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 T 10 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 A1 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 immunofluorescence 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 A1 astrocytes polarization.
目的: 探讨M2型小胶质细胞（M2 microglia，M2-MG）移植促进小鼠脊髓损伤（spinal cord injury，SCI）修复的效果。.
方法: 取15只新生2～3 d C57BL/6乳鼠大脑皮质，通过胰蛋白酶消化法分离原代细胞并进行Iba1免疫荧光染色鉴定为MG后，以IL-4极化诱导培养48 h（实验组），通过精氨酸酶 1（Arginase 1，Arg-1）、Iba1免疫荧光染色鉴定其是否极化为M2-MG；以正常培养MG作为对照组。另取5只新生1周C57BL/6乳鼠背根神经节（dorsal root ganglion，DRG）与M2-MG共培养5 d，观测轴突长度；以单纯DRG作为对照。取42只6周龄雌性C57BL/6小鼠随机分为假手术组（ n=6）、SCI组（ n=18）及SCI+M2-MG组（ n=18）。手术暴露脊柱T 10节段后，假手术组仅切除椎板，SCI组及SCI+M2-MG组行SCI造模后，SCI+M2-MG组同时注射M2-MG。术后观测各组小鼠存活情况，并于术后当天（0）、3、7、14、21、28 d采用BMS（Basso Mouse Scale）评分评价小鼠运动功能恢复情况，28 d行足迹实验评价小鼠步态。术后取SCI组及SCI+M2-MG组脊髓组织行免疫荧光染色，其中7、14、28 d 胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）染色观测SCI损伤区域面积，28 d 神经元核抗原染色观察神经元存活情况，7、14 d GFAP/C3双重荧光染色观察表示A1星形胶质细胞数量变化。.
结果: 免疫荧光染色鉴定体外培养细胞为MG且纯度可达90%，经IL-4诱导培养后Arg-1免疫荧光染色示极化为M2表型。M2-MG与DRGs体外共培养5 d后可促进轴突生长（ P<0.05）。动物实验示，术后各组小鼠均存活至实验完成。SCI组及SCI+M2-MG组小鼠术后后肢运动功能随时间延长逐渐恢复，其中21、28 d SCI+M2-MG组BMS评分较SCI组更高（ P<0.05），28 d时拖曳步态明显减轻，但尚未达假手术组水平。免疫荧光染色示，与SCI组相比，SCI+M2-MG 组在7、14、28 d损伤区域面积均缩小，28 d时存活神经元数量增加，7、14 d时A1星形胶质细胞减少，差异均有统计学意义（ P<0.05）。.
结论: M2-MG移植可促进小鼠SCI神经元存活和轴突再生，改善小鼠后肢运动功能，分析这种神经保护作用与A1星形胶质细胞极化被抑制有关。.
Keywords: M2 microglia; Spinal cord injury; astrocytes; cell transplantation; mouse.