Effects Of Integrated Fetal Spinal Cord Transplantation Associated With BDNF On Repair Of Complete Spinal Cord Transection In Rats

Spinal cord injury (SCI)is a kind of frequent injury in nervous system,with serous consequence,bringing much suffering to the patients and muchburden for the society.The repair mechanism of spinal cord injury is verycomplicated,usually including the vassular mechanism and the nervous-biochemical mechanism simultaneously after SCI,which influence eachother.Among many therapeutic tools of SCI,it is proved that the tissuetransplantation is best for the functional recovery of injured spinalcord.Transplants include peripheral nerve graft ,embryonic central neural graftand cellular transplant.Of the total,the effect of embryonic central neural graftis most ideal for repairing the spinal injury.Spinal cord belongs to immune privilige tissue.Embryo stems frommother nuclide,and its reject reaction cann't happen.Studies of SCI mostly usefetal myeloid tissue.Its mechanism of action: the neurones of the transplantform synapse connection with host spinal cord after growth anddifferentiation.The glial cells of the transplant contribute to regenerate axonalmyelinizate;make newborn axon pass through as "bridge";suppress glial cellproliferation, and lessen glial scar. The merits of FSC transplantationinclude :embeded fetal spinal cord is easy to survive and substitute injuredspinal cord;plentiful nervous nutrient substance stimulates host nervouspotential energy for growth;lessen nervous retrograde degeneration andnecrosis;promote axonal regeneration and remyelinization;inhibit postinjuryglial scarring;support and guide regenerated axon to pass through injuredregion or set up synapse contact and signal transduction and so on.The transplanting methods usually contain segmented myeloid tissuedirective graft or fetal myeloid composite cell suspension infusion.Those abovehave same defects,which are that they also ruin proper anatomic structure andhabitat of the transplant in great extent and take disadvantage of reparation andreunion of the injured spinal cord.Recently SCI models for the studies of spinalcord graft commonly use these several kinds:1.contusion SCI;2.Concis orresorption SCI;3 . clip compression SCI;4 . photo-chemlly inducedSCI.However whatever incomplete or complete lesion animal models hind limbmotor functional recovery spontaneously occured.To the so-called completespinal cord transaction even greatly sparing descent fiber residual contributes toevident functional recovery, so it is most difficult to judge spinal cordfunctional recovery derived from personal repair of the vestigial myeloid tissueor repair effect of spinal cord graft.Thus warrant the completeness of spinalcord transaction is very important for the studies of functional recoveryfollowing transverse lesion.Thus to establish a new type of effective animalmodel and to choose proper transplanting method and dependable evaluationcriterion are extremely significant to research further regulatory factor ofdifferent spinal cord regeneration.Our experiment wanted to establish complete spinal cord transverse ratmodelsand enhanced the survival rate of animal models.We made use ofseveral integrated fetal spinal cord grafts,simultaneously used advanced Alzetosmotic pumps to inject BDNF by timing and quantitation,then assessed spinalcord regeneration and functional recovery through evaluating the ethologicalchange of animal models with spinal cord transplantation;investigated theeffects of fetal spinal cord transplantation and BDNF on the expression ofGAP-43 and c-Jun during repairing injured spinal cord via in-situ hybridizationand immunocytochemical stain;determined the dependability and availabilityof the animal models of repairing SCI with several integrated fetal spinal cordstransplantation and established foundation for further researching completespinal cord traverse lesion and repair mechanism.We modified the complete spinal cord transection models of rats bylooking straightly at the operating microscope. We cut down a whole spinalcord segment of 3～4mm with sharp corectome,and ensured completemutilation microscopically.During operation retaining main blood-supplyasand regurgitant vessels of spinal cord of rats as far as possible materiallydecreased primary and secondum injury derived from haemorrhage andischemic,and provided favourable nutritional support for recovery as soon aspossible and regenetation of the injured region and adjacent myeloidtissue. Because our SCI models chose to cut down a whole spinal cordsegment and resulted in big defect, which made transplant choice difficult. Tosolve the problem we took the lead in choosing several integrated fetal spinalcords transplantion and studied the repair effect of fetal spinal cordtransplantion.Many experiments verified that neurotrophic factor played a significantrole in the repair and restitution of spinal cord injury and retrieving moribundnerve cells.Of the total brain derived neurotrophic factor(BDNF)is especiallyeffective for maintaining neuronal survival and promoting the functionalrecovery of nerve cells.BDNF is correlated closely with neurotrophic factorfamily β-transforming growth factor, and produces an conspicuous nutritionalsupport effect on dynamoneure of myelonic cortex. It can maintain Ca2+steadystate and prevent apoptosis. Even if the effect of BDNF is obvious, it is notenough to support the restitution of neuronic circuit loop as far as completespinal cord transaction and far distance of mutilation are concerned.We chosemodified CBS to evaluate hind limb motor functional recovery.In this study themotor function of rat models with BDNF only didn't recover ( CBS score91.68±1.34 at the week 6 postoperation), however , the motor function ofrats recovered well in the group with complete fetal spinal cord transplantationassociated with BDNF (CBS score 31.75±4.29 at the week 6 postoperation).That illustrated BDNF played an important role in maintaining neuronalsurvival and promoting the functional recovery of nerve cells.The study of functional proteins may analyze the change of proteinexpression in different developmental stage,to hint the generation anddevelopment rule of some disease.The previous experiments approached thenerve cells regeneration and repair effect of all kinds of therapeutic regimenusually following injury via detecting the expression of the proteins related withneuronal growth.Among the total GAP-43 and c-Jun were received notateproteins reflecting myelonic regeneration and most representative and utilizeduniversally.Growth associated proteins 43 (GAP-43) is a specific nerve tissuegrowth-associated protein and a fast transport cytolemma phosphorylatedprotein linked closely with the neural development ,axonal regeneration andsynaptic restitution processes . GAP-43 protein and GAP-43 mRNA isspecifically expressed and distributed extensively in the nervous system,andespecially in the nerve cells.It is expressed along the integral axon in thedeveloping nerve cell and especially abundant in the growth cone.GAP-43 geneis at least 50kb in length, located at the chromosome 16 of the rat. During theneural development and regeneration,GAP-43 shows up-regulation in the entireperiod of axon elongation and synapse formation,and regulate the signal actionof the axon induced by the neurone.GAP-43 may affect the axnonal energy forgrowth through conducting the axon growth and adjusting now unionoccurrence ,so it is considered to be an endogenic determinative factor in theperiod of the neurone growth, as a mark of the nerve regeneration. Digoxin-signed GAP-43 cDNA probe is listed in the neural plastic and preferredmolecular probes to research nerve growth and development injury repairinginternationally.According to the feature that GAP-43 expression can reflect the nerveregeneration,in Exp.2 we randomly divided the rat models into three groups:normal control;A group (complete spinal cord transaction + integrated fetalspinal cord transplantation);B group (complete spinal cord transaction +integrated fetal spinal cord transplantation + BDNF) .We took hybridization insitu to detect the GAP-43 neuron positive cell numbers of spinal cord tissuemicrotome section of transplant area in three groups of animal models at theweek 1,2,3,4,5,6 postoperation.GAP-43 primer sequence is F5′-GCAGGACGAGGGTAAAGA-3′,R5′-CACGCACCAGAT-CAAATAA-3′,we useddigoxin labelled cDNA probe.The result showed :the GAP-43 neuron positivecell numbers increased after operation in group A,the number peak occurred atthe second week, and began to descend at the third week. The one of groupB also increased, but the peak occured at the third week, and began todescend at the forth week.The neuron positive cell numbers of the group Bwere mostly higher than the one of the group A at the week 3,4,5postoperation(P<0.01).Both neuron positive cell numbers decreased obviouslyand were roughly equal at the sixth week in group A and B.c–Jun is an inducible transcription factor encoded by immediate earlygene (IEG),which regulates other target genetic transcription,and plays aconsiderable role in cell proliferation , activation and differentiationprocesses.c-Jun family has c-Jun,Jun-B,Jun-D.c-Jun gene is 315kb longwithout intron. c-Jun mRNA is 312kb in length. Its codogenic neucleoproteinJun is composed of 334 amino acid, and its molecular mass is 39kD.c-Junexpression might be taken as a mark of neurone survival and regenerationcapacity after nerve fiber lesion.Its expression up-regulating is a probably aprecondition of nerve regeneration.More literatures have reported that theperipheral nerve division or crushing and the axon binding all can make c-Junexpression increase considerably,however,after nerve regeneration c-Junprotein level becomes common.Previous studies showed c-Jun expression wasup-regulated obviously via utilizing fetal spinal cord transplantation and/orBDNF at the injured region following SCI according to the mechanism ofaction of fetal spinal cord transplantation,the regenerating energy of centralnervous system was enhanced and the neuraxial sprout and regenerate of centralnervous system were promoted.So they considered transcription factor c-Junwas connected with the regenerate of central nervous system, embeddedembryonic myeloid tissue might encourage c-Jun expression at the injuredspinal cord,thus it was reason of stimulating axon regeneration of centralneuron.According to the feature that c-Jun expression can be taken as a symbol ofthe neurone regeneration,in Exp.3 we took immunocytochemistry SP stain toevaluate the c-Jun positive cell area density of spinal cord tissue microtome sec-tion of transplant area in three groups of animal models ( grouping same asExp.2)at the week 1,2,3,4,5,6 postoperation (cells/mm2 ).The result showed the increasing extent of c-Jun positive cell area densityin group B was greatly much stronger than the one in group A ( P<0.01).Thepeak of cell density occurred at the third week,last for two weeks, then beganto descend in both group A and group B.The rate of decay in group A was slowcompared with group B.Our result showed that several integrated fetal spinal cords transplantta-tion simultaneously would up-regulate GAP-43 expression and enhanced c-Junimmune reaction of the injured region.The high-peak of GAP-43 and c-Junexpression appeared at the third week,and the argument of CBS sore changewas greatest at the third week too.That suggested it took 2~3 weeks that fetalspinal cord graft and the injured myeloid tissue completed repairing and linkingwith each other.Comparing spinal cord transplantation only with spinal cordtransplant action associated with BDNF,it pointed out that BDNF couldn'tmake the motor function recover evidently earlier,even if it might preservespinal nerve cells,encourage nerve cells from impairment and facilitate nervecells regenerationOur experiment established complete spinal cord transaction model of ratssuccessfully,which raised the survival rate of rats (80% or so) and providedgood environment for spinal cord regeneration. We chose several integratedfetal spinal cords,and succeeded in transplanting them simultaneously into thesame SCI model.That method would offer a novel opinion for the futureexperiments to make animal models with bigger myelonic mutilation.In the testwe explored initially the repair effect of the integrated fetal spinal cord andbrain derived neurotrophic factor for transverse spinal cord and took the lead infounding a new method to research spinal cord transplantation, and suppliedrationale and practice guidance for ulterior scientific studies.