| Objective: To establish the model of T10 acute thoracic cord injury with clip compression in rats and stimulate different spinal cords with magnetic stimulation,through observing behavior and histomorphology changes within eight weeks and detecting the Growth associated protein-43,to explore the effects of repetitive trans-spinal magnetic stimulation(rTSMS)act on different segments of the spinal cord on movement function and expression of GAP-43 in rats after acute cord injury,and to preliminarily discusses the best treatment site of rTSMS.Providing theoretical foundation and experimental evidence for the application of spinal cord injury with rTSMS.Methods: Sixteen rats were randomly selected from 80 SD rats as group E(sham-operated group,only exposed spinal cord).The spinal cord of the remaining 64 rats were damaged with a cerebral aneurysm clip(calibration force 70 g)to establish the model of T10 acute thoracic cord injury.The injured rats were randomly divided into group A: T6 segment stimulation group,B group: T10 segment stimulation group,C group: L2 segment stimulation group,D group: control group.There were 16 rats in each group which the sex ratio is 1:1.In group A,B,and C,5 Hz and 75% of the maximum output intensity(1.5T)of magnetic stimulation were given in the corresponding spinal segments from the 4th day after surgery.Each sequence was 5s,intermittent 2s,continuous 10 sequences,once a day,5 days every week,continuous treatment 8 weeks.The behavioral BBB scores were performed one day before surgery and one day,one week,2 week,4 week,6 week,and 8 week after operation.After 8 week,with the injury segment as the center,the spinal cord tissue about 1cm length was taken,and the injured spinal cord was observed by transmission electron microscope.Tissue ultrastructure changes were detected.The expression of GAP-43 protein was detected by immunohistochemistry and Western Blot.Real-time PCR was used to detect the expression of GAP-43 mRNA in the injured spinal cord.Result: 1.Effects of rTSMS on Motor Function in Rats with Acute Spinal Cord Injury(1)BBB score Rat BBB scores were 21 points in all groups 1 day before surgery.In group E,the BBB score was not significantly decreased after spinal cord injury.The BBB score in group E was significantly higher than that in other groups at each time point(P<0.05);the BBB scores of A,B,C and D group rapidly decreased to 0-2 points after SCI.During the crawling process,they all showed typical signs of paraplegia and no visible hindlimb movement.Over time,BBB scores have increased to some extent.There was no significant difference between groups A and B at all time points after operation(P>0.05).In one day and one week after surgery,the motor function began to recover,but the recovery was slower.Compared with group D,the BBB scores of rats in groups A,B,and C were slightly higher,but the difference was not statistically significant(P>0.05),and there was no significant difference between the groups of A,B and C(P>0.05);BBB scores of group A and B were significantly higher than group D at weeks 2,4,6 and 8 postoperatively(P<0.05);at weeks 4,6 and 8 postoperatively,the BBB score in group C was significantly higher than that in group D(P<0.05);The BBB scores in group A and B were significantly higher than those in group C(P<0.05)at weeks 4 postoperatively.However,the BBB scores in group C were higher than those in group A and group B at weeks 6 and 8 postoperatively(P<0.05).(2)TEM observation Group A: Part of the myelin sheath showed a loose and swelling lamellar structure,and the structure of myelin has changed.The mild demyelination could be observed.Group B: Large gaps,myelin sheath swelling and myelin sheath lamina could be observed between myelin sheaths.Group C: Loose lamellar structure is less,and the arrangement between myelin sheath and axon was dense,which showing mild demyelination.Group D: huge gaps,myelin swelling,disorder structure could be observed between myelin sheaths,and the normal structure of the axons disappeared.Part of the myelin showing severe demyelination.Group E: The structure of myelin sheath was complete and clear,and the myelin sheath and axon were normal.The mitochondria were evenly distributed and the structure was clear.2.Effect of rTSMS on Expression of GAP-43 protein in Rats with Acute Spinal Cord Injury(1)Immunohistochemistry Eight weeks after spinal cord injury,a large number of yellow or brown-yellow positive cell structures were observed in the cytoplasm of spinal cord neurons in groups A,B,C,and D.In the E group,blue negative cells were mostly absent because the spinal cord was not injured.Compared with group E,the expression of GAP-43 protein in the spinal cord of groups A,B,C and D was significantly increased(P<0.05).The expression of GAP-43 protein in group A and group C was higher than that in group D.The amount was significantly increased(P<0.05).Compared with group A and group B,the expression of GAP-43 protein in group C was higher(P<0.05);the expression of GAP-43 protein in group A was higher than that in group B(P<0.05).(2)Western Blot Detection(Western Blot)After 8 weeks of spinal cord injury,compared with E group,the relative expression of GAP-43 protein in spinal cord tissue of groups A,B,C and D was significantly increased(P<0.05);In group A,B and C,the relative expression of GAP-43 protein was significantly higher than that of group D(P<0.05);the relative expression of GAP-43 protein of treatment group A,B,and C was compared,and there was no significant difference in relative expression(P>0.05).(3)Real-time PCR Detection After 8 weeks of spinal cord injury,compared with E group,the expression of GAP-43 mRNA in the spinal cord injury areas of A,B,C and D groups was significantly increased(P<0.05);The expression of GAP-43 mRNA in A,B and C groups was significantly higher than that in group D(P<0.05).The expression of GAP-43 mRNA in spinal cord injury areas of group C was significantly higher than that in group A and B(P<0.05).There was no significant difference in GAP-43 mRNA expression between group A and group B(P>0.05).Conclusion: 1.The effect of rTSMS on the motor function and the expression of GAP-43 in different spinal cord segments after spinal cord injury were different.When rTSMS acted on the L2 spinal cord segment,the motor function improved most obviously and the expression level of GAP-43 was the highest.2.rTSMS can promote the expression of GAP-43 and axonal regeneration in the spinal cord which is beneficial to the recovery of motor function after acute spinal cord injury. |
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