Effects of Ch ABC, NEP1-40 combined with neural stem cell transplantation on the recovery of neurological function in rats with spinal cord injurey
Author(s): YU Yongtao, LIAO Yehui, TANG Qiang, Department of Spinal Surgery, Affiliated Hospital of Sichuan Medical university
Pages: 527-536
Year: 2016 Issue: 6
Journal: Chinese Journal of Spine and Spinal Cord
Keyword: Spinal cord injury; ATRA; Neural stem cells; Ch ABC; NEP1-40; Cell transplantation; Rat;
Abstract: Objectives: To investigate the effects of combined transplantation of chondroitinase ABC(Ch ABC),Nogo-66(1-40) antagonist peptide(NEP1-40) and rat embryonic neural stem cells(NSCs) on the recovery of neurological function in rats with spinal cord injury. Methods: The NSCs cryopreserved in our preliminary experiment were cultured and induced by all-trans-retinoic acid(ATRA)(the concentration of 1.0 μmol/L) in vitro, the immunofluorescence techniques were used to detect the expressions of NSCs specific markers of glial fibrillary acidic protein(GFAP) and neuron-specific enolase(NSE). The NSCs were marked by 5-Bromo-2-deoxy Uridine(Brd U) before implantation. 60 adult Sprague-Daw female rats were randomly divided into six groups, namely, the sham operation group(group A, n=10), the injury control group(group B, n=10), the NSCs treatment group(group C, n=10), the NSCs combined with Ch ABC treatment group(group D, n=10), the NSCs combined with NEP1-40 treatment group(group E, n=10), and NSCs combined with Ch ABC and NEP1-40 treatment group(group F, n=10). Complete thoracic spinal cord transected injury models of rats were performed in all rats except group A before transplantation. On the third day after surgery, group E and group F were injected with NEP1-40 for 20μl/d through the indwelling catheter for 28 days consecutively; on the eighth day after surgery, group C, D, E and F were injected with 10μl of NSCs that were intervened with ATRA and labeled with Brd U through the indwelling catheter; and on the eighth day after surgery, group D and F were injected with Ch ABC for 10μl/d through the indwelling catheter for 7 days consecutively. Equal quantity of transplant liquid was kept through injection with normal saline through the indwelling catheter at all time points. Basso, Beattie, Bresnahan(BBB) score and somatosensory evoked potential(SEP) test were used to evaluate the functional changes preoperatively, postoperatively as well as 2 weeks, 5 weeks and 8 weeks after transplantation, respectively. The injured spinal cords were sampled for observing 8 weeks after transplantation and HE staining were performed to observe the morphological alteration in the injured spinal cord. The number, distribution and differentiation of transplanted NSCs were examined by double immunofluorescence staining, by using microtubule-associated protein 2(MAP-2) and Brd U. Results: ATRA could remarkably promote the differentiation of NSCs into neurons. 2 weeks after operation, the recovery of the BBB score and the SEP latent period were observed in SCI groups, and the recovery of all transplanted treatment groups was better than that of SCI model group. The BBB score and SEP latent period showed significant difference at any observing point after transplantation(P<0.05). BBB score of group B at each time point was significantly lower than that of group C, D, E and F, and SEP latent period was significantly longer than that of group C,D, E and F(P<0.05); the outcome of neurological functional recovery in group F remarkably surpassed than that of group C, D and E among all post-transplantation observing time points, and group F had higher BBB score and shorter SEP latent period than group C, D, E(P<0.05). 8 weeks after transplantation, HE staining in group A showed complete morphological structure and regular arrangement of cells; while the morphological structure was seriously damaged in group B, where irregular arrangement of cells as well as a large number of scar and cysts could be seen; and there were obvious cells and capillary hyperplasia and a few small cysts in group C, D, E, and group F in particular. The viable positive cells labeled by Brd U could only be observed in group C, D, E and F, and the number of Brdu-positive cells in group F were greater than that of group C, D and E(P<0.05), with the difference being statistically significant. Using Fluorescence microscope,the double positive cells for MAP-2 and Brd U could be detected in group C, D, E and F. The axonal regeneration and growth was distinctly promoted in group F. The number and surface area of positive cells labeled by MAP-2 showed significant difference(P<0.05), and group F had greater number of cells and larger surface area when compared with group A, B, C, D and E(P<0.05). Conclusions: The transplantation of NSCs induced by ATRA can improve the functional recovery of the SCI in rats, moreover, combined chondroitinase ABC and NEP1-40 has a synergistic effect on the recovery of neurological function in rats with spinal cord injury.
Pages: 527-536
Year: 2016 Issue: 6
Journal: Chinese Journal of Spine and Spinal Cord
Keyword: Spinal cord injury; ATRA; Neural stem cells; Ch ABC; NEP1-40; Cell transplantation; Rat;
Abstract: Objectives: To investigate the effects of combined transplantation of chondroitinase ABC(Ch ABC),Nogo-66(1-40) antagonist peptide(NEP1-40) and rat embryonic neural stem cells(NSCs) on the recovery of neurological function in rats with spinal cord injury. Methods: The NSCs cryopreserved in our preliminary experiment were cultured and induced by all-trans-retinoic acid(ATRA)(the concentration of 1.0 μmol/L) in vitro, the immunofluorescence techniques were used to detect the expressions of NSCs specific markers of glial fibrillary acidic protein(GFAP) and neuron-specific enolase(NSE). The NSCs were marked by 5-Bromo-2-deoxy Uridine(Brd U) before implantation. 60 adult Sprague-Daw female rats were randomly divided into six groups, namely, the sham operation group(group A, n=10), the injury control group(group B, n=10), the NSCs treatment group(group C, n=10), the NSCs combined with Ch ABC treatment group(group D, n=10), the NSCs combined with NEP1-40 treatment group(group E, n=10), and NSCs combined with Ch ABC and NEP1-40 treatment group(group F, n=10). Complete thoracic spinal cord transected injury models of rats were performed in all rats except group A before transplantation. On the third day after surgery, group E and group F were injected with NEP1-40 for 20μl/d through the indwelling catheter for 28 days consecutively; on the eighth day after surgery, group C, D, E and F were injected with 10μl of NSCs that were intervened with ATRA and labeled with Brd U through the indwelling catheter; and on the eighth day after surgery, group D and F were injected with Ch ABC for 10μl/d through the indwelling catheter for 7 days consecutively. Equal quantity of transplant liquid was kept through injection with normal saline through the indwelling catheter at all time points. Basso, Beattie, Bresnahan(BBB) score and somatosensory evoked potential(SEP) test were used to evaluate the functional changes preoperatively, postoperatively as well as 2 weeks, 5 weeks and 8 weeks after transplantation, respectively. The injured spinal cords were sampled for observing 8 weeks after transplantation and HE staining were performed to observe the morphological alteration in the injured spinal cord. The number, distribution and differentiation of transplanted NSCs were examined by double immunofluorescence staining, by using microtubule-associated protein 2(MAP-2) and Brd U. Results: ATRA could remarkably promote the differentiation of NSCs into neurons. 2 weeks after operation, the recovery of the BBB score and the SEP latent period were observed in SCI groups, and the recovery of all transplanted treatment groups was better than that of SCI model group. The BBB score and SEP latent period showed significant difference at any observing point after transplantation(P<0.05). BBB score of group B at each time point was significantly lower than that of group C, D, E and F, and SEP latent period was significantly longer than that of group C,D, E and F(P<0.05); the outcome of neurological functional recovery in group F remarkably surpassed than that of group C, D and E among all post-transplantation observing time points, and group F had higher BBB score and shorter SEP latent period than group C, D, E(P<0.05). 8 weeks after transplantation, HE staining in group A showed complete morphological structure and regular arrangement of cells; while the morphological structure was seriously damaged in group B, where irregular arrangement of cells as well as a large number of scar and cysts could be seen; and there were obvious cells and capillary hyperplasia and a few small cysts in group C, D, E, and group F in particular. The viable positive cells labeled by Brd U could only be observed in group C, D, E and F, and the number of Brdu-positive cells in group F were greater than that of group C, D and E(P<0.05), with the difference being statistically significant. Using Fluorescence microscope,the double positive cells for MAP-2 and Brd U could be detected in group C, D, E and F. The axonal regeneration and growth was distinctly promoted in group F. The number and surface area of positive cells labeled by MAP-2 showed significant difference(P<0.05), and group F had greater number of cells and larger surface area when compared with group A, B, C, D and E(P<0.05). Conclusions: The transplantation of NSCs induced by ATRA can improve the functional recovery of the SCI in rats, moreover, combined chondroitinase ABC and NEP1-40 has a synergistic effect on the recovery of neurological function in rats with spinal cord injury.
Related Articles