| objective:The effects of neural stem cell?NSC?transplantation on repair in spinal cord injury are not ideal.All-trans-retinoic acid?ATRA?can promote nerve regeneration and repair,and has been observed to act as a mediator of inflammation-induced sensitization.Its potential therapeutic use in spinal cord injury is therefore of great interest,but its effects on neuroinflammation need to be identified.Previous studies have confirmed that the differentiation ratio of neurons from NSCs in vitro increases with ATRA intervention.Nerve growth factor?NGF?is one of the most important nerve growth regulators in the nervous system,and it is characterized by its dual biological functions of facilitating neuron nutrition and protruding growth.It has important clinical significance for the development,differentiation,growth,and expression of functional properties of neurons,as well as the regeneration and repair of injured nerves.In this study,we observed the effects of transplanting NSCs induced by ATRA combined with NGF on the neurological functional recovery of injured spinal cords in Sprague Dawley?SD?rats.Methods:?1?Frozen mouse embryonic neural stem cells,which had been extracted by our research group at the early stage,were resuscitated.Then,DMEM/F12?1:1?complete culture medium was added to adjust the cell density.The cells were inoculated in a culture bottle and placed in a C02 saturated humidity and constant temperature incubator for subculture.The third generation of NSCs were divided into two batches,one was induced in culture medium containing ATRA for 7 days,and the other was given normal culture without ATRA induction for 7 days.The expression of Nestin,NSE and GFAP antibodies were identified by immunofluorescence staining.?2?Female SD rats?n=72,age,10 weeks old,weight,200–250 g?were provided,12 SD rats for each group were used for the sham-operation group,injury control group,non-treated NSCs transplant group,NGF-only group,and transplant ATRA groups that either did or did not receive NGF injection.A complete spinal cord injury model was established by intraperitoneal injection of 2%pentobarbital sodium(30 mg kg-1)given to SD rats in each group.A median incision in the back was made with the T10–T12 spinous process as the center,followed by dissection of the bilateral paravertebral muscles.Rats in the sham operation group underwent laminectomy.Surgical transplantation was performed again 7days after the establishment of the complete spinal cord transection model.NSCs were divided into two groups,one that received ATRA treatment and the other that did not.BrdU was used to mark ATRA-treated NSCs 1 day before transplantation.The non-treated NSCs transplant group was injected with 10?L of non-ATRA-treated NSCs,and the other transplant groups were injected with 10?L of NSCs induced by ATRA.DMEM/F12?Hyclone,USA?liquid medium?10?L?was injected respectively in the sham operation,injury control,and NGF-only groups.Transcatheter injection of NGF(3?g kg-1 d-1)?Staidson,China?was conducted simultaneously in the NGF-only and ATRA+NGF groups for 7 consecutive days.?3?BBB score was used to evaluate the changes in the motor function of the hind limbs of rats in each group 1 day before modeling,7 days after modeling,and 1,2,5,and 8 weeks after transplantation.Obtuse manipulation test was conducted 1,2,5,and 8 weeks after transplantation as a supplement to the motor function evaluation.The electrophysiological examinations of both lower limbs were performed 1 day before modeling,7 days after modeling,and 1,2,5,and 8 weeks after transplantation,and the somatosensory evoked potential waveforms and amplitudes of each group of rats were recorded at 8 weeks after transplantation to further detect the sensory and motor function recovery of rats.At 8 weeks after transplantation after the neural electrophysiological examination for cardiac perfusion,wood grain-made of spinal cord tissue damage area in HE staining,histologic observation and 5-bromine deoxidization uracil nucleoside/collagen fibre acidic protein?BrdU/GFAP?and microtubule associated protein-2/collagen fibre acidic protein?MAP-2/GFAP?immunofluorescence double standard to detect and track markers of transplanted cells differentiation and distribution of nerve fiber regeneration conditions.?4?Statistical analysis was performed using SPSS 17.0.The Kolmogorov–Smirnov goodness-of-fit test was used to determine the normality?Gaussian-shaped distribution?of the data.One-way ANOVA was used for group comparisons,and pairwise comparisons were performed with the SNK-q test.A level of P<0.05 was considered statistically significant.Results:?1?The resuscitated NSCs were positive for Nestin after subculture,and the interventional NSCs induced by ATRA in vitro were positive for NSE and GFAP antibodies.?2?Locomotor testing indicated that the BBB scores in the non-treated NSCs and NGF-only groups were higher than that in the injury control group,and the ATRA-induced groups were higher than those observed in the injury control,non-treated NSCs,and NGF-only groups at 2,5,and 8weeks after transplantation.After 5 weeks,the BBB score in the ATRA+NGF group was higher than that in the ATRA intervention-only group.A statistical difference of inclined test was also found between the ATRA+NGF group and ATRA intervention-only group after 5 weeks post-transplantation.The effect on the ATRA+NGF group was better than that of the ATRA intervention-only group,and the ATRA intervention-only group was better than those of the non-treated and NGF-only groups.?3?Nerve electrophysiological examination indicated that at 8 weeks after transplantation,the latency of the ATRA+NGF group was significantly shorter than that observed in the ATRA intervention-only group;the latencies of both the ATRA-induced groups were gradually shortened compared with those of the non-treated NSCs and NGF-only groups;and those of the non-treated NSCs and NGF-only group were shortened compared with that of the injury control group.?4?HE staining in each group observed by optical microscopy at 8 weeks after transplantation indicated that in the sham operation group,the demarcation of gray and white matter was clear and the bundles of nerve fibers were well-arranged.In the injury control group,spinal cord tissue was fragmented in the injury site with connected glial scars,neuron necrosis,and a large number of cavities and cystic cavities.In the ATRA intervention-only group,a large number of glial scars at the juncture between the intact spinal cord and injured spinal cord were detected.Cavities and cystic cavities were smaller than those in the injury control group,but larger than those observed in the ATRA+NGF group.In the ATRA+NGF group,cell proliferation and smaller cavity and capsular spaces were found.In the non-treated NSCs group,cell proliferation and a large number of astrocytes were observed and some glial scars were found.In the NGF-only group,some cavities and cystic cavities were observed,but these cavities were smaller than those in the injury control group.?5?Immunofluorescence indicated that no BrdU-positive cells were found in the sham operation,injury control,non-treated NSCs,or NGF-only groups.In the transplant groups,the positive cells were labeled orange-red in the cell body and were mainly distributed in the gray and white matter.Under high magnification,NSCs were observed as round or elliptical and distributed around the fiber bundles and cavity.The number of BrdU-positive cells was significantly greater in the ATRA+NGF group than in the ATRA intervention-only group.In all groups,GFAP stained cells were distributed in the cavities of gray and white matter at the transected area of the spinal cord,and the neuronal cell bodies extended to the periphery with radial projections,some of which were interwoven into nets.There were fewer GFAP-positive cells in the ATRA-induced groups than in the other groups.Positive cells in both the non-treated NSCs and NGF groups were fewer than in the sham-operation and injury control groups,but higher than in the ATRA-induced groups.There were fewer cells observed in the ATRA+NGF group compared to the ATRA intervention-only group.There were more positive cells in the ATRA-induced groups than in the other groups,and more in the ATRA+NGF group than in the ATRA intervention-only group.Positive cells in both the non-treated NSCs and NGF groups were more than in the sham-operation and injury control groups,but fewer than in the ATRA intervention groups.Conclusion:In animal experiments of rats:?1?Single introduction of NSCs transplantation or NGF injection in the area of spinal cord injury can promote the recovery of motor function after injury.?2?The differentiation of transplanted NSCs into neurons was promoted by ATRA intervention,and its effect on the recovery of motor function after injury was better than that of the single application of NSCs transplantation without intervention or NGF injection.?3?NSCs combined with NGF transplantation induced by ATRA intervention can promote the survival and migration of neuronal differentiation of neural stem cells,and promote the growth of local axons,as well as hinders the growth of glial scars.The effect of ATRA-induced NSCs combined with NGF transplantation on the recovery of motor function in rats with spinal cord injury was significantly better than that of ATRA intervention NSCs alone. |
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