Experimental study of tetramethylpyrazine-loaded electroconductive hydrogel on angiogenesis and neuroprotection after spinal cord injury_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DENG Bowen ¹ , JIANG Shengyuan ¹ , LIU Gang ¹ , LI Xiaoye ¹ , BAI Huizhong ¹ , HUO Luyao ¹ , XU Jie ² , XU Lin ¹ ,  MU Xiaohong ¹

1. Department of Orthopedics, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, P. R. China;
2. Department of Microsurgery of the Hands and Feet, Jinhua Hospital of Traditional Chinese Medicine, Jinhua Zhejiang, 321017, P. R. China;

Corresponding author：

MU Xiaohong, Email: muxiaohong2006@126.com

Keywords：

Spinal cord injury; tetramethylpyrazine; hydrogel; neuroprotection; angiogenesis

DOI：

10.7507/1002-1892.202311009

Video：

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Objective To explore the mechanisms for repairing spinal cord injury (SCI) with tetramethylpyrazine-loaded electroconductive hydrogel (hereinafter referred to as “TGTP”). Mehtods A total of 72 female Sprague-Dawley rats were randomly divided into 4 groups: sham operation group (group A), SCI group (group B), SCI+electroconductive hydrogel group (group C), and SCI+TGTP group (group D). Only the vertebral plate was removed in group A, while the remaining groups were subjected to a whole transection model of spinal cord with a 2 mm gap in the lesions. The recovery of hindlimb motor function was evaluated by Basso, Beattie, Bresnahan (BBB) score and modified Rivlin-Tator inclined plate test before operation and at 1, 3, 7, 14, and 28 days after operation, respectively. Animals were sacrificed at 7 days and 28 days after modeling. Neovascularisation was observed by immunofluorescence staining of CD31 and the expression levels of angiopoietin 1 (Ang-1) and Tie-2 were assessed by Western blot assay. At 28 days postoperatively, the expression levels of pro-angiogenic related proteins, including platelet-derived growth factor B (PDGF-B), PDGF receptor β (PDGFR-β), vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2), were also assessed by Western blot. The fibrous scar in the injured area was assessed using Masson staining, while neuronal survival was observed through Nissl staining. Furthermore, LFB staining was utilized to detect myelin distribution and regeneration. Immunofluorescence and Western blot assay were employed to evaluate the expression of neurofilament 200 (NF200). Results The hindlimb motor function of rats in each group gradually recovered from the 3rd day after operation. The BBB score and climbing angle in group D were significantly higher than those in group B from 3 to 28 days after operation, and significantly higher than those in group C at 14 days and 28 days after operation (P<0.05). Masson staining showed that the collagen volume fraction in groups B-D were significantly higher than that in group A, and that in group D was significantly lower than that in groups B and C (P<0.05); a small amount of black conductive particles were scattered at the broken end in group D, and the surrounding collagen fibers were less than those in group C. Nissl and LFB staining showed that the structure of neurons and myelin sheath in the injured area of spinal cord in group D was relatively complete and continuous, and the number of Nissl bodies and the positive area of myelin sheath in group D were significantly better than those in groups B and C (P<0.05). NF200 immunofluorescence staining and Western blot assay results showed that the relative expression of NF200 protein in group D was significantly higher than that in groups B and C (P<0.05). CD31 immunofluorescence staining showed that the fluorescence intensity of group D was better than that of groups B and C at 28 days after operation, and tubular or linear neovascularization could be seen. The relative expressions of Ang-1 and Tie-2 proteins in group D were significantly higher than those in groups B and C at 7 and 28 days after operation (P<0.05). The relative expressions of PDGF-B and PDGFR-β proteins in group D were significantly higher than those in groups B and C, and group B was significantly higher than group C at 28 days after operation (P<0.05). The relative expressions of VEGF-A and VEGFR2 proteins in group D were higher than those in groups B and C, showing significant difference when compared with group B (P<0.05), but only the expression of VEGF-A protein was significantly higher than that in group C (P<0.05). There was significant difference only in VEGFR-2 protein between groups B and C (P<0.05). Conclusion TGTP may enhance the revascularization of the injured area and protect the neurons, thus alleviating the injury of spinal cord tissue structure and promoting the recovery of neurological function after SCI in rats.

Citation： DENG Bowen, JIANG Shengyuan, LIU Gang, LI Xiaoye, BAI Huizhong, HUO Luyao, XU Jie, XU Lin, MU Xiaohong. Experimental study of tetramethylpyrazine-loaded electroconductive hydrogel on angiogenesis and neuroprotection after spinal cord injury. Chinese Journal of Reparative and Reconstructive Surgery, 2024, 38(2): 189-197. doi: 10.7507/1002-1892.202311009 Copy

1.	Li L, Mu J, Zhang Y, et al. Stimulation by exosomes from hypoxia preconditioned human umbilical vein endothelial cells facilitates mesenchymal stem cells angiogenic function for spinal cord repair. ACS Nano, 2022, 16(7): 10811-10823.
2.	You Z, Gao X, Kang X, et al. Microvascular endothelial cells derived from spinal cord promote spinal cord injury repair. Bioact Mater, 2023, 29: 36-49.
3.	Dray C, Rougon G, Debarbieux F. Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord. Proc Natl Acad Sci U S A, 2009, 106(23): 9459-9464.
4.	Fedorova J, Kellerova E, Bimbova K, et al. The histopathology of severe graded compression in lower thoracic spinal cord segment of rat, evaluated at late post-injury phase. Cell Mol Neurobiol, 2022, 42(1): 173-193.
5.	Figley SA, Khosravi R, Legasto JM, et al. Characterization of vascular disruption and blood-spinal cord barrier permeability following traumatic spinal cord injury. J Neurotrauma, 2014, 31(6): 541-552.
6.	Cao Y, Wu T, Yuan Z, et al. Three-dimensional imaging of microvasculature in the rat spinal cord following injury. Sci Rep, 2015, 5: 12643.
7.	Li Y, Lucas-Osma AM, Black S, et al. Pericytes impair capillary blood flow and motor function after chronic spinal cord injury. Nat Med, 2017, 23(6): 733-741.
8.	Liu SM, Xiao ZF, Li X, et al. Vascular endothelial growth factor activates neural stem cells through epidermal growth factor receptor signal after spinal cord injury. CNS Neurosci Ther, 2019, 25(3): 375-385.
9.	Tsai HH, Niu J, Munji R, et al. Oligodendrocyte precursors migrate along vasculature in the developing nervous system. Science, 2016, 351(6271): 379-384.
10.	陶经纬, 周婧雅, 赵毅, 等. 川芎嗪对脊髓损伤大鼠铁死亡的调控作用及机制. 中国组织工程研究, 2024, 28(26): 4158-4163.
11.	张厚君, 蒋昇源, 邓博文, 等. 川芎嗪改善脊髓损伤模型大鼠炎性微环境的机制. 中国组织工程研究, 2023, 27(11): 1701-1707.
12.	蒋昇源, 邓博文, 徐林, 等. 川芎嗪修复脊髓损伤的作用及机制. 中国组织工程研究, 2022, 26(11): 1799-1804.
13.	Li G, Sng KS, Shu B, et al. Effects of tetramethylpyrazine treatment in a rat model of spinal cord injury: A systematic review and meta-analysis. Eur J Pharmacol, 2023, 945: 175524.
14.	Li J, Wei J, Wan Y, et al. TAT-modified tetramethylpyrazine-loaded nanoparticles for targeted treatment of spinal cord injury. J Control Release, 2021, 335: 103-116.
15.	Zhou L, Fan L, Yi X, et al. Soft conducting polymer hydrogels cross-linked and doped by tannic acid for spinal cord injury repair. ACS Nano, 2018, 12(11): 10957-10967.
16.	Fan L, Liu C, Chen X, et al. Exosomes-loaded electroconductive hydrogel synergistically promotes tissue repair after spinal cord injury via immunoregulation and enhancement of myelinated axon growth. Adv Sci (Weinh), 2022, 9(13): e2105586.
17.	蒋昇源, 邓博文, 刘港, 等. 携载川芎嗪缓释微粒导电水凝胶修复脊髓损伤实验研究. 中国修复重建外科杂志, 2023, 37(1): 65-73.
18.	贺丰, 俞兴, 穆晓红, 等. 新型脊髓完全横断缺损模型大鼠的建立. 中国组织工程研究, 2016, 20(5): 635-639.
19.	Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. J Neurosurg, 1977, 47(4): 577-581.
20.	Wang Y, Lv HQ, Chao X, et al. Multimodal therapy strategies based on hydrogels for the repair of spinal cord injury. Mil Med Res, 2022, 9(1): 16.
21.	Fan L, Liu C, Chen X, et al. Directing induced pluripotent stem cell derived neural stem cell fate with a three-dimensional biomimetic hydrogel for spinal cord injury repair. ACS Appl Mater Interfaces, 2018, 10(21): 17742-17755.
22.	Sun X, Liu H, Tan Z, et al. Remodeling microenvironment for endogenous repair through precise modulation of chondroitin sulfate proteoglycans following spinal cord injury. Small, 2023, 19(6): e2205012.
23.	Ruschel J, Hellal F, Flynn KC, et al. Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury. Science, 2015, 348(6232): 347-352.
24.	Hu X, Xu W, Ren Y, et al. Spinal cord injury: molecular mechanisms and therapeutic interventions. Signal Transduct Target Ther, 2023, 8(1): 245.
25.	Wheaton BJ, Sena J, Sundararajan A, et al. Identification of regenerative processes in neonatal spinal cord injury in the opossum (Monodelphis domestica): A transcriptomic study. J Comp Neurol, 2021, 529(5): 969-986.
26.	Liu D, Huang Y, Jia C, et al. Administration of antagomir-223 inhibits apoptosis, promotes angiogenesis and functional recovery in rats with spinal cord injury. Cell Mol Neurobiol, 2015, 35(4): 483-491.
27.	Ouellette J, Lacoste B. From neurodevelopmental to neurodegenerative disorders: The vascular continuum. Front Aging Neurosci, 2021, 13: 749026.
28.	Simons M, Gordon E, Claesson-Welsh L. Mechanisms and regulation of endothelial VEGF receptor signalling. Nat Rev Mol Cell Biol, 2016, 17(10): 611-625.
29.	Ruhrberg C, Gerhardt H, Golding M, et al. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev, 2002, 16(20): 2684-2698.
30.	Duan YY, Chai Y, Zhang NL, et al. Microtubule stabilization promotes microcirculation reconstruction after spinal cord injury. J Mol Neurosci, 2021, 71(3): 583-595.
31.	Duran CL, Howell DW, Dave JM, et al. Molecular regulation of sprouting angiogenesis. Compr Physiol, 2017, 8(1): 153-235.
32.	Borges E, Jan Y, Ruoslahti E. Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain. J Biol Chem, 2000, 275(51): 39867-39873.
33.	Dias DO, Kalkitsas J, Kelahmetoglu Y, et al. Pericyte-derived fibrotic scarring is conserved across diverse central nervous system lesions. Nat Commun, 2021, 12(1): 5501.
34.	Menezes K, Rosa BG, Freitas C, et al. Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats. Sci Rep, 2020, 10(1): 19604.
35.	Wallace RG, Rochfort KD, Barabas P, et al. COMP-Ang1: Therapeutic potential of an engineered Angiopoietin-1 variant. Vascul Pharmacol, 2021, 141: 106919.
36.	Lee SW, Kim WJ, Jun HO, et al. Angiopoietin-1 reduces vascular endothelial growth factor-induced brain endothelial permeability via upregulation of ZO-2. Int J Mol Med, 2009, 23(2): 279-284.
37.	Herrera JJ, Sundberg LM, Zentilin L, et al. Sustained expression of vascular endothelial growth factor and angiopoietin-1 improves blood-spinal cord barrier integrity and functional recovery after spinal cord injury. J Neurotrauma, 2010, 27(11): 2067-2076.
38.	Li Y, Zhou X, Sarkar B, et al. Recent progress on self-healable conducting polymers. Adv Mater, 2022, 34(24): e2108932.
39.	Kim J, Joshi HP, Sheen SH, et al. Resolvin D3 promotes inflammatory resolution, neuroprotection, and functional recovery after spinal cord injury. Mol Neurobiol, 2021, 58(1): 424-438.
40.	许子星, 许卫红, 陈薛敏, 等. 急性脊髓损伤大鼠血管重构与炎症反应的相关性研究. 中国修复重建外科杂志, 2020, 34(11): 1429-1437.

1. Li L, Mu J, Zhang Y, et al. Stimulation by exosomes from hypoxia preconditioned human umbilical vein endothelial cells facilitates mesenchymal stem cells angiogenic function for spinal cord repair. ACS Nano, 2022, 16(7): 10811-10823.
2. You Z, Gao X, Kang X, et al. Microvascular endothelial cells derived from spinal cord promote spinal cord injury repair. Bioact Mater, 2023, 29: 36-49.
3. Dray C, Rougon G, Debarbieux F. Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord. Proc Natl Acad Sci U S A, 2009, 106(23): 9459-9464.
4. Fedorova J, Kellerova E, Bimbova K, et al. The histopathology of severe graded compression in lower thoracic spinal cord segment of rat, evaluated at late post-injury phase. Cell Mol Neurobiol, 2022, 42(1): 173-193.
5. Figley SA, Khosravi R, Legasto JM, et al. Characterization of vascular disruption and blood-spinal cord barrier permeability following traumatic spinal cord injury. J Neurotrauma, 2014, 31(6): 541-552.
6. Cao Y, Wu T, Yuan Z, et al. Three-dimensional imaging of microvasculature in the rat spinal cord following injury. Sci Rep, 2015, 5: 12643.
7. Li Y, Lucas-Osma AM, Black S, et al. Pericytes impair capillary blood flow and motor function after chronic spinal cord injury. Nat Med, 2017, 23(6): 733-741.
8. Liu SM, Xiao ZF, Li X, et al. Vascular endothelial growth factor activates neural stem cells through epidermal growth factor receptor signal after spinal cord injury. CNS Neurosci Ther, 2019, 25(3): 375-385.
9. Tsai HH, Niu J, Munji R, et al. Oligodendrocyte precursors migrate along vasculature in the developing nervous system. Science, 2016, 351(6271): 379-384.
10. 陶经纬, 周婧雅, 赵毅, 等. 川芎嗪对脊髓损伤大鼠铁死亡的调控作用及机制. 中国组织工程研究, 2024, 28(26): 4158-4163.
11. 张厚君, 蒋昇源, 邓博文, 等. 川芎嗪改善脊髓损伤模型大鼠炎性微环境的机制. 中国组织工程研究, 2023, 27(11): 1701-1707.
12. 蒋昇源, 邓博文, 徐林, 等. 川芎嗪修复脊髓损伤的作用及机制. 中国组织工程研究, 2022, 26(11): 1799-1804.
13. Li G, Sng KS, Shu B, et al. Effects of tetramethylpyrazine treatment in a rat model of spinal cord injury: A systematic review and meta-analysis. Eur J Pharmacol, 2023, 945: 175524.
14. Li J, Wei J, Wan Y, et al. TAT-modified tetramethylpyrazine-loaded nanoparticles for targeted treatment of spinal cord injury. J Control Release, 2021, 335: 103-116.
15. Zhou L, Fan L, Yi X, et al. Soft conducting polymer hydrogels cross-linked and doped by tannic acid for spinal cord injury repair. ACS Nano, 2018, 12(11): 10957-10967.
16. Fan L, Liu C, Chen X, et al. Exosomes-loaded electroconductive hydrogel synergistically promotes tissue repair after spinal cord injury via immunoregulation and enhancement of myelinated axon growth. Adv Sci (Weinh), 2022, 9(13): e2105586.
17. 蒋昇源, 邓博文, 刘港, 等. 携载川芎嗪缓释微粒导电水凝胶修复脊髓损伤实验研究. 中国修复重建外科杂志, 2023, 37(1): 65-73.
18. 贺丰, 俞兴, 穆晓红, 等. 新型脊髓完全横断缺损模型大鼠的建立. 中国组织工程研究, 2016, 20(5): 635-639.
19. Rivlin AS, Tator CH. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. J Neurosurg, 1977, 47(4): 577-581.
20. Wang Y, Lv HQ, Chao X, et al. Multimodal therapy strategies based on hydrogels for the repair of spinal cord injury. Mil Med Res, 2022, 9(1): 16.
21. Fan L, Liu C, Chen X, et al. Directing induced pluripotent stem cell derived neural stem cell fate with a three-dimensional biomimetic hydrogel for spinal cord injury repair. ACS Appl Mater Interfaces, 2018, 10(21): 17742-17755.
22. Sun X, Liu H, Tan Z, et al. Remodeling microenvironment for endogenous repair through precise modulation of chondroitin sulfate proteoglycans following spinal cord injury. Small, 2023, 19(6): e2205012.
23. Ruschel J, Hellal F, Flynn KC, et al. Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury. Science, 2015, 348(6232): 347-352.
24. Hu X, Xu W, Ren Y, et al. Spinal cord injury: molecular mechanisms and therapeutic interventions. Signal Transduct Target Ther, 2023, 8(1): 245.
25. Wheaton BJ, Sena J, Sundararajan A, et al. Identification of regenerative processes in neonatal spinal cord injury in the opossum (Monodelphis domestica): A transcriptomic study. J Comp Neurol, 2021, 529(5): 969-986.
26. Liu D, Huang Y, Jia C, et al. Administration of antagomir-223 inhibits apoptosis, promotes angiogenesis and functional recovery in rats with spinal cord injury. Cell Mol Neurobiol, 2015, 35(4): 483-491.
27. Ouellette J, Lacoste B. From neurodevelopmental to neurodegenerative disorders: The vascular continuum. Front Aging Neurosci, 2021, 13: 749026.
28. Simons M, Gordon E, Claesson-Welsh L. Mechanisms and regulation of endothelial VEGF receptor signalling. Nat Rev Mol Cell Biol, 2016, 17(10): 611-625.
29. Ruhrberg C, Gerhardt H, Golding M, et al. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev, 2002, 16(20): 2684-2698.
30. Duan YY, Chai Y, Zhang NL, et al. Microtubule stabilization promotes microcirculation reconstruction after spinal cord injury. J Mol Neurosci, 2021, 71(3): 583-595.
31. Duran CL, Howell DW, Dave JM, et al. Molecular regulation of sprouting angiogenesis. Compr Physiol, 2017, 8(1): 153-235.
32. Borges E, Jan Y, Ruoslahti E. Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain. J Biol Chem, 2000, 275(51): 39867-39873.
33. Dias DO, Kalkitsas J, Kelahmetoglu Y, et al. Pericyte-derived fibrotic scarring is conserved across diverse central nervous system lesions. Nat Commun, 2021, 12(1): 5501.
34. Menezes K, Rosa BG, Freitas C, et al. Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats. Sci Rep, 2020, 10(1): 19604.
35. Wallace RG, Rochfort KD, Barabas P, et al. COMP-Ang1: Therapeutic potential of an engineered Angiopoietin-1 variant. Vascul Pharmacol, 2021, 141: 106919.
36. Lee SW, Kim WJ, Jun HO, et al. Angiopoietin-1 reduces vascular endothelial growth factor-induced brain endothelial permeability via upregulation of ZO-2. Int J Mol Med, 2009, 23(2): 279-284.
37. Herrera JJ, Sundberg LM, Zentilin L, et al. Sustained expression of vascular endothelial growth factor and angiopoietin-1 improves blood-spinal cord barrier integrity and functional recovery after spinal cord injury. J Neurotrauma, 2010, 27(11): 2067-2076.
38. Li Y, Zhou X, Sarkar B, et al. Recent progress on self-healable conducting polymers. Adv Mater, 2022, 34(24): e2108932.
39. Kim J, Joshi HP, Sheen SH, et al. Resolvin D3 promotes inflammatory resolution, neuroprotection, and functional recovery after spinal cord injury. Mol Neurobiol, 2021, 58(1): 424-438.
40. 许子星, 许卫红, 陈薛敏, 等. 急性脊髓损伤大鼠血管重构与炎症反应的相关性研究. 中国修复重建外科杂志, 2020, 34(11): 1429-1437.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study of tetramethylpyrazine-loaded electroconductive hydrogel on angiogenesis and neuroprotection after spinal cord injury

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