Co-transplantation Of GDNF And Rat Embryonic Neural Stem Cells Intervened By ATRA For Repair And Regeneration Of Spinal Cord Injury

Objective: To investigate the co-transplantation of glial cell linederived neurotrophic factor (GDNF) and rat embryonic neural stem cellsintervened by all-trans-retinoic acid (ATRA,1.0μmol/L) into spinal cordtransection model of rats in vitro, and to explore the effect of repair andregeneration for spinal cord injury. Methods:1.NSCs were isolated fromcerebral cortex tissue of rat embryos (embryonic day2weeks), cellssuspension culture were carried out in serum-free medium containing growthfactor (20ng/ml bFGF,20ng/ml EGF) at the cells inoculum density of l×106/ml,and per five-seven days were generated once. At the end of the third cellpassage, the cells were intervened by ATRA (1.0μmol/L) in vitro with sevendays. Immunofluorescence techniques were used to detected the expressions ofNSCs specific markers of glial fibrillary acidic protein (GFAP) andneuron-specific enolase (NSE) and Nestin. After identification, choose the highconcentration of cells marked by BrdU to be preparing for transplantation.2.Experiments were performed on sixty Sprague-Dawley rats weighingbetween250and350g. The rats were initially anesthetized with anintraperitoneal injection of2%pentobarbital sodium (30mg/kg) to Preparationof thoracic (T9-T11) spinal cord transection model. The rats were randomlydivided into six groups:(1)Normal control group (n=10);(2)Sham operationgroup (n=10) in which the rats were performed of Laminectomy, then did not receive any intervention;(3)Injury control group (n=10) in which the spinalcord was transected, then treatment of saline by perfusion(15μl/d, for7consecutive days);(4)ATRA intervention group (n=10) in which the spinalcord was transected, then transplant of NSCs (2×105/μl,10μl total) marked byBrdU through micro-injector;(5)GDNF group (n=10) in which the spinal cordwas transected, then PE-10tube is placed in subarachnoid space below theinjury level for perfusion of GDNF (15μl/d, for7consecutive days);(6)ATRA+GDNF group (n=10) in which the spinal cord was transected, thenCo-transplantation of NSCs and GDNF through micro-injector(15μl/d, for7consecutive days). After operation behavioral evaluation, electrophysiologicalexamination, grids climbing tests were performed at1day pre-transplantationand1,2,5,8weeks post-transplantation to further assess the animals’ motorand sensory function. HE staining of spinal cord, Fluorescence double stainingof BrdU/MAP-2and BrdU/GFAP of the tissues isolated from Formaldehydeheart perfusion fixation were performed, which can trace on distribution anddifferentiation of transplantation cells labeled by BrdU, and test forregeneration of nerve fibers, at2,5,8weeks post-transplantation respectively.Statistical analysis was conducted by spss20.0statistic software with themethod of one-way ANOVA. Results:1.Rat embryonic neural stem cellsintervened by ATRA(1.0μmol/L) in vitro whose Nestin, GFAP and NSEshowed positive expression. The cell proliferation was significantly changedand cell differentiation into neurons was increased.2. The mean score of behavior in ATRA+GDNF group was significantly higher than that in ATRAintervention group and GDNF group after2weeks transplantation,（P<0.01）5weeks later in the stabilization and8weeks later the mean score was for thebest（P<0.01）.3. Before transplantation, all rats were completely paralyzed,when they were walking in the grid, the hind limb was weakness and frettingoccasionally, easily false step, the body can only rely on the forelimb moveddragging, except the normal control group and sham operation group. At theend of the8weeks, in ATRA+GNDF group the rats were coordination betweenfore and hind limb gait, the sole of the feet can be loaded, but cannot supportthe body.4. At2,5,8weeks after transplantation, the latent of SEP and MEPwas gradually shortened, and the waveform was emerged. There was astatistically significant difference in ATRA intervention group and GDNF groupcompare with injury control group（P<0.05）. The ATRA intervention groupcompare with GDNF group was no statistically significant difference（P>0.05）.There was a statistically significant difference in ATRA+GNDF group andATRA intervention group and GDNF group respectively（P<0.05），but comparewith Normal control group and Sham operation group still have a gap（P<0.05）.5. It can be observed that there were many different cavities dispersivedistribution in the white matter and neuronal degeneration, nerve cell apoptosis,at2weeks after spinal cord transaction. At5weeks later, most of the structuraldamage in the region of injuries with forming scar tissue and the morphology ofneuronal tended to be normal. At8weeks later, there were many of hyperplasia of glial cell aggregates in the scar tissue and part of the axon regeneration andinterlaced, the holes were reduced.6. At2weeks after transplantation, therewas a large number of cells marked by BrdU, mainly distributed in the gray andwhite matter, the hyperplasia of NSCs were round or oval, cell migration fromthe transplant center to the periphery, mostly gathered in the fiber bundle andaround the void. At2weeks later, there were many NSCs marked by BrdU inthe injured segment, but in the gray matter the number of cell was survival andsignificantly less than that in the white matter. At8weeks later, in ATRAintervention group the NSCs marked by BrdU were markedly reduced, but inATRA+GNDF group there was a large number of NSCs were still alive. Thepositive cells marked by BrdU were not found in GDNF group.7. It can be seenthat the GFAP immuno-positive cells were distribution in the gray and whitematter of the spinal cord impaired loci, part of the cells could be moved to thearound, and the cell bodies showed green fluorescence that radial processreaches out to every direction in ATRA intervention group and ATRA+GNDFgroup at2,5weeks after transplantation. At8weeks later, the positive cells weresignificantly reduced, and part of cells were spread processes interwoven into anetwork. The BrdU/GFAP positive cells by double labeling were not found inNormal control group, Sham operation group, Injury control group and GDNFgroup.The MAP-2immuno-positive cells were mainly distributed in the graymatter of the neurons soma and process, and glial cells without staining at2weeks after transplantation. At5weeks later, the positive cells were significantly reduced. The BrdU/MAP-2positive cells by double labeling werenot found in Normal control group, Sham operation group, Injury control groupand GDNF group.8. At the2,5, and8weeks After transplantation, GFAP andMAP-2expression in each group. Image analysis showed that theATRA+GDNF group GFAP, MAP-2Positive Cells expression area wassignificantly expanded compared with ATRA intervention group and GDNFgroup respectively. Conclusion:1.The NSCs were intervened byATRA(1.0μmol/L) in vitro that could promote the proliferation of cellssignificantly and remarkably promoted the differentiation of cells into neurons.2. After transplantation, the cells survived in injured spinal cord and migrated tothe periphery, and that cells integrates of peripheral tissue, and the cells candifferentiate into neurons, astroglia. The differentiation ratio of neurons andastroglia in injured spinal cord was increased by Co-transplantation.3. Theco-transplantation of GDNF and rat embryonic neural stem cells intervened byATRA in vitro can effectively supply with deficient nerve cells, up-regulate theexpressions of GFAP and MAP-2, improve the micro environment, andpromote the motor and sensory functional recovery of the spinal cord in rats.4.The functional recovery of rat hind limb by Combine transplantation is betterthan the single application of cell transplantation or neurotrophic factor.