Experimental study of M2 microglia transplantation promoting spinal cord injury repair in mice_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Jing ¹ , ZHANG Xiaoyue ¹ , JIANG Qi ¹ , QU Di ¹ , HU Yusheng ¹ ,  QI Chao ² ,  FU Haitao ²

1. Qingdao Medical College of Qingdao University, Qingdao Shandong, 266073, P. R. China;
2. Department of Sports Medicine, the Affiliated Hospital of Qingdao University, Qingdao Shandong, 266103, P. R. China;

Corresponding author：

QI Chao, Email: qichao2002@163.com; FU Haitao, Email: fuhaitao@qdu.edu.cn

Keywords：

Spinal cord injury; M2 microglia; astrocytes; cell transplantation; mouse

DOI：

10.7507/1002-1892.202311093

Video：

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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 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.

Citation： ZHANG Jing, ZHANG Xiaoyue, JIANG Qi, QU Di, HU Yusheng, QI Chao, FU Haitao. Experimental study of M2 microglia transplantation promoting spinal cord injury repair in mice. Chinese Journal of Reparative and Reconstructive Surgery, 2024, 38(2): 198-205. doi: 10.7507/1002-1892.202311093 Copy

1.	Van Broeckhoven J, Sommer D, Dooley D, et al. Macrophage phagocytosis after spinal cord injury: when friends become foes. Brain, 2021, 144(10): 2933-2945.
2.	Kawai M, Imaizumi K, Ishikawa M, et al. Long-term selective stimulation of transplanted neural stem/progenitor cells for spinal cord injury improves locomotor function. Cell Rep, 2021, 37(8): 110019.
3.	Zipser CM, Cragg JJ, Guest JD, et al. Cell-based and stem-cell-based treatments for spinal cord injury: evidence from clinical trials. Lancet Neurol, 2022, 21(7): 659-670.
4.	Zeng YS, Ding Y, Xu HY, et al. Electro-acupuncture and its combination with adult stem cell transplantation for spinal cord injury treatment: A summary of current laboratory findings and a review of literature. CNS Neurosci Ther, 2022, 28(5): 635-647.
5.	Sun X, Huang LY, Pan HX, et al. Bone marrow mesenchymal stem cells and exercise restore motor function following spinal cord injury by activating PI3K/AKT/mTOR pathway. Neural Regen Res, 2023, 18(5): 1067-1075.
6.	Yamazaki K, Kawabori M, Seki T, et al. Clinical trials of stem cell treatment for spinal cord injury. Int J Mol Sci, 2020, 21(11): 3994.
7.	Chang J, Qian Z, Wang B, et al. Transplantation of A2 type astrocytes promotes neural repair and remyelination after spinal cord injury. Cell Commun Signal, 2023, 21(1): 37.
8.	Jiang Y, Guo J, Tang X, et al. The immunological roles of olfactory ensheathing cells in the treatment of spinal cord injury. Front Immunol, 2022 May 20: 13: 881162.
9.	Pearse DD, Bastidas J, Izabel SS, et al. Schwann cell transplantation subdues the pro-inflammatory innate immune cell response after spinal cord injury. Int J Mol Sci, 2018, 19(9): 2550.
10.	Ren J, Zhu B, Gu G, et al. Schwann cell-derived exosomes containing MFG-E8 modify macrophage/microglial polarization for attenuating inflammation via the SOCS3/STAT3 pathway after spinal cord injury. Cell Death Dis, 2023, 14(1): 70.
11.	Wu Y, Hirschi KK. Tissue-resident macrophage development and function. Front Cell Dev Biol, 2021, 8: 617879.
12.	Zhan L, Krabbe G, Du F, et al. Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain. PLoS Biol, 2019, 17(2): e3000134.
13.	Walker DG, Lue LF. Immune phenotypes of microglia in human neurodegenerative disease: challenges to detecting microglial polarization in human brains. Alzheimers Res Ther, 2015, 7(1): 56.
14.	Akhmetzyanova E, Kletenkov K, Mukhamedshina Y, et al. Different approaches to modulation of microglia phenotypes after spinal cord injury. Front Syst Neurosci, 2019, 13: 37.
15.	Kuboyama T, Kominato S, Nagumo M, et al. Recovery from spinal cord injury via M2 microglial polarization induced by Polygalae Radix. Phytomedicine, 2021, 82: 153452.
16.	Pepe G, De Maglie M, Minoli L, et al. Selective proliferative response of microglia to alternative polarization signals. J Neuroinflammation, 2017, 14(1): 236.
17.	Kobashi S, Terashima T, Katagi M, et al. Transplantation of M2-deviated microglia promotes recovery of motor function after spinal cord injury in mice. Mol Ther, 2020, 28(1): 254-265.
18.	Yu G, Zhang Y, Ning B. Reactive astrocytes in central nervous system injury: subgroup and potential therapy. Front Cell Neurosci, 2021, 15: 792764.
19.	Wang J, Cheng C, Liu Z, et al. Inhibition of A1 astrocytes and activation of A2 astrocytes for the treatment of spinal cord injury. Neurochem Res, 2023, 48(3): 767-780.
20.	Hou J, Bi H, Ge Q, et al. Heterogeneity analysis of astrocytes following spinal cord injury at single-cell resolution. FASEB J, 2022, 36(8): e22442.
21.	Greenhalgh AD, David S, Bennett FC. Immune cell regulation of glia during CNS injury and disease. Nat Rev Neurosci, 2020, 21(3): 139-152.
22.	Fujita Y, Yamashita T. Mechanisms and significance of microglia-axon interactions in physiological and pathophysiological conditions. Cell Mol Life Sci, 2021, 78(8): 3907-3919.
23.	Wang J, Xing H, Wan L, et al. Treatment targets for M2 microglia polarization in ischemic stroke. Biomed Pharmacother, 2018, 105: 518-525.
24.	Goshi N, Morgan RK, Lein PJ, et al. A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation. J Neuroinflammation, 2020, 17(1): 155.
25.	Lv B, Zhang X, Yuan J, et al. Biomaterial-supported MSC transplantation enhances cell-cell communication for spinal cord injury. Stem Cell Res Ther, 2021, 12(1): 36.
26.	Kigerl KA, Gensel JC, Ankeny DP, et al. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci, 2009, 29(43): 13435-13444.
27.	Zhao Q, Ren YL, Zhu YJ, et al. The origins and dynamic changes of C3- and S100A10-positive reactive astrocytes after spinal cord injury. Front Cell Neurosci, 2023, 17: 1276506.

1. Van Broeckhoven J, Sommer D, Dooley D, et al. Macrophage phagocytosis after spinal cord injury: when friends become foes. Brain, 2021, 144(10): 2933-2945.
2. Kawai M, Imaizumi K, Ishikawa M, et al. Long-term selective stimulation of transplanted neural stem/progenitor cells for spinal cord injury improves locomotor function. Cell Rep, 2021, 37(8): 110019.
3. Zipser CM, Cragg JJ, Guest JD, et al. Cell-based and stem-cell-based treatments for spinal cord injury: evidence from clinical trials. Lancet Neurol, 2022, 21(7): 659-670.
4. Zeng YS, Ding Y, Xu HY, et al. Electro-acupuncture and its combination with adult stem cell transplantation for spinal cord injury treatment: A summary of current laboratory findings and a review of literature. CNS Neurosci Ther, 2022, 28(5): 635-647.
5. Sun X, Huang LY, Pan HX, et al. Bone marrow mesenchymal stem cells and exercise restore motor function following spinal cord injury by activating PI3K/AKT/mTOR pathway. Neural Regen Res, 2023, 18(5): 1067-1075.
6. Yamazaki K, Kawabori M, Seki T, et al. Clinical trials of stem cell treatment for spinal cord injury. Int J Mol Sci, 2020, 21(11): 3994.
7. Chang J, Qian Z, Wang B, et al. Transplantation of A2 type astrocytes promotes neural repair and remyelination after spinal cord injury. Cell Commun Signal, 2023, 21(1): 37.
8. Jiang Y, Guo J, Tang X, et al. The immunological roles of olfactory ensheathing cells in the treatment of spinal cord injury. Front Immunol, 2022 May 20: 13: 881162.
9. Pearse DD, Bastidas J, Izabel SS, et al. Schwann cell transplantation subdues the pro-inflammatory innate immune cell response after spinal cord injury. Int J Mol Sci, 2018, 19(9): 2550.
10. Ren J, Zhu B, Gu G, et al. Schwann cell-derived exosomes containing MFG-E8 modify macrophage/microglial polarization for attenuating inflammation via the SOCS3/STAT3 pathway after spinal cord injury. Cell Death Dis, 2023, 14(1): 70.
11. Wu Y, Hirschi KK. Tissue-resident macrophage development and function. Front Cell Dev Biol, 2021, 8: 617879.
12. Zhan L, Krabbe G, Du F, et al. Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain. PLoS Biol, 2019, 17(2): e3000134.
13. Walker DG, Lue LF. Immune phenotypes of microglia in human neurodegenerative disease: challenges to detecting microglial polarization in human brains. Alzheimers Res Ther, 2015, 7(1): 56.
14. Akhmetzyanova E, Kletenkov K, Mukhamedshina Y, et al. Different approaches to modulation of microglia phenotypes after spinal cord injury. Front Syst Neurosci, 2019, 13: 37.
15. Kuboyama T, Kominato S, Nagumo M, et al. Recovery from spinal cord injury via M2 microglial polarization induced by Polygalae Radix. Phytomedicine, 2021, 82: 153452.
16. Pepe G, De Maglie M, Minoli L, et al. Selective proliferative response of microglia to alternative polarization signals. J Neuroinflammation, 2017, 14(1): 236.
17. Kobashi S, Terashima T, Katagi M, et al. Transplantation of M2-deviated microglia promotes recovery of motor function after spinal cord injury in mice. Mol Ther, 2020, 28(1): 254-265.
18. Yu G, Zhang Y, Ning B. Reactive astrocytes in central nervous system injury: subgroup and potential therapy. Front Cell Neurosci, 2021, 15: 792764.
19. Wang J, Cheng C, Liu Z, et al. Inhibition of A1 astrocytes and activation of A2 astrocytes for the treatment of spinal cord injury. Neurochem Res, 2023, 48(3): 767-780.
20. Hou J, Bi H, Ge Q, et al. Heterogeneity analysis of astrocytes following spinal cord injury at single-cell resolution. FASEB J, 2022, 36(8): e22442.
21. Greenhalgh AD, David S, Bennett FC. Immune cell regulation of glia during CNS injury and disease. Nat Rev Neurosci, 2020, 21(3): 139-152.
22. Fujita Y, Yamashita T. Mechanisms and significance of microglia-axon interactions in physiological and pathophysiological conditions. Cell Mol Life Sci, 2021, 78(8): 3907-3919.
23. Wang J, Xing H, Wan L, et al. Treatment targets for M2 microglia polarization in ischemic stroke. Biomed Pharmacother, 2018, 105: 518-525.
24. Goshi N, Morgan RK, Lein PJ, et al. A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation. J Neuroinflammation, 2020, 17(1): 155.
25. Lv B, Zhang X, Yuan J, et al. Biomaterial-supported MSC transplantation enhances cell-cell communication for spinal cord injury. Stem Cell Res Ther, 2021, 12(1): 36.
26. Kigerl KA, Gensel JC, Ankeny DP, et al. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci, 2009, 29(43): 13435-13444.
27. Zhao Q, Ren YL, Zhu YJ, et al. The origins and dynamic changes of C3- and S100A10-positive reactive astrocytes after spinal cord injury. Front Cell Neurosci, 2023, 17: 1276506.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study of M2 microglia transplantation promoting spinal cord injury repair in mice

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