An Experimental Study Of Synapse's Formation And Function When Transplanting Neural Stem Cells Into PNS To Delay The Denervated Muscle Atrophy

PartⅠThe study of Culture,Increase and Transplant Neural StemCells in Delaying Denervated Muscle Atrophy.Objective To Verify the Process validity in obtain,culture and increase Neural StemCells.Study the effect of Neural Stem Cells in delaying the denervated muscleatrophy when it was transplanted into peripheral nerveous system.Methods Spinal Neural Stem Cells (NSCs) were obtained from Pregnant 10-12th dayembryo spinal cords of SD rats mechanically.The NSCs were cultured in no serumculture medium,after 2-3 generations,the neural stem cells were blew into singlecells,suspensioned and transplanted into the distal part of tibial nerve.Cell culturemedium was injected into the distal tibial nerve in contral group.3,5 and 7 monthspost-transplantation,the distal part of the tibial nerve and muscles were harvested andidentified with specific markers,by means of indirect immunofluorescent stainingrespectively.To evaluate the survival and differentiation of neural stem cells inperipheral nerve,The gastrocnemius muscles were excised and immediately weighedand the muscle fibers' area was measured and compaired to see whether thetransplanted NSCs can delay the denervated muscle atrophy.Results The process of Spinal NSCs obtain and culture can get enough NSCs,andthese NSCs are active enough for transplantation.3,5 and 7 months after NSCstransplanted into tibial nerves,the immunofluorescent staining showed that NSCscould survive and differentiate into neurons.As the time go on,there were moreneurons apaired.What's more,these neurons can synthesis and secrete considerablesynaptophysin.Both of the humid weight and muscle fibers' cross section area oftriceps surae showed that the muscle atrophy was more serious in the control groupthan in the NSCs transplanted group.Conclusions 1.The process of Spinal NSCs obtain and culture was suitable for theexperiment.2.Embryonic spinal stem cells can differentiate into different neural cells in vivo after transplantation; 3.NSCs' transplantate into PNS can delay thedenervated muscle atrophy.PartⅡThe Experiment of New Formed Neuromuscular Junctionbetween Transplanted Neurol Stem Cells and Denervated MuscleObjective Obtain enough neural stem cells and make the same animal models basedon experiment methods verified in PartⅠ,then study the new neuromuscularformation and detect its function.Methods Based on the verified methods in partⅠ,we obtained enough activityseperated neural stem cells,made the same NSCs transplanted models and controlgroups.3,5,7 months after transplantation,we perform the different detect.Four andtwo weeks before the nerve and gastrocnemius muscle excision,the distal part of thetibial nerve was dissociated respectively to prevent potential nerve regeneration fromother nerves or tissues.Then the gastrocnemius muscle adjacent to the nerve entryplace were exsected and were carried out immunohistochemistry,to detect the changeof the neuromuscular Junction and the new formed Junction.We also usedElectrophysiology and Direct and Retrograde Tracing to detect the function of thenew formed neuromuscular Junction.Results 3 months after fetal NSCs transplantation,the pre-menbrane dispersed,thepost-menbrane atrophy and cataplasia,no electrophysiology conductive ability; thesimilar results in the control models.5 months later,in 5 of 8 fetal NSCs transplanted rats,the transplant-derived colocalization of premenbrane reappeared and the host-derivedpostmenbrane became more mature than ever.While some of the premenbrane were lesscontinuous and in dot profile,the morphology of these new NMJs were simpler than thenormal NMJs.The CMAP waves were detected and the latent period was 12.5 ms,waveamplitude was 0.9 mv; In the same time,the pre-membrane disappeared and thepost-membrane fell into pieces in control rats,no CMAP can be detected.7 monthsposttransplantation,in 5 of 8 rats,the pre- and post-membrane were detected and moremature than ever,in some specimens,we detected the mature NMJs and thepostmembrane without the pre-menbrane (denervated muscle fiber) simultaneity,suggesting that the muscle reinnervation was incomplete.The latent period of CMAPwas 5 ms and wave amplitude was 1.5 mv.In the control group,we cannot see the shapeof NMJs,nigher pre-menbrane nor the post-menbrane,no CMAP can be detected.Inanterograde and retrograde tracing,the NSC transplant can transfer the true blue chloride while the control group cannot.Conclusion 1,The transplanted NSCs can protect and delay the atrophy and cataplasia ofthe post-membrane; 2,new neuromuscular Junction can be formed between thetransplanted NSCs and the denervated muscle; 3,new formed NMJs are biologicallyactive.PartⅢThe Formation of Synapse between Transplanted NSCs andHost's Regenerate Neural AxonsObjective To study the relationship between transplanted NSCs and host's regenerateneural axons after the NSCs transplanted into the impaired PNS.Methods NSCs were separated from pregnancy 10-12th day spinal cord of F344 ratsmechanically and cultured in no serum culture medium.Transplante 1.Scm F344nerve segment to the distal part of the GFP gene transferred rats' tibial nerve.Meanwhile,inject 10~6/ul×5ul F344 NSCs into the transplanted F344 nerve segment.2 weeks later,suture the proximal tibial nerve stump with the transplanted F344 nervesegment.Another 2 weeks later,the tibial nerve and transplanted nerve segment wereharvested and identified with specific markers,by means of indirectimmunofluorescent staining respectively.To evaluate the survival and differentiationof neural stem cells and to see the relationship between transplanted NSCs and host'sregenerate neural axons.Results 4 weeks after NSCs transplantation,NSCs survived and differentiated intoneurons.The regenerate axons can form synapse with neurons differentiated from thestem cells.Conclusions In PNS,the host's regenerate axons can form synapse with the neuronsdifferentiated from the stem cells.