| Objective:This study investigates the role and possible mechanism of microglia after dimethylaminetetracycline activation in promoting neurological recovery in mice with spinal cord injury,providing a new idea and theoretical basis for the treatment of spinal cord injury.Methods:Microglia were divided into experimental and control groups,and microglia in the experimental group were treated with 10mmol/L dimethylaminotetracycline for 24 h and their differentiation was identified by immunofluorescence and flow-through,while the control group was not specially treated.Microglia in the experimental and control groups were co-cultured with dorsal root neuron cells for 24 h,and the axonal growth of dorsal root neuron cells was observed,and the expression of cellular markers was determined by enzyme-linked immunosorbent assay(ELISA)by taking the supernatant separately.Ninety-six 6-week-old mice were randomly divided into blank control group,simple spinal cord injury group,unactivated group and activated group,24 mice in each group:the T10 spinal cord injury model was constructed using the clamp method for the simple spinal cord injury group,unactivated group and activated group,and after successful modeling,microglia without dimethylamine-tetracycline activation were implanted in the unactivated group,and laminectomy alone was performed in the blank control group without other treatments.Motor function was assessed using the BMS score at 1d,7d,14 d,and 28 d after modeling.Mice in each group were executed at 4w,and spinal cord tissues were fixed,embedded,frozen,and sectioned on ice.HE staining was performed separately to observe the morphological changes of spinal cord tissue in each group.Immunofluorescence staining was performed in each group to determine microglia survival,differentiation and nerve fiber recovery.Results:Immunofluorescence showed that the expression of M1 microglia marker INOS in the experimental group was significantly lower than that in the control group,while the expression of M2 microglia marker ARG1 in the experimental group was significantly higher than that in the control group.The expression of M1 microglia marker CD16/32 in the experimental group was significantly lower than that in the control group,and the expression of M2 microglia marker CD206 in the experimental group was significantly higher than that in the control group.After 24 h co-culture,the length and number of axon growth of dorsal root neurons in the experimental group were significantly stronger than those in the uncontrolled group.The results showed that the M1 microglia marker IL-6 in the supernatant of the experimental group was significantly lower than that of the control group,and the M2 microglia marker IL-10 in the supernatant of the experimental group was significantly higher than that of the control group.At 1 d after modeling,the motor function scores of mice were not statistically different between the simple spinal cord injury group,the unactivated group and the activated group(P > 0.05),and the scores of the blank control group were significantly higher than those of the other three groups(P < 0.05).At 14 d and 28 d after modeling,the motor function scores of mice in the activated microglia group were significantly higher than those in the simple spinal cord injury group and the unactivated group(P < 0.05).At 4 w after modeling,HE staining showed that the spinal cord tissues of mice in the blank control group had no obvious necrotic tissue cells,normal tissue morphology and structure,and a small amount of inflammatory cell infiltration;the spinal cord tissues of mice in the simple spinal cord injury group showed a large number of necrotic tissue cavities and disorganized tissue morphology and structure;the spinal cord tissues of mice in the activation group showed a small number of tissue cavities,and the tissue morphology and structure were more regular,and the degree of necrosis was lower than that in the simple spinal cord injury group.The degree of necrosis was lower than that of the spinal cord injury group alone,but higher than that of the blank control group.Immunofluorescence showed that both the unactivated group and the activated group had transplanted microglia at 4w.The recovery of nerve fibers was positively correlated with the recovery of motor function in mice and the blank control group was better than the activated group at 4w.The activated group was better than the unactivated group and the simple injury group.Conclusion : This research demonstrated that minocycline activation can promote the differentiation of microglia to the M2 type,and transplantation of minocycline activated microglia can effectively promote the recovery of neurological function in mice after spinal cord injury. |
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