic nerve could be observed through HE staining.The changes of myelin sheath thickness in rats were observed by transmission electron microscopy.The changes of MIF positive expression at the damaged sciatic nerve were detected by immunohistochemistry and immunofluorescence..Result: 1 Sciatic nerve function index(SFI)The SFI was measured after modeling and before treatment,and the SFI of the sham group was significantly higher than that of the other two groups(P<0.05),suggesting that the model was established successfully.After treatment,the SFI of the treatment group of each subgroup at each time period was better than that of the model group(P<0.05).The comparison on transverse injury time suggested that the 14 d group was better than that of the 7d group,and the 7d group was better than that of the 3d group.It can be seen that the recovery of treatment group was better than that of model group,and the recovery effect was the best after 14 d treatment,which indicated that the longer the treatment,the better the recovery effect of damaged nerves.2 Neural Electrophysiology(MNCV)The motor nerve conduction velocity test(MNCV)showed that,after modeling,the MNCV of the model group was decreased compared with that of the sham operation group(P<0.05),suggesting that the model was established successfully.After treatment,the MNCV of the treatment group of each subgroup at each time period was higher than that of the model group(P<0.05).The comparison on transverse injury time suggested that the 14 d group was better than that of the 7d group,and the 7d group was better than that of the 3d group,which indicated that the longer the treatment,the better the recovery effect of damaged nerves.3 HE stainingIn the rats of sham-operated group,the direction of the nerve fibers were normal,the axons were clear,the nerve myelin sheath tightly wrapped the axons,with good continuity,and the Schwann cells were distributed evenly.In the rats of model group,the nerve fibers were disordered,no axons and myelin sheaths were seen,and the surrounding tissues of the nerve fibers were infiltrated with a large number of inflammatory cells.In the rats of treatment group,the nerve fibers were irregular and appeared edema on the 3rd day,axons were broken,myelin sheaths were detached,and inflammatory cells were increased;on the 7th day,the edema of the nerve fibers was more obvious,and the axons and myelin sheaths disappeared;on the 14 th day,the recovery of morphological structure of the nerve fibers was seen,and the distribution of the nerve fibers was more regular than before.4 Immunohistochemistry detectionThe average optical density of MIF in the sham-operated group,3d group,7d group,and 14 d group all remained at a low level,with no significant difference.The average optical density of the treatment group on the 3rd and 7th days were higher than that of the model group,but the value on the 14 th day was dropped rapidly and was close to that of the sham-operated group.However,the value of model group on the 14 th day was increased significantly.5 Transmission Electron Microscope Observation(TEM)In the rats of sham-operated group,the myelin sheaths of the sciatic nerve fibers were regular,with uniform density and certain thickness,and the axons were full.In the rats of model group,the myelin sheaths protruded toward the axons and invaded the axons,the myelin sheath layers were splited and easily disintegrated,some of which were fuzzy or disappeared,and the axons formed a cavity.After several days of comparison,the remodelling of myelin sheath in the model group progressed slowly.The treatment group changed significantly compared with the model group.On the 14 th day,it can be seen that the myelin sheaths were almost completedly reconstructed,with increased thickness,ordered arrangement and even texture,and the nerve fibers were full.The improvement of the pathological changes in the treatment group on the 7th day was better than that in the 3d group,and the 14 d group was better than that of the 7d group.6 Immunofluorescence detectionA small amount of expression was observed in the sham-operated group on 3d,7d,and 14 d.The nerve fibers were cord-like shape,which direction was normal.The positive expression in the model group and the treatment group were significantly higher than that in the sham-operated group.Compared with the sham-operated group,the positive expression in the model group was increased significantly,the nerve fibers were disordered,and the positive expression remained unchanged until the 14 th days.The positive expression of the treatment group was significantly increased on the 3rd and 7th days,but the positive expression on the 14 th day was significantly decreased,which was close to that of the sham-operated group.Conclusion: 1 Electro-acupuncture of Huantiao point can accelerate the recovery of motor function and sciatic nerve conduction velocity in rats with sciatic nerve injury.2 Electro-acupuncture of Huantiao point can shorten the pathological secretion period of MIF.3 Electro-acupuncture of Huantiao point can speed up the repair of nerve fibers and myelin sheaths.4 Electro-acupuncture of Huantiao point,the 7d group was better than the 3d group,the 14 d group was significantly better than the 7d group,suggesting that the electro-acupuncture of Huantiao point can accelerate the repair time of the sciatic nerve. |
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