TRANSPLANTATION OF NEURAL STEM CELLS INDUCED BY ALL-TRANS-RETINOIC ACID COMBINED WITH GLIAL CELL LINE DERIVED NEUROTROPHIC FACTOR AND CHONDROITINASE ABC FOR REPAIRING SPINAL CORD INJURY OF RATS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIAOYehui ¹ ,  ZHONGDejun ¹ , KANGMin ² , YAOShuaihui ¹ , ZHANGYi ¹ , YUYongtao ¹

1. Department of Spinal Surgery Affiliated Hospital of Luzhou Medical College, Luzhou Sichuan, 646000, P. R. China;
2. Department of Gastroenterology, Affiliated Hospital of Luzhou Medical College, Luzhou Sichuan, 646000, P. R. China;

Corresponding author：

ZHONGDejun, Email: zdj_1974@163.com

Keywords：

Neural stem cells; Glial cell line derived neurotrophic factor; Chondroitinase ABC; Spinal cord injury; Rat

DOI：

10.7507/1002-1892.20150216

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Objective To observe the effect of transplantation of neural stem cells (NSCs) induced by all-trans-retinoic acid (ATRA) combined with glial cell line derived neurotrophic factor (GDNF) and chondroitinase ABC (ChABC) on the neurological functional recovery of injured spinal cord in Sprague Dawley (SD) rats. Methods Sixty adult SD female rats, weighing 200-250 g, were randomly divided into 5 groups (n=12): sham operation group (group A), SCI model group (group B), NSCs+GDNF treatment group (group C), NSCs+ChABC treatment group (group D), and NSCs+GDNF+ChABC treatment group (group E). T10 segmental transversal injury model of the spinal cord was established except group A. NSCs induced by ATRA and marked with BrdU were injected into the site of injury at 8 days after operation in groups C-E. Groups C-E were treated with GDNF, ChABC, and GDNF+ChABC respectively at 8-14 days after operation;and group A and B were treated with the same amount of saline solution. Basso Beattie Bresnahan (BBB) score and somatosensory evoked potentials (SEP) test were used to study the functional improvement at 1 day before remodeling, 7 days after remodeling, and at 1, 2, 5, and 8 weeks after transplantation. Immunofluorescence staining and HE staining were performed to observe the cells survival and differentiation in the spinal cord. Results Five mouse died but another rats were added. At each time point after modeling, BBB score of groups B, C, D, and E was significantly lower than that of group A, and SEP latent period was significantly longer than that of group A (P<0.05), but no difference was found among groups B, C, D, and E at 7 days after remodeling and 1 week after transplantation (P>0.05). BBB score of groups C, D, and E was significantly higher than that of group B, and SEP latent period was significantly shorter than that of group B at 2, 5, and 8 weeks after transplantation (P<0.05);group E had higher BBB score and shorter SEP latent period than groups C and D at 5 and 8 weeks, showing significant difference (P<0.05). HE staining showed that there was a clear boundary between gray and white matter of spinal cord and regular arrangement of cells in group A;there were incomplete vascular morphology, irregular arrangement of cells, scar, and cysts in group B;there were obvious cell hyperplasia and smaller cysts in groups C, D, and E. BrdU positive cells were not observed in groups A and B, but could be found in groups C, D and E. Group E had more positive cells than groups C and D, and difference was significant (P<0.05). The number of glial fibrillary acidic protein positive cells of groups C, D, and E was significantly less than that of groups A and B, and it was significantly less in group E than groups C and D (P<0.05). The number of microtubule-associated protein 2 positive cells of groups C, D, and E was significantly more than that of groups A and B, and it was significantly more in group E than groups C and D (P<0.05). Conclusion The NSCs transplantation combined with GDNF and ChABC could significantly promote the functional recovery of spinal cord injury, suggesting that GDNF and ChABC have a synergistic effect in the treatment of spinal cord injury.

Citation： LIAOYehui, ZHONGDejun, KANGMin, YAOShuaihui, ZHANGYi, YUYongtao. TRANSPLANTATION OF NEURAL STEM CELLS INDUCED BY ALL-TRANS-RETINOIC ACID COMBINED WITH GLIAL CELL LINE DERIVED NEUROTROPHIC FACTOR AND CHONDROITINASE ABC FOR REPAIRING SPINAL CORD INJURY OF RATS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(8): 1009-1015. doi: 10.7507/1002-1892.20150216 Copy

1.	Vroemen M,Aigner L,Winkler J,et al.Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways.Eur J Neurosci,2003,18(4):743-751.
2.	Salewski RP,Mitchell RA,Shen C,et al.Transplantation of neural stem cells clonally derived from embryonic stem cells promotes recovery after murine spinal cord injury.Stem Cells Dev,2015,24(1):36-50.
3.	Yao ZG,Sun XL,Li P,et al.Neural stem cells transplantation alleviate the hyperalgesia of spinal cord injured (SCI) associated with down-regulation of BDNF.Int J Clin Exp Med,2015,8(1):404-412.
4.	Hashimoto M,Nitta A,Fukumitsu H,et al.Inflammation-induced GDNF improves locomotor function after spinal cord injury.Neuroreport,2005,16(2):99-102.
5.	Hashimoto M,Ito T,Fukumitsu H,et al.Stimulation of production of glial cell line-derived neurotrophic factor and nitric oxide by lipopolysaccharide with different dose-responsiveness in cultured rat macrophages.Biomed Res,2005,26(5):223-229.
6.	Bunge MB.Novel combination strategies to repair the injured mammalian spinal cord.J Spinal Cord Med,2008,31(3):262-269.
7.	Zhang L,Ma Z,Smith GM,et al.GDNF-enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons.Glia,2009,57(11):1178-1191.
8.	Nielsen J,Gotfryd K,Li S,et al.Role of glial cell line-derived neurotrophic factor (GDNF)-neural cell adhesion molecule (NCAM) interactions in induction of neurite outgrowth and identification of a binding site for NCAM in the heel region of GDNF.J Neurosci,2009,29(36):11360-11376.
9.	Paratcha G,Ledda F,Ibáñez CF.The neural cell adhesion molecule NCAM is an alternative signaling receptor for GDNF family ligands.Cell,2003,113(7):867-879.
10.	Siebert JR,Steencken AC,Osterhout DJ.Chondroitin sulfate proteoglycans in the nervous system:inhibitors to repair.Biomed Res Int,2014,2014:845323.
11.	Zhang C,He X,Li H,et al.Chondroitinase ABC plus bone marrow mesenchymal stem cells for repair of spinal cord injury.Neural Regen Res,2013,8(11):965-974.
12.	Starkey ML,Bartus K,Barritt AW,et al.Chondroitinase ABC promotes compensatory sprouting of the intact corticospinal tract and recovery of forelimb function following unilateral pyramidotomy in adult mice.Eur J Neurosci,2012,36(12):3665-3678.
13.	钟德君,张德盛,宋跃明.全反式维甲酸对鼠胚神经干细胞增殖和分化的作用.中国修复重建外科杂志,2008,22(2):206-211.
14.	姚帅辉,钟德君,康敏,等.ATRA干预培养鼠胚神经干细胞联合GDNF移植对脊髓损伤再生修复的作用.山东医药,2014,54(22):29-31.
15.	Basso DM,Beattie MS,Bresnahan JC.A sensitive and reliable locomotor rating scale for open field testing in rats.J Neurotrauma,1995,12(1):1-21.
16.	钟德君,张德盛,宋跃明.鼠胚神经干细胞体外培养方法的优化.中国组织工程研究与临床康复,2007,11(50):10088-10092.
17.	Ricci-Vitiani L,Casalbore P,Petrucci G,et al.Influence of local environment on the differentiation of neural stem cells engrafted onto the injured spinal cord.Neurol Res,2006,28(5):488-492.
18.	Yasuda A,Tsuji O,Shibata S,et al.Significance of remyelination by neural stem/progenitor cells transplanted into the injured spinal cord.Stem Cells,2011,29(12):1983-1994.
19.	Holstege JC,Jongen JL,Kennis JH,et al.Immunocytochemical localization of GDNF in primary afferents of the lumber dorsal horn.Neuroreport,1998,9(12):2893-2897.
20.	Abdanipour A,Tiraihi T,Taheri T.Intraspinal transplantation of motoneuron-like cell combined with delivery of polymer-based glial cell line-derived neurotrophic factor for repair of spinal cord contusion injury.Neural Regen Res,2014,9(10):1003-1013.
21.	Soleman S,Yip PK,Duricki DA,et al.Delayed treatment with chondroitinase ABC promotes sensorimotor recovery and plasticity after stroke in aged rats.Brain,2012,135(Pt 4):1210-1223.
22.	Hill JJ,Jin K,Mao XO,et al.Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats.Proc Natl Acad Sci U S A,2012,109(23):9155-9160.
23.	Walker BA,Ji SJ,Jaffrey SR.Intra-axonal translation of RhoA promotes axon growth inhibition by CSPG.J Neurosci,2012,32(41):14442-14447.
24.	Crespo D,Asher RA,Lin R,et al.How does chondroitinase promote functional recovery in the damaged CNS? Exp Neurol,2007,206(2):159-171.
25.	Wang D,Ichiyama RM,Zhao R,et al.Chondroitinase combined with rehabilitation promotes recovery of forelimb function in rats with chronic spinal cord injury.J Neurosci,2011,31(25):9332-9344.

1. Vroemen M,Aigner L,Winkler J,et al.Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways.Eur J Neurosci,2003,18(4):743-751.
2. Salewski RP,Mitchell RA,Shen C,et al.Transplantation of neural stem cells clonally derived from embryonic stem cells promotes recovery after murine spinal cord injury.Stem Cells Dev,2015,24(1):36-50.
3. Yao ZG,Sun XL,Li P,et al.Neural stem cells transplantation alleviate the hyperalgesia of spinal cord injured (SCI) associated with down-regulation of BDNF.Int J Clin Exp Med,2015,8(1):404-412.
4. Hashimoto M,Nitta A,Fukumitsu H,et al.Inflammation-induced GDNF improves locomotor function after spinal cord injury.Neuroreport,2005,16(2):99-102.
5. Hashimoto M,Ito T,Fukumitsu H,et al.Stimulation of production of glial cell line-derived neurotrophic factor and nitric oxide by lipopolysaccharide with different dose-responsiveness in cultured rat macrophages.Biomed Res,2005,26(5):223-229.
6. Bunge MB.Novel combination strategies to repair the injured mammalian spinal cord.J Spinal Cord Med,2008,31(3):262-269.
7. Zhang L,Ma Z,Smith GM,et al.GDNF-enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons.Glia,2009,57(11):1178-1191.
8. Nielsen J,Gotfryd K,Li S,et al.Role of glial cell line-derived neurotrophic factor (GDNF)-neural cell adhesion molecule (NCAM) interactions in induction of neurite outgrowth and identification of a binding site for NCAM in the heel region of GDNF.J Neurosci,2009,29(36):11360-11376.
9. Paratcha G,Ledda F,Ibáñez CF.The neural cell adhesion molecule NCAM is an alternative signaling receptor for GDNF family ligands.Cell,2003,113(7):867-879.
10. Siebert JR,Steencken AC,Osterhout DJ.Chondroitin sulfate proteoglycans in the nervous system:inhibitors to repair.Biomed Res Int,2014,2014:845323.
11. Zhang C,He X,Li H,et al.Chondroitinase ABC plus bone marrow mesenchymal stem cells for repair of spinal cord injury.Neural Regen Res,2013,8(11):965-974.
12. Starkey ML,Bartus K,Barritt AW,et al.Chondroitinase ABC promotes compensatory sprouting of the intact corticospinal tract and recovery of forelimb function following unilateral pyramidotomy in adult mice.Eur J Neurosci,2012,36(12):3665-3678.
13. 钟德君,张德盛,宋跃明.全反式维甲酸对鼠胚神经干细胞增殖和分化的作用.中国修复重建外科杂志,2008,22(2):206-211.
14. 姚帅辉,钟德君,康敏,等.ATRA干预培养鼠胚神经干细胞联合GDNF移植对脊髓损伤再生修复的作用.山东医药,2014,54(22):29-31.
15. Basso DM,Beattie MS,Bresnahan JC.A sensitive and reliable locomotor rating scale for open field testing in rats.J Neurotrauma,1995,12(1):1-21.
16. 钟德君,张德盛,宋跃明.鼠胚神经干细胞体外培养方法的优化.中国组织工程研究与临床康复,2007,11(50):10088-10092.
17. Ricci-Vitiani L,Casalbore P,Petrucci G,et al.Influence of local environment on the differentiation of neural stem cells engrafted onto the injured spinal cord.Neurol Res,2006,28(5):488-492.
18. Yasuda A,Tsuji O,Shibata S,et al.Significance of remyelination by neural stem/progenitor cells transplanted into the injured spinal cord.Stem Cells,2011,29(12):1983-1994.
19. Holstege JC,Jongen JL,Kennis JH,et al.Immunocytochemical localization of GDNF in primary afferents of the lumber dorsal horn.Neuroreport,1998,9(12):2893-2897.
20. Abdanipour A,Tiraihi T,Taheri T.Intraspinal transplantation of motoneuron-like cell combined with delivery of polymer-based glial cell line-derived neurotrophic factor for repair of spinal cord contusion injury.Neural Regen Res,2014,9(10):1003-1013.
21. Soleman S,Yip PK,Duricki DA,et al.Delayed treatment with chondroitinase ABC promotes sensorimotor recovery and plasticity after stroke in aged rats.Brain,2012,135(Pt 4):1210-1223.
22. Hill JJ,Jin K,Mao XO,et al.Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats.Proc Natl Acad Sci U S A,2012,109(23):9155-9160.
23. Walker BA,Ji SJ,Jaffrey SR.Intra-axonal translation of RhoA promotes axon growth inhibition by CSPG.J Neurosci,2012,32(41):14442-14447.
24. Crespo D,Asher RA,Lin R,et al.How does chondroitinase promote functional recovery in the damaged CNS? Exp Neurol,2007,206(2):159-171.
25. Wang D,Ichiyama RM,Zhao R,et al.Chondroitinase combined with rehabilitation promotes recovery of forelimb function in rats with chronic spinal cord injury.J Neurosci,2011,31(25):9332-9344.

Chinese Journal of Reparative and Reconstructive Surgery

TRANSPLANTATION OF NEURAL STEM CELLS INDUCED BY ALL-TRANS-RETINOIC ACID COMBINED WITH GLIAL CELL LINE DERIVED NEUROTROPHIC FACTOR AND CHONDROITINASE ABC FOR REPAIRING SPINAL CORD INJURY OF RATS

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