| Objective:To establish an acute spinal cord injury(ASCI)rat model of clamp type thoracic 10 with aneurysm clamp,and to intervene with magnetic stimulation of different frequencies,through evaluating the behavior of rats,observing the general morphological structure of spinal cord tissue and detecting the expression of growth associated protein-43(GAP-43)in spinal cord tissue,to explore the effects of repetitive trans-spinal magnetic stimulation(rTSMS)with different frequence on movement function of ASCI rats,and to explore the most effective treatment frequency of rTSMS,so as to provide theoretical support and experimental data support for the application of rTSMS in clinical treatment of SCI.Methods: The model of temporary cerebral aneurysm clip was established.A total of 96 SD rats were randomly divided into 1Hz,5Hz,10Hz、15Hz group,control group and sham operation group.On the 4th day after operation,interventions were performed at frequencies of 1Hz,5Hz,10 Hz and 15 Hz,respectively.The maximum output intensity(1.5T)was 35%.Each sequence was 5s,and the interval between two stimulation sequences was 10 s.There were 450 pulses,once a day,five days a week.The total experimental period was 8w.BBB score(Basso,Beattie and Bresnahan score,BBB score)and inclined plate test were performed at 1 day before and 1,2,4,6 and 8 weeks after the establishment of the model in each group.After the completion of the experiment,the tissue of spinal cord injury was taken and observed by hematoxylin-eosin(HE)staining and Luxol Fast Blue(LFB)myelin sheath staining.Immunohistochemistry and Western Blot were used to detect the expression of GAP-43 protein in spinal cord tissue.Result:1.The effect of rTSMS on the improvement of hindlimb motor function in rats after spinal cord injury (1)BBB score The BBB scores of rats before modeling were 21(P > 0.05).The BBB score of sham operation group was higher than other groups(P < 0.05);the BBB score of control group,1Hz,5Hz,10 Hz and 15 Hz groups decreased,showing typicalparaplegia signs.One week after operation,BBB scores of rats in 1Hz,5Hz,10 Hz and15Hz groups were no difference in the control group(P > 0.05),and no difference in the among these groups(P > 0.05);BBB scores in 1Hz group were higher than control group(P < 0.05);BBB scores in 5Hz,10 Hz and 15 Hz groups were no difference in the control group(P > 0.05),and BBB scores in 1Hz group were higher than 15 Hz group(P< 0.05).Four weeks after operation,BBB scores in 1Hz,5Hz,10 Hz and 15 Hz groups were higher than control group(P < 0.05),and BBB scores in 1Hz group were higher than 5Hz,10 Hz and 15 Hz groups(P < 0.05);BBB scores in 10 Hz and 15 Hz groups were higher than 1Hz group(P < 0.05);BBB scores in 15 Hz group were higher than1 Hz,5Hz and 10 Hz groups(P < 0.05),and BBB scores in 10 Hz and 15 Hz groups were higher than 1Hz group.(P < 0.05).(2)inclined plate test There was no difference in the angle of the anterior inclined plate test(P > 0.05).The angle of inclined plate test in sham operation group was higher than other groups at different time points after operation(P < 0.05).One week after operation,there was no difference in the angle of inclined plate test between the 1Hz,5Hz,10 Hz and 15 Hz groups(P > 0.05),and there was no difference in the among these groups(P > 0.05).Two weeks after operation,the angle of inclined plate test in 1Hz group was higher than control group(P < 0.05),and there was no difference in the among the 5Hz,10 Hz and 15 Hz groups(P > 0.05),and the angle of inclined plate test in1 Hz group was higher than 15 Hz group(P > 0.05).Four weeks after operation,the inclined test angle of 1Hz group was higher than 5Hz,10 Hz and 15 Hz group(P < 0.05),but there was no difference among 5Hz,10 Hz and 15 Hz groups(P > 0.05);the inclined test angle of 1Hz,5Hz,10 Hz and 15 Hz group was higher than control group(P < 0.05),and the inclined test angle of 15 Hz group was higher than 1Hz group(P < 0.05);8weeks after operation,the inclined test angle of 15 Hz group was higher than that of 1Hz group(P < 0.05).The angle values of inclined plate test in 5Hz,10 Hz and 15 Hz groups were higher than 1Hz,5Hz and 10 Hz groups(P < 0.05).The angle values of inclined plate test in 5Hz,10 Hz and 15 Hz groups were higher than 1Hz groups(P < 0.05).2.The effect of rTSMS on the pathology of spinal cord tissue(1)HE staining The severity of spinal cord lesion in control group was the most serious.The severity of spinal cord lesion in sham-operated group,1Hz,5Hz,10 Hz and15Hz group was lighter than that in control group,which indicated that rTSMS had a certain effect on the repair of spinal cord injury.(2)LFB myelin staining After 8 weeks of intervention,the integral optical density of spinal cord tissue in the control group was lower than that sham-operated group(P <0.05);the integral optical density of spinal cord tissue in the 1Hz,5Hz,10 Hz and 15 Hz groups was higher than control group(P < 0.05);there was no difference among the intervention groups(P > 0.05).3.The effect of rTSMS on the expression of GAP-43 protein in spinal cord tissue(1)immunohistochemistry After 8 weeks of intervention,the expression of GAP-43 protein in spinal cord tissue of 1Hz,5Hz,10 Hz and 15 Hz groups was higher than sham operation group(P < 0.05);the expression of GAP-43 protein in spinal cord tissue of 15 Hz group was higher than control group,1Hz group,5Hz group and 10 Hz group(P < 0.05);there was no difference in the expression of GAP-43 protein among other intervention groups(P > 0.05).(2)Western Blot After 8 weeks of intervention,the expression of GAP-43 protein in spinal cord tissues of control group and 1Hz,5Hz,10 Hz and 15 Hz groups was higher than sham operation group(P < 0.05);the expression of GAP-43 protein in spinal cord tissues of 10 Hz and 15 Hz groups was higher than control group(P < 0.05);the expression of GAP-43 protein in spinal cord tissues of 10 Hz and 15 Hz groups was higher than 1Hz and 5Hz groups(P < 0.05),and the expression of GAP-43 protein in spinal cord tissues of 10 Hz and 15 Hz groups was higher than 1Hz and there was no difference in the expression of GAP-43 protein in medullary tissue(P > 0.05).Conclusion:1.rTSMS can improve the movement function of ASCI rats,promote the expression of GAP-43 protein in spinal cord tissue.2.There are differences in the improvement of movement function between different frequencies of rTSMS,the improvement of early movement function of rTSMS withfrequency of 1Hz is more obvious after SCI,the improvement of movement function of rTSMS with frequency of 15 Hz was more obvious in the middle and late stages after SCI. |
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