Effect of M2-like macrophage/microglia-derived mitochondria transplantation in treatment of mouse spinal cord injury_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HUANG Tengli , SHEN Junjie , LIN Junqing ,  ZHENG Xianyou

Department of Orthopedic Surgery, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai, 200233, P. R. China;

Corresponding author：

ZHENG Xianyou, Email: zhengxianyou@126.com

Keywords：

Spinal cord injury; mitochondria transplantation; microglia; macrophage; angiogenesis; mice

DOI：

10.7507/1002-1892.202201040

Video：

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Objective To investigate the effect of M2-like macrophage/microglia-derived mitochondria transplantation in treatment of mouse spinal cord injury (SCI). Methods 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 T₁₀ laminectomy. After the T₁₀ 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. Results 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). Conclusion 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.

Citation： HUANG Tengli, SHEN Junjie, LIN Junqing, ZHENG Xianyou. Effect of M2-like macrophage/microglia-derived mitochondria transplantation in treatment of mouse spinal cord injury. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(6): 751-759. doi: 10.7507/1002-1892.202201040 Copy

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8.	Weixler V, Lapusca R, Grangl G, et al. Autogenous mitochondria transplantation for treatment of right heart failure. J Thorac Cardiovasc Surg, 2021, 162(1): e111-e121.
9.	Kubat GB, Ozler M, Ulger O, et al. The effects of mesenchymal stem cell mitochondrial transplantation on doxorubicin-mediated nephrotoxicity in rats. J Biochem Mol Toxicol, 2021, 35(1): e22612. doi: 10.1002/jbt.22612.
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11.	Dechandt CRP, Couto-Lima CA, Alberici LC. Triglyceride depletion of brown adipose tissue enables analysis of mitochondrial respiratory function in permeabilized biopsies. Anal Biochem, 2016, 515: 55-60.
12.	Chen CL, Lin CY, Kung HJ. Targeting mitochondrial OXPHOS and their regulatory signals in prostate cancers. Int J Mol Sci, 2021, 22(24): 13435. doi: 10.3390/ijms222413435.
13.	García-Bartolomé A, Peñas A, Illescas M, et al. Altered expression ratio of actin-binding gelsolin isoforms is a novel hallmark of mitochondrial OXPHOS dysfunction. Cells, 2020, 9(9): 1922. doi: 10.3390/cells9091922.
14.	Zhang BL, Gao YL, Li QF, et al. Effects of brain-derived mitochondria on the function of neuron and vascular endothelial cell after traumatic brain injury. World Neurosurg, 2020, 138: e1-e9.
15.	Gu CJ, Li LW, Huang YF, et al. Salidroside ameliorates mitochondria-dependent neuronal apoptosis after spinal cord ischemia-reperfusion injury partially through onhibiting oxidative stress and promoting mitophagy. Oxid Med Cell Longev, 2020, 2020: 3549704.
16.	Han Q, Xie Y, Ordaz JD, et al. Restoring cellular energetics promotes axonal regeneration and functional recovery after spinal cord injury. Cell Metab, 2020, 31(3): 623-641.
17.	Ge XH, Tang PY, Rong YL, et al. Exosomal miR-155 from M1-polarized macrophages promotes EndoMT and impairs mitochondrial function via activating NF-κB signaling pathway in vascular endothelial cells after traumatic spinal cord injury. Redox Biol, 2021, 41: 101932. doi: 10.1016/j.redox.2021.101932.
18.	Kim YM, Kim SJ, Tatsunami R, et al. ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis. Am J Physiol Cell Physiol, 2017, 312(6): C749-C764.

1. Tran AP, Warren PM, Silver J. The biology of regeneration failure and success after spinal cord injury. Physiol Rev, 2018, 98(2): 881-917.
2. Rabchevsky AG, Michael FM, Patel SP. Mitochondria focused neurotherapeutics for spinal cord injury. Exp Neurol, 2020, 330: 113332. doi: 10.1016/j.expneurol.2020.113332.
3. Abbaszadeh F, Fakhri S, Khan H. Targeting apoptosis and autophagy following spinal cord injury: Therapeutic approaches to polyphenols and candidate phytochemicals. Pharmacol Res, 2020, 160: 105069. doi: 10.1016/j.phrs.2020.105069.
4. Abate M, Festa A, Falco M, et al. Mitochondria as playmakers of apoptosis, autophagy and senescence. Semin Cell Dev Biol, 2020, 98: 139-153.
5. Simmons EC, Scholpa NE, Schnellmann RG. FDA-approved 5-HT 1F receptor agonist lasmiditan induces mitochondrial biogenesis and enhances locomotor and blood-spinal cord barrier recovery after spinal cord injury. Exp Neurol, 2021, 341: 113720. doi: 10.1016/j.expneurol.2021.113720.
6. 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.
7. Scholpa NE, Williams H, Wang W, et al. Pharmacological stimulation of mitochondrial biogenesis using the food and drug administration-approved β2-adrenoreceptor agonist formoterol for the Treatment of Spinal Cord Injury. J Neurotrauma, 2019, 36(6): 962-972.
8. Weixler V, Lapusca R, Grangl G, et al. Autogenous mitochondria transplantation for treatment of right heart failure. J Thorac Cardiovasc Surg, 2021, 162(1): e111-e121.
9. Kubat GB, Ozler M, Ulger O, et al. The effects of mesenchymal stem cell mitochondrial transplantation on doxorubicin-mediated nephrotoxicity in rats. J Biochem Mol Toxicol, 2021, 35(1): e22612. doi: 10.1002/jbt.22612.
10. Van den Bossche J, O’Neill LA, Menon D. Macrophage immunometabolism: Where are we (going)? Trends Immunol, 2017, 38(6): 395-406.
11. Dechandt CRP, Couto-Lima CA, Alberici LC. Triglyceride depletion of brown adipose tissue enables analysis of mitochondrial respiratory function in permeabilized biopsies. Anal Biochem, 2016, 515: 55-60.
12. Chen CL, Lin CY, Kung HJ. Targeting mitochondrial OXPHOS and their regulatory signals in prostate cancers. Int J Mol Sci, 2021, 22(24): 13435. doi: 10.3390/ijms222413435.
13. García-Bartolomé A, Peñas A, Illescas M, et al. Altered expression ratio of actin-binding gelsolin isoforms is a novel hallmark of mitochondrial OXPHOS dysfunction. Cells, 2020, 9(9): 1922. doi: 10.3390/cells9091922.
14. Zhang BL, Gao YL, Li QF, et al. Effects of brain-derived mitochondria on the function of neuron and vascular endothelial cell after traumatic brain injury. World Neurosurg, 2020, 138: e1-e9.
15. Gu CJ, Li LW, Huang YF, et al. Salidroside ameliorates mitochondria-dependent neuronal apoptosis after spinal cord ischemia-reperfusion injury partially through onhibiting oxidative stress and promoting mitophagy. Oxid Med Cell Longev, 2020, 2020: 3549704.
16. Han Q, Xie Y, Ordaz JD, et al. Restoring cellular energetics promotes axonal regeneration and functional recovery after spinal cord injury. Cell Metab, 2020, 31(3): 623-641.
17. Ge XH, Tang PY, Rong YL, et al. Exosomal miR-155 from M1-polarized macrophages promotes EndoMT and impairs mitochondrial function via activating NF-κB signaling pathway in vascular endothelial cells after traumatic spinal cord injury. Redox Biol, 2021, 41: 101932. doi: 10.1016/j.redox.2021.101932.
18. Kim YM, Kim SJ, Tatsunami R, et al. ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis. Am J Physiol Cell Physiol, 2017, 312(6): C749-C764.

Chinese Journal of Reparative and Reconstructive Surgery

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

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