| Objective: To observe the effect that transplanted neural stem cells(NSCs), which was induced by ATRA, and combined with Glial cell line-Derived neurotrophic factor(GDNF) and chondroitinase ABC(Ch ABC) into injured site of spinal cord of rats, on the hind limb motor as well as sensory function recovery. And the survival and differentiation of the transplanted NSCs were also observed.Methods : The NSCs, induced by ATRA, had been cultured and cryopreserved during the preliminary work. NSCs was remarked with 5-Brdu-2-deoxy-uridine(Brd U) one day before transplantation after anabiosis. 60 adult Sprague-Daw female mice, weighing from 210~250g, were randomly divided into five groups: sham operation group(group A, n=12), SCI model group(group B, n=12), NSCs with GDNF treatment group(group C, n=12), NSCs with Ch ABC treatment group(group D), NSCs with GDNF and Ch ABC group(group E, n=12). Rats were anaesthetized with intraperitoneally administered 2% sodium pentobarbital(30mg/Kg). Under aseptic conditions and with the aid of an operative microscope, T9-T11 laminectomy was performed. Then, the dura mater was opened, and the spinal cord were transected at thoracic leverT10 and for the rats of SCI model group and treatment group. Rats of the sham operation group only received T9-T11 laminectomy and PE-10 catheterization. The mice were evaluated for Basso-Beattie-Bresnahan(BBB) rating 1 day before the surgery, and the animals with BBB scores lower than 21 points were excluded any experiment group. 8 days after the operation, the rats of sham operation group and SCI model group only received 0.9% normal saline(NS, 20μL/d) treatment though PE-10 catheter for 7 days. Rats of the treatment groups received GDNF(10μL/d) with 0.9%NS(10μL/d)(group C), or Ch ABC(10μL/d) with 0.9%NS(10μL/d)(group D), or GDNF(10μL/d) with Ch ABC(10μL/d)(group E) for 7 days after NSCs transplantation. BBB score and SEP test were used to study the functional improvement at the time of 7 days after the operation and 1, 2, 5 and 8 weeks after the treatment. At eighth weeks post-treatment, the rats were anaesthetized with intraperitoneally administered 2% sodium pentobarbital(30mg/Kg) again and transcardially perfused with 4% paraformaldehyde in phosphate buffer. Spinal cord(SC) was dissected and the SC segment that redeived the graft was removed. HE staining and Single/double labelling(such as Brd U, GFAP and MAP-2) immunofluorescence techniques were performed to assess cell survival, differentiation and axons regeneration of the graft NSCs. SPSS 19.0 statistic software was used to the statistical analysis. Differences are statistically significant when P<0.05.Results:(1).Basso-Beattie-Bresnahan(BBB) Locomotor Scale: 7 daysafter operation, there was a significant difference between sham operation group and SCI model group or treatment group(P<0.05), but with no difference among the SCI model group or treatment group(P>0.05). 2 weeks after treatment, the locomotor scores of the rats began to recovery from the second week after the treatment. The BBB score were increased in treatment group from the second week compared with that of the SCI model group(P<0.05). The BBB scores of group E was much higher than that of the group C and D after fifth week, and the difference has statistically significant(P<0.05).(2).Somatosensory Evoked Potential(SEP) examination: Comapred with sham operation group, the SEP reaction latency of SCI model group and treatment group was increased after 7 days of surgery, and the difference was statistically significant(P<0.05), but there was no difference among the SCI model group and treatment group(P>0.05). The SEP reaction latency was shortened and had significant difference compared with sham operation group from second week post-treatment(P<0.05). The SEP reaction latency of group E was shorter than those of group C and D in fifth and eighth week post-treatment, and the difference has statistically significant(P<0.05).(3).HE staining observation: In the sham group, the dividing line of white and grey matter were clear, and the structure of cells was integrity. The spinal cord structure was disorganization, and more scar and cysts was formed during the SCI model group. Compared with SCI model group, the treatment group had more hyperplasia of glial cellaggregates in the scar tissue, and the holes were reduced.(4). Immunofluorescence and positive cells counting: No Brd U-positive cell had been detected in the group A and B, but could be found in the treatment group. The mean number of Brd U-positive cell were 95.2±10.2(group C), 92.7±8.9(group D) and 109.0±10.8(group E). The group E has more cells than group C and D, and the difference has statistically significant(P<0.05). The mean number of GFAP-positive cell were 13.5±2.5(group C), 12.9±1.9(group D) and 10.3±2.1(group E). The group E has smaller cells than group C and D, and the difference has statistically significant(P<0.05). The mean number of MAP-2-positive cell were 11.7±2.2(group C), 10.6±2.4(group D) and 14.1±2.3(group E). The group E has more cells than group C and D, and the difference has statistically significant(P<0.05).Conclusion:1.NSCs, which had been induced by ATRA, could promote the recovery of the damaged spinal cord; 2.The effect of NSCs combined with GDNF and Ch ABC was much better than that of NSCs combined with GDNF or Ch ABC; 3.GDNF and Ch ABC have a synergistic effect in the treatment of spinal cord injury. |
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