EFFECTS OF BONE MARROW MESENCHYMAL STEM CELLS TRANSPLANTATION FOR TREATING RAT SPINAL CORD INJURY AND CYTOKINE EXPRESSION AT INJURY SITES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

MOCuiping ¹ , RENLijie ^2△ , ZHAOZhenfu ¹ , ZHOUGuangqian ¹ , YAOXiaolu ³ , GONGFeipeng ³ ,  CHENGang ³

1. Key Laboratory of Anti-aging and Regenerative Medicine, Medicine School, Shenzhen University, Shenzhen Guangdong, 518054, P.R.China;
2. Department of Neurology, Shenzhen No.2 People's Hospital, the First Affiliated Hospital of Shenzhen University;
3. No.1 Department of Orthopaedics, Jiangxi Provincial People's Hospital;

Corresponding author：

CHENGang, Email: doctorchengang@163.com

Keywords：

Spinal cord injury; Bone marrow mesenchymal stem cells; Cytokine; Neural regeneration; Rat

DOI：

10.7507/1002-1892.20160054

Video：

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Objective To investigate the effects of bone marrow mesenchymal stem cells (BMSCs) transplantation for treating spinal cord injury (SCI) in rat and the cytokine expression changes in the local injury tissues. Methods BMSCs were separated from Sprague Dawley (SD) rat and cultured with the whole bone marrow culture method. rAd-EGFP was used to transfect the 5th generation BMSCs for green fluorescent protein (GFP) label. Twelve SD rats were randomly divided into experimental group (n=6) and control group (n=6). After the T₁₀ SCI model was established with Allen's impact device in 2 groups, 1×10⁶ GFP-labeled BMSCs and PBS were administered by subarachnoid injection in situ in experimental group and control group, respectively. Basso-Beattie-Bresnahan (BBB) score was used to detect the motor function at immediat, 1, 2, 3, 4, and 5 weeks after SCI. At 5 weeks, the spinal cord tissues were harvested for the histological and immunofluorescent staining examinations to measure the expressions of neural marker molecules, including Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific nuclear protein (NeuN). Cytokine was analyzed with antibody array. Results At 5 weeks, 2 rats died of urinary tract infection in 2 groups respectively, the other rats survived to the end of experiment. BBB score of experimental group was significantly higher than that of control group at 1, 2, 3, 4, and 5 weeks (P < 0.05). At 5 weeks, histological results showed that there were many cells with regular arrangement in the experimental group; there were less cells with irregular arrangement in the control group. Compared with the control group, Nestin and NeuN expressions significantly increased (P < 0.05), and GFAP expression significantly decreased (P < 0.05) in the experimental group. Leptin and ciliary neurotrophic factor levels were higher in the experimental group than the control group, but granulocyte-macrophage colony-stimulating factor, tumor necrosis factorα, interleukin 1β, and tissue inhibitor of metalloproteinases 1 levels were lower in the experimental group than the control group. Conclusion BMSCs transplantation can improve survival and regeneration of nerve cells and enhances the recovery of nerve function by regulating secretion of cytokines from grafted BMSCs.

Citation： MOCuiping, RENLijie, ZHAOZhenfu, ZHOUGuangqian, YAOXiaolu, GONGFeipeng, CHENGang. EFFECTS OF BONE MARROW MESENCHYMAL STEM CELLS TRANSPLANTATION FOR TREATING RAT SPINAL CORD INJURY AND CYTOKINE EXPRESSION AT INJURY SITES. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(3): 265-271. doi: 10.7507/1002-1892.20160054 Copy

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2.	Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg, 1991, 75(1): 15-26.
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6.	Sjöberg G, Jiang WQ, Ringertz NR, et al. Colocalization of nestin and vimentin/desmln in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging mieroseopy. Exp Cell Res, 1994, 214(2): 447-458.
7.	Maslov AY, Barone TA, Plunkett RJ, et al. Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J Neurosci, 2004, 24(7): 1726-1733.
8.	Wolf HK, Buslei R, Schmidt-Kastner R, et al. NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem, 1996, 44(10): 1167-1171.
9.	Magistretti PJ, Pellerin L, Rothman DL, et al. Energy on Demand. Science, 1999, 283(5401): 496-497.
10.	Doetsch F, Caillé I, Lim DA, et al. Subventricular zone astroeytes are neural stem cells in the adult manunalian brain. Cell, 1999, 97(6): 703-716.
11.	Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci, 2009, 32(12): 638-647.
12.	Nagamoto-Combs K, Morecraft RJ, Darling WG, et al. Long-term gliosis and molecular changes in the cervical spinal cord of the rhesus monkey after traumatic brain injury. J Neurotrauma, 2010, 27(3): 565-585.
13.	Li ZW, Tang RH, Zhang JP, et al. Inhibiting epidermal growth factor receptor attenuates reactive astrogliosis and improves functional outcome after spinal cord injury in rats. Neurochem Int, 2011, 58(7): 812-819.
14.	苏国辉.促进中枢神经损伤再生的探索之路.中国处方药, 2004, (2): 31-33.
15.	Glenn Y, Zhigang H. Glial inhibition of CNS axon regeneration. Nature Reviews Neuroscience, 2006, 7: 617-627.
16.	Lin J, Yan CT, Wang LH, et al. Leptin fluctuates in intestinal ischemia-reperfusion injury as inflammatory cytokine. Peptides, 2004, 25(12): 2187-2193.
17.	段世博, 闫华, 刘睽, 等.瘦素与创伤.中华创伤杂志, 2011, 27(2): 187-189.
18.	Forger NG, Wong V, Breedlove SM. Ciliary neurotrophic factor arrests muscle and motoneuron degeneration in androgen-insensitive rats. J Neurobio, 1995, 28(3): 354-362.
19.	谢步章.胸腰椎手术后脑脊液漏的防治.山西医药杂志, 2008, 37(2): 132-133.
20.	Lee YL, Shih K, Bao P, et al. Cytokine chemokine expression in contused rat spinal cord. Neurochem Int, 2000, 36(4-5): 417-425.

1. Fehlings MG, Vawda R. Cellular treatments for spinal cord injury: the time is right for clinical trials. Neurotherapeutics, 2011, 8(4): 704-720.
2. Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg, 1991, 75(1): 15-26.
3. Figley SA, Austin JW, Rowland JW, et al. Pathophysiology of spinal cord injury//The Cervical Spinal. 5th eds. New York: Lippincott Williams & Wilkins, 2011: 372-379.
4. Stachowiak MK, Mofett J, Maher P, et al. Growth factor regulation of cell growth and proliferation in the nervous system. A new intracrinmuclear mechanism. Mol Neurobiol, 1997, 15(3): 257-283.
5. Ciccolim F, Svendsen CN. Fibroblast growth factor 2 (FGF-2) promotes acquisition of epidermal growth factor (EGF) responsiveness in mouse striatal precursor cell: identification of neural precursors responding to both EGF and FGF-2. J Neurosci, 1998, 18(19): 7869-7880.
6. Sjöberg G, Jiang WQ, Ringertz NR, et al. Colocalization of nestin and vimentin/desmln in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging mieroseopy. Exp Cell Res, 1994, 214(2): 447-458.
7. Maslov AY, Barone TA, Plunkett RJ, et al. Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J Neurosci, 2004, 24(7): 1726-1733.
8. Wolf HK, Buslei R, Schmidt-Kastner R, et al. NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem, 1996, 44(10): 1167-1171.
9. Magistretti PJ, Pellerin L, Rothman DL, et al. Energy on Demand. Science, 1999, 283(5401): 496-497.
10. Doetsch F, Caillé I, Lim DA, et al. Subventricular zone astroeytes are neural stem cells in the adult manunalian brain. Cell, 1999, 97(6): 703-716.
11. Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci, 2009, 32(12): 638-647.
12. Nagamoto-Combs K, Morecraft RJ, Darling WG, et al. Long-term gliosis and molecular changes in the cervical spinal cord of the rhesus monkey after traumatic brain injury. J Neurotrauma, 2010, 27(3): 565-585.
13. Li ZW, Tang RH, Zhang JP, et al. Inhibiting epidermal growth factor receptor attenuates reactive astrogliosis and improves functional outcome after spinal cord injury in rats. Neurochem Int, 2011, 58(7): 812-819.
14. 苏国辉.促进中枢神经损伤再生的探索之路.中国处方药, 2004, (2): 31-33.
15. Glenn Y, Zhigang H. Glial inhibition of CNS axon regeneration. Nature Reviews Neuroscience, 2006, 7: 617-627.
16. Lin J, Yan CT, Wang LH, et al. Leptin fluctuates in intestinal ischemia-reperfusion injury as inflammatory cytokine. Peptides, 2004, 25(12): 2187-2193.
17. 段世博, 闫华, 刘睽, 等.瘦素与创伤.中华创伤杂志, 2011, 27(2): 187-189.
18. Forger NG, Wong V, Breedlove SM. Ciliary neurotrophic factor arrests muscle and motoneuron degeneration in androgen-insensitive rats. J Neurobio, 1995, 28(3): 354-362.
19. 谢步章.胸腰椎手术后脑脊液漏的防治.山西医药杂志, 2008, 37(2): 132-133.
20. Lee YL, Shih K, Bao P, et al. Cytokine chemokine expression in contused rat spinal cord. Neurochem Int, 2000, 36(4-5): 417-425.

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Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF BONE MARROW MESENCHYMAL STEM CELLS TRANSPLANTATION FOR TREATING RAT SPINAL CORD INJURY AND CYTOKINE EXPRESSION AT INJURY SITES

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