Effect of removing microglia from spinal cord on nerve repair after spinal cord injury in mice_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

JIANG Qi ¹ , QI Chao ² , SUN Yuerong ² , XUE Shiyuan ¹ , WEI Xinyi ¹ ,  FU Haitao ²

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

Corresponding author：

FU Haitao, Email: fuhaitao@qdu.edu.cn

Keywords：

Microglia; spinal cord injury; nerve injury; mouse

DOI：

10.7507/1002-1892.202503099

Video：

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Objective To investigate the effects of removing microglia from spinal cord on nerve repair and functional recovery after spinal cord injury (SCI) in mice. Methods Thirty-nine 6-week-old female C57BL/6 mice were randomly divided into control group (n=12), SCI group (n=12), and PLX3397+SCI group (n=15). The PLX3397+SCI group received continuous feeding of PLX3397, a colony-stimulating factor 1 receptor inhibitor, while the other two groups were fed a standard diet. After 14 days, both the SCI group and the PLX3397+SCI group were tested for ionized calcium binding adapter molecule 1 (Iba1) to confirm that the PLX3397+SCI group had completely depleted the spinal cord microglia. The SCI model was then prepared by clamping the spinal cord in both the SCI group and the PLX3397+SCI group, while the control group underwent laminectomy. Preoperatively and at 1, 3, 7, 14, 21, and 28 days postoperatively, the Basso Mouse Scale (BMS) was used to assess the hind limb function of mice in each group. At 28 days, a footprint test was conducted to observe the gait of the mice. After SCI, spinal cord tissue from the injury site was taken, and Iba1 immunofluorescence staining was performed at 7 days to observe the aggregation and proliferation of microglia in the spinal cord. HE staining was used to observe the formation of glial scars at the injury site at 28 days; glial fibrillary acidic protein (GFAP) immunofluorescence staining was applied to astrocytes to assess the extent of the injured area; neuronal nuclei antigen (NeuN) immunofluorescence staining was used to evaluate neuronal survival. And 5-hydroxytryptamine (5-HT) immunofluorescence staining was performed to assess axonal survival at 60 days. Results All mice survived until the end of the experiment. Immunofluorescence staining revealed that the microglia in the spinal cord of the PLX3397+SCI group decreased by more than 95% compared to the control group after 14 days of continuous feeding with PLX3397 (P<0.05). Compared to the control group, the BMS scores in the PLX3397+SCI group and the SCI group significantly decreased at different time points after SCI (P<0.05). Moreover, the PLX3397+SCI group showed a further decrease in BMS scores compared to the SCI group, and exhibited a dragging gait. The differences between the two groups were significant at 14, 21, and 28 days (P<0.05). HE staining at 28 days revealed that the SCI group had formed a well-defined and dense gliotic scar, while the PLX3397+SCI group also developed a gliotic scar, but with a more blurred and loose boundary. Immunofluorescence staining revealed that the number of microglia near the injury center at 7 days increased in the SCI group than in the control group, but the difference between groups was not significant (P>0.05). In contrast, the PLX3397+SCI group showed a significant reduction in microglia compared to both the control and SCI groups (P<0.05). At 28 days after SCI, the area of spinal cord injury in the PLX3397+SCI group was significantly larger than that in SCI group (P<0.05); the surviving neurons significantly reduced compared with the control group and SCI group (P<0.05). The axonal necrosis and retraction at 60 days after SCI were more obvious. Conclusion The removal of microglia in the spinal cord aggravate the tissue damage after SCI and affecte the recovery of motor function in mice, suggesting that microglia played a neuroprotective role in SCI.

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2.	Sunshine MD, Bindi VE, Nguyen BL, et al. Oxygen therapy attenuates neuroinflammation after spinal cord injury. J Neuroinflammation, 2023, 20(1): 303. doi: 10.1186/s12974-023-02985-6.
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5.	Deng J, Meng F, Zhang K, et al. Emerging roles of microglia depletion in the treatment of spinal cord injury. Cells, 2022, 11(12): 1871. doi: 10.3390/cells11121871.
6.	Li F, Wang Y, Zheng K. Microglial mitophagy integrates the microbiota-gut-brain axis to restrain neuroinflammation during neurotropic herpesvirus infection. Autophagy, 2023, 19(2): 734-736.
7.	Li X, Yu Z, Zong W, et al. Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury. J Neuroinflammation, 2020, 17(1): 263. doi: 10.1186/s12974-020-01942-x.
8.	Tan Z, Guo Y, Shrestha M, et al. Microglia depletion exacerbates retinal ganglion cell loss in a mouse model of glaucoma. Exp Eye Res, 2022, 225: 109273. doi: 10.1016/j.exer.2022.109273.
9.	Baaklini CS, Ho MFS, Lange T, et al. Microglia promote remyelination independent of their role in clearing myelin debris. Cell Rep, 2023, 42(12): 113574. doi: 10.1016/j.celrep.2023.113574.
10.	Orihuela R, McPherson CA, Harry GJ. Microglial M1/M2 polarization and metabolic states. Br J Pharmacol, 2016, 173(4): 649-665.
11.	Hsu CH, Pan YJ, Zheng YT, et al. Ultrasound reduces inflammation by modulating M1/M2 polarization of microglia through STAT1/STAT6/PPARγ signaling pathways. CNS Neurosci Ther, 2023, 29(12): 4113-4123.
12.	Long Y, Li XQ, Deng J, et al. Modulating the polarization phenotype of microglia—A valuable strategy for central nervous system diseases. Ageing Res Rev, 2024, 93: 102160. doi: 10.1016/j.arr.2023.102160.
13.	Guo H, Du M, Yang Y, et al. Sp1 Regulates the M1 polarization of microglia through the HuR/NF-κB axis after spinal cord injury. Neuroscience, 2024, 544: 50-63.
14.	Wang C, Wang Q, Lou Y, et al. Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation. J Cell Mol Med, 2018, 22(2): 1148-1166.
15.	Shen Q, Zhang G. Depletion of microglia mitigates cerebrovascular dysfunction in diet-induced obesity mice. Am J Physiol Endocrinol Metab, 2021, 321(3): E367-E375.
16.	Asai H, Ikezu S, Tsunoda S, et al. Depletion of microglia and inhibition of exosome synthesis halt tau propagation. Nat Neurosci, 2015, 18(11): 1584-1593.
17.	Elmore MR, Najafi AR, Koike MA, et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron, 2014, 82(2): 380-397.
18.	Rice RA, Spangenberg EE, Yamate-Morgan H, et al. Elimination of microglia improves functional outcomes following extensive neuronal loss in the hippocampus. J Neurosci, 2015, 35(27): 9977-9989.
19.	Bellver-Landete V, Bretheau F, Mailhot B, et al. Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury. Nat Commun, 2019, 10(1): 518. doi: 10.1038/s41467-019-08446-0.
20.	Brennan FH, Li Y, Wang C, et al. Microglia coordinate cellular interactions during spinal cord repair in mice. Nat Commun, 2022, 13(1): 4096. doi: 10.1038/s41467-022-31797-0.
21.	Liu K, Lu Y, Lee JK, et al. PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat Neurosci, 2010, 13(9): 1075-1081.
22.	Au NPB, Ma CHE. Neuroinflammation, microglia and implications for retinal ganglion cell survival and axon regeneration in traumatic optic neuropathy. Front Immunol, 2022, 13: 860070. doi: 10.3389/fimmu.2022.860070.
23.	Hu X, Huang J, Li Z, et al. Lactate promotes microglial scar formation and facilitates locomotor function recovery by enhancing histone H4 lysine 12 lactylation after spinal cord injury. J Neuroinflammation, 2024, 21(1): 193. doi: 10.1186/s12974-024-03186-5.
24.	Okojie AK, Uweru JO, Coburn MA, et al. Distinguishing the effects of systemic CSF1R inhibition by PLX3397 on microglia and peripheral immune cells. J Neuroinflammation, 2023, 20(1): 242. doi: 10.1186/s12974-023-02924-5.
25.	Bisht K, Okojie KA, Sharma K, et al. Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling in mice. Nat Commun, 2021, 12(1): 5289. doi: 10.1038/s41467-021-25590-8.
26.	Tran AP, Warren PM, Silver J. New insights into glial scar formation after spinal cord injury. Cell Tissue Res. 2022 Mar;387(3): 319-336.
27.	Bradbury EJ, Burnside ER. Moving beyond the glial scar for spinal cord repair. Nat Commun, 2019, 10(1): 3879. doi: 10.1038/s41467-019-11707-7.
28.	Vidal-Itriago A, Radford RAW, Aramideh JA, et al. Microglia morphophysiological diversity and its implications for the CNS. Front Immunol, 2022, 13: 997786. doi: 10.3389/fimmu.2022.997786.
29.	Silver J, Miller JH. Regeneration beyond the glial scar. Nat Rev Neurosci, 2004, 5(2): 146-156.
30.	Silver J. The glial scar is more than just astrocytes. Exp Neurol, 2016, 286: 147-149.
31.	Lu L, Ye J, Yi D, et al. Runx2 suppresses astrocyte activation and astroglial scar formation after spinal cord injury in mice. Mol Neurobiol, 2024, 61(12): 10820-10829.
32.	Qian D, Dong Y, Liu X, et al. Salidroside promotes the repair of spinal cord injury by inhibiting astrocyte polarization, promoting neural stem cell proliferation and neuronal differentiation. Cell Death Discov, 2024, 10(1): 224. doi: 10.1038/s41420-024-01989-2.
33.	Hemati-Gourabi M, Cao T, Romprey MK, et al. Capacity of astrocytes to promote axon growth in the injured mammalian central nervous system. Front Neurosci, 2022, 16: 955598. doi: 10.3389/fnins.2022.955598.
34.	Dias DO, Göritz C. Fibrotic scarring following lesions to the central nervous system. Matrix Biol, 2018. doi: 10.1016/j.matbio.2018.02.009.

1. Cowan H, Lakra C, Desai M. Autonomic dysreflexia in spinal cord injury. BMJ, 2020, 371: m3596. doi: 10.1136/bmj.m3596.
2. Sunshine MD, Bindi VE, Nguyen BL, et al. Oxygen therapy attenuates neuroinflammation after spinal cord injury. J Neuroinflammation, 2023, 20(1): 303. doi: 10.1186/s12974-023-02985-6.
3. Sterner RC, Sterner RM. Immune response following traumatic spinal cord injury: Pathophysiology and therapies. Front Immunol, 2023, 13: 1084101. doi: 10.3389/fimmu.2022.1084101.
4. Sun C, Deng J, Ma Y, et al. The dual role of microglia in neuropathic pain after spinal cord injury: Detrimental and protective effects. Exp Neurol, 2023, 370: 114570. doi: 10.1016/j.expneurol.2023.114570.
5. Deng J, Meng F, Zhang K, et al. Emerging roles of microglia depletion in the treatment of spinal cord injury. Cells, 2022, 11(12): 1871. doi: 10.3390/cells11121871.
6. Li F, Wang Y, Zheng K. Microglial mitophagy integrates the microbiota-gut-brain axis to restrain neuroinflammation during neurotropic herpesvirus infection. Autophagy, 2023, 19(2): 734-736.
7. Li X, Yu Z, Zong W, et al. Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury. J Neuroinflammation, 2020, 17(1): 263. doi: 10.1186/s12974-020-01942-x.
8. Tan Z, Guo Y, Shrestha M, et al. Microglia depletion exacerbates retinal ganglion cell loss in a mouse model of glaucoma. Exp Eye Res, 2022, 225: 109273. doi: 10.1016/j.exer.2022.109273.
9. Baaklini CS, Ho MFS, Lange T, et al. Microglia promote remyelination independent of their role in clearing myelin debris. Cell Rep, 2023, 42(12): 113574. doi: 10.1016/j.celrep.2023.113574.
10. Orihuela R, McPherson CA, Harry GJ. Microglial M1/M2 polarization and metabolic states. Br J Pharmacol, 2016, 173(4): 649-665.
11. Hsu CH, Pan YJ, Zheng YT, et al. Ultrasound reduces inflammation by modulating M1/M2 polarization of microglia through STAT1/STAT6/PPARγ signaling pathways. CNS Neurosci Ther, 2023, 29(12): 4113-4123.
12. Long Y, Li XQ, Deng J, et al. Modulating the polarization phenotype of microglia—A valuable strategy for central nervous system diseases. Ageing Res Rev, 2024, 93: 102160. doi: 10.1016/j.arr.2023.102160.
13. Guo H, Du M, Yang Y, et al. Sp1 Regulates the M1 polarization of microglia through the HuR/NF-κB axis after spinal cord injury. Neuroscience, 2024, 544: 50-63.
14. Wang C, Wang Q, Lou Y, et al. Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation. J Cell Mol Med, 2018, 22(2): 1148-1166.
15. Shen Q, Zhang G. Depletion of microglia mitigates cerebrovascular dysfunction in diet-induced obesity mice. Am J Physiol Endocrinol Metab, 2021, 321(3): E367-E375.
16. Asai H, Ikezu S, Tsunoda S, et al. Depletion of microglia and inhibition of exosome synthesis halt tau propagation. Nat Neurosci, 2015, 18(11): 1584-1593.
17. Elmore MR, Najafi AR, Koike MA, et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron, 2014, 82(2): 380-397.
18. Rice RA, Spangenberg EE, Yamate-Morgan H, et al. Elimination of microglia improves functional outcomes following extensive neuronal loss in the hippocampus. J Neurosci, 2015, 35(27): 9977-9989.
19. Bellver-Landete V, Bretheau F, Mailhot B, et al. Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury. Nat Commun, 2019, 10(1): 518. doi: 10.1038/s41467-019-08446-0.
20. Brennan FH, Li Y, Wang C, et al. Microglia coordinate cellular interactions during spinal cord repair in mice. Nat Commun, 2022, 13(1): 4096. doi: 10.1038/s41467-022-31797-0.
21. Liu K, Lu Y, Lee JK, et al. PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat Neurosci, 2010, 13(9): 1075-1081.
22. Au NPB, Ma CHE. Neuroinflammation, microglia and implications for retinal ganglion cell survival and axon regeneration in traumatic optic neuropathy. Front Immunol, 2022, 13: 860070. doi: 10.3389/fimmu.2022.860070.
23. Hu X, Huang J, Li Z, et al. Lactate promotes microglial scar formation and facilitates locomotor function recovery by enhancing histone H4 lysine 12 lactylation after spinal cord injury. J Neuroinflammation, 2024, 21(1): 193. doi: 10.1186/s12974-024-03186-5.
24. Okojie AK, Uweru JO, Coburn MA, et al. Distinguishing the effects of systemic CSF1R inhibition by PLX3397 on microglia and peripheral immune cells. J Neuroinflammation, 2023, 20(1): 242. doi: 10.1186/s12974-023-02924-5.
25. Bisht K, Okojie KA, Sharma K, et al. Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling in mice. Nat Commun, 2021, 12(1): 5289. doi: 10.1038/s41467-021-25590-8.
26. Tran AP, Warren PM, Silver J. New insights into glial scar formation after spinal cord injury. Cell Tissue Res. 2022 Mar;387(3): 319-336.
27. Bradbury EJ, Burnside ER. Moving beyond the glial scar for spinal cord repair. Nat Commun, 2019, 10(1): 3879. doi: 10.1038/s41467-019-11707-7.
28. Vidal-Itriago A, Radford RAW, Aramideh JA, et al. Microglia morphophysiological diversity and its implications for the CNS. Front Immunol, 2022, 13: 997786. doi: 10.3389/fimmu.2022.997786.
29. Silver J, Miller JH. Regeneration beyond the glial scar. Nat Rev Neurosci, 2004, 5(2): 146-156.
30. Silver J. The glial scar is more than just astrocytes. Exp Neurol, 2016, 286: 147-149.
31. Lu L, Ye J, Yi D, et al. Runx2 suppresses astrocyte activation and astroglial scar formation after spinal cord injury in mice. Mol Neurobiol, 2024, 61(12): 10820-10829.
32. Qian D, Dong Y, Liu X, et al. Salidroside promotes the repair of spinal cord injury by inhibiting astrocyte polarization, promoting neural stem cell proliferation and neuronal differentiation. Cell Death Discov, 2024, 10(1): 224. doi: 10.1038/s41420-024-01989-2.
33. Hemati-Gourabi M, Cao T, Romprey MK, et al. Capacity of astrocytes to promote axon growth in the injured mammalian central nervous system. Front Neurosci, 2022, 16: 955598. doi: 10.3389/fnins.2022.955598.
34. Dias DO, Göritz C. Fibrotic scarring following lesions to the central nervous system. Matrix Biol, 2018. doi: 10.1016/j.matbio.2018.02.009.

Chinese Journal of Reparative and Reconstructive Surgery

Latest ArticlesEffect of removing microglia from spinal cord on nerve repair after spinal cord injury in mice

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