| Purpose:To explore the specific action of microcurrent stimulation on the survival,proliferation and differentiation of spinal cord endogenous neural stem cells and to explore the relevant mechanism of action,to provide theoretical support for the application of biophysical techniques to intervene spinal cord injury and spinal cord reconstructionMethods:According to the spinal cord diameter of Sprague-Dawley rats,an implantable microelectrode conforming to the spinal cord size of SD rats was tailored.All rats were randomly divided into 3 different groups,Group A(normal group,only laminectomy and exposed spinal cord),Group B(spinal cord injury group,Allen method,microelectrode was implanted into the spinal cord),Group C(spinal cord injury+electrical stimulation group,implantable microelectrode fixed on the surface of the spinal cord,and electrical stimulation).There were 50 rats in each group,Brdu(50 mg/kg)was injected intraperitoneally 5 days before the start of the experiment to label the spinal cord to maintain the differentiated cells.Immunofluorescence and confocal laser scanning microscopy were taken to detect the number,morphology and distribution of specific nerve cells in each group.Western Blot and PCR were used to detect the expression of specific proteins and genes.The recovery of motor function of hindlimbs was evaluated by the BBB scoring method.Results:Nestin-positive cells were scattered in the gray matter of H-type spinal cord in the spinal cord of normal SD rats,mainly distributed in the area around the central canal of the spinal cord,but no Nestin-positive cells in the white matter.The number of Nestin positive cells increased after spinal cord injury(P <0.05).At 14 days after spinal cord injury,the number of Nestin-positive cells in group B reached the peak,but it was still lower than that in group C(P <0.05)(P <0.01).Nestin-positive cells in group B began to descend 21 days after SCI,but still higher than that in group A(p <0.01).Nestin-positive cells in group C showed an increasing tendency until 21 days after spinal cord injury(P<0.01,p <0.05),but decreased gradually after reaching a plateau stage.The number ofGFAP positive cells in group B and C increased significantly at 3 days after spinal cord injury compared with group A(all p <0.05),but the expression of GFAP positive cells in group B and C was significantly higher than that in group B and C(P> 0.05).At 14 days,the difference was more obvious,GFAP-positive cells in group B peaked,but GFAP-positive cells in group C decreased markedly(P <0.05),and the difference was statistically significant(p <0.05)(P <0.05).However,the number of GFAP-positive cells in group B increased from 21 days to 28 days,but decreased significantly in group C until reaching 28 days.The difference was not statistically significant(p> 0.05).The expression of MAP-2 and Olig-2 in B and C groups decreased significantly at 3 days after spinal cord injury,but there was no significant difference between group B and group C(p> 0.05)2,Olig-2 positive cells continued to decline,but group C began to rise,significantly higher than the B group(p <0.01,p <0.05),but still lower than the A group(p < The number of MAP-2 and Olig-2 positive cells in group B decreased to a stable level in group B,but remained stable in group C until 28 days,and the values of group A and C were close to 28 days after spinal cord injury.Real-time PCR and Western-Blot showed that the expression of Notch1,Notch3,Notch4 and Hes1 in group A had no significant change at each time point(p> 0.05);Notch1,Notch3,Notch4,Hes1 The expression level of Notch1,Notch3,Notch4 and Hes1 in group C was lower than that in group B,but it was still higher than that in group A(P <0.05).The expression of Notch1,Notch4,Hes1 in group C was significantly lower than that in group A at 2 weeks after spinal cord injury(P> 0.05).The expression of Notch1,Notch3,Notch4 and Hes1 genes in B and C groups were significantly higher than those in B and C groups at 2 and 4 weeks after spinal cord injury(P<0.05).Conclusion: The level of serum creatinine in group C is lower than that in group B(P <0.05).For BBB score: when compared to Group A,BBB score of Group B and C at every time points after the injury decreased statistically significant(p=0.00).SD rats of Group B and C were bilateral hind limb and movements were obstacles,drag and other symptoms.1 d after injury,animal double hind limb function loss,muscle strength decreased to 0,double hind legs mopping crawling.There was no significant difference between Group B and C(p=0.41).At 1 week after operation,most of the animals showed lower extremity traction(5.00±0.67),(5.90±1.10),the difference was not statistically significant(p=0.066);to 2 weeks after the operation,the lower limb joints(8.00±1.08),(9.70±0.95),the difference was statistically significant(p<0.01);at 3 weeks after the operation,most of the rats were occasionally recuperated with micturition reflex,BBB score were(9.80±0.79)and(10.70±1.06),respectively.There were statistically significant differences between the two groups(p=0.018).At 4 weeks after the operation,some rats were able to stand on their weights and BBB scores were(10.50±1.08),(11.40±0.84),the difference between the two groups was statistically significant(p=0.00),but the difference between the two groups gradually narrowed to 4 weeks after operation BBB score reached a relatively stable(P = 0.017),BBB score in Group C and Group B was still on the rise.Conclusion:There were a few of endogenous neural stem cells in spinal gray matter.Endogenous neural stem cells proliferated after spinal cord injury,but were mainly differentiated into neurons.Micro-current stimulation could activate endogenous neural stem cells,and can inhibit the Notch signaling pathway to promote endogenous neural stem cells differentiate to neurons and oligodendrocyte,and inhibit its differentiation to astrocytes,and promote the self-repair of spinal cord injury.This study provides a theoretical basis for the application of electrical stimulation in the repair of spinal cord injury and its mechanism. |
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