Experimental study of M2 microglia transplantation promoting spinal cord injury repair in mice
Objective To investigate the effect of M2 microglia(M2-MG)transplantation on spinal cord injury(SCI)repair in mice.Methods Primary MG were obtained from the cerebral cortex of 15 C57BL/6 mice born 2-3 days old by pancreatic enzyme digestion and identified by immunofluorescence staining of Iba1.Then the primary MG were co-cultured with interleukin 4 for 48 hours(experimental group)to induce into M2 phenotype and identified by immunofluorescence staining of Arginase 1(Arg-1)and Iba1.The normal MG were harvested as control(control group).The dorsal root ganglion(DRG)of 5 C57BL/6 mice born 1 week old were co-cultured with M2-MG for 5 days to observe the axon length,the DRG alone was used as control.Forty-two 6-week-old female C57BL/6 mice were randomly divided into sham group(n=6),SCI group(n=18),and SCI+M2-MG group(n=18).In sham group,only the laminae of T10 level were removed;SCI group and SCI+M2-MG group underwent SCI modeling,and SCI+M2-MG group was simultaneously injected with M2-MG.The survival of mice in each group was observed after operation.At immediate(0),3,7,14,21,and 28 days after operation,the motor function of mice was evaluated by Basso Mouse Scale(BMS)score,and the gait was evaluated by footprint experiment at 28 days.The spinal cord tissue was taken after operation for immunofluorescence staining,in which glial fibrillary acidic protein(GFAP)staining at 7,14,and 28 days was used to observe the injured area of the spinal cord,neuronal nuclei antigen staining at 28 days was used to observe the survival of neurons,and GFAP/C3 double staining at 7 and 14 days was used to observe the changes in the number of Al astrocytes.Results The purity of MG in vitro reached 90％,and the most of the cells were polarized into M2 phenotype identified by Arg-1 immuno-fluorescence staining.M2-MG promoted the axon growth when co-cultured with DRGs in vitro(P＜0.05).All groups of mice survived until the experiment was completed.The hind limb motor function of SCI group and SCI+M2-MG group gradually recovered over time.Among them,the SCI+M2-MG group had significantly higher BMS scores than the SCI group at 21 and 28 days(P＜0.05),and the dragging gait significantly improved at 28 days,but it did not reach the level of the sham group.Immunofluorescence staining showed that compared with the SCI group,the SCI+M2-MG group had a smaller injury area at 7,14,and 28 days,an increase in neuronal survival at 28 days,and a decrease in the number of A1 astrocytes at 7 and 14 days,with significant differences(P＜0.05).Conclusion M2-MG transplantation improves the motor function of the hind limbs of SCI mice by promoting neuron survival and axon regeneration.This neuroprotective effect is related to the inhibition of Al astrocytes polarization.
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