Effect of M2-like macrophage/microglia-derived mitochondria transplantation in treatment of mouse spinal cord injury

Abstract

To investigate the effect of M2-like macrophage/microglia-derived mitochondria transplantation in treatment of mouse spinal cord injury (SCI). BV2 cells were classified into M1 (LPS treatment), M2 (IL-4 treatment), and M0 (no treatment) groups. After receiving M1 and M2 polarization, BV2 cells received microscopic observation, immunofluorescence staining [Arginase-1 (Arg-1)] and flow cytometry [inducible nitric oxide synthase (iNOS), Arg-1] to determine the result of polarization. MitoSox Red and 2, 7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) stainings were used to evaluate mitochondrial function difference. Mitochondria was isolated from M2-like BV2 cells through differential velocity centrifugation for following transplantation. Then Western blot was used to measure the expression levels of the relevant complexes (complexes Ⅱ, Ⅲ, Ⅳ, and Ⅴ) in the oxidative phosphorylation (OXPHOS), and compared with M2-like BV2 cells to evaluate whether the mitochondria were obtained. Thirty-six female C57BL/6 mice were randomly divided into 3 groups ( n=12). Mice from sham group were only received the T 10 laminectomy. After the T 10 spinal cord injury (SCI) model was prepared in the SCI group and mitochondria transplantation (MT) group, mitochondrial storage solution and mitochondria (100 μg) derived from M2-like BV2 cells were injected into the injured segment, respectively. After operation, the Basso Mouse Scale (BMS) score was performed to evaluate the motor function recovery. And immunofluorescence staining, lycopersicon esculentum agglutinin (LEA)-FITC staining, and ELISA [vascular endothelial growth factor A (VEGFA)] were also performed. After polarization induction, BV2 cells in M1 and M2 groups showed specific morphological changes of M1-like and M2-like macrophages, respectively. Immunofluorescence staining showed that the positive expression of M2-like macrophages marker (Arg-1) was significantly higher in M2 group than in M0 group and M1 group ( P<0.05). Flow cytometry showed that the expression of M1-like macrophage marker (iNOS) was significantly higher in M1 group than in M0 group and M2 group ( P<0.05), and the expression of Arg-1 was significantly higher in M2 group than in M0 group and M1 group ( P<0.05). MitoSox Red and DCFH-DA stainings showed that the fluorescence intensity of the M2 group was significantly lower than that of the M1 group ( P<0.05), and there was no significant difference with the M0 group ( P>0.05). The M2-like BV2 cells-derived mitochondria was identified through Western blot assay. Animal experiments showed that the BMS scores of MT group at 21 and 28 days after operation were significantly higher than those of SCI group ( P<0.05). At 14 days after operation, the number of iNOS-positive cells in MT group was significantly lower than that in SCI group ( P<0.05), but still higher than that in sham group ( P<0.05); the number of LEA-positive cells and the expression of VEGFA in MT group were significantly more than those in the other two groups ( P<0.05). M2-like macrophage/microglia-derived mitochondria transplantation can promote angiogenesis and inhibit inflammatory M1-like macrophage/microglia polarization after mouse SCI to improve function recovery.