Synergetic Effects Of Ciliary Neurotrophic Factor And Olfactory Ensheathing Cells On Optic Nerve Reparation

[Objective]The optic nerves lose their ability to regenerate after injury,and up to now there are still no effective methods to fully restore the damaged optic nerves. Recent research shows that olfactory ensheathing cells(OECs) and ciliary neurotrophic factor(CNTF) can be effective in promoting nerve injury repair and regeneration.In this study,it is the first time to combine the olfactory ensheathing cells transplantation that is still in the experimental stage and intravitreous injection of ciliary neurotrophic factor that has entered the clinical application in order to achieve better results in treating optic nerve injury,so as to provide new ideas and experimental evidence for the clinical treatment of optic nerves injury or even the central nervous system injury.[Methods]In this study,we use the OECs original culture technique to purify OECs in vitro.And in this experiment,the supraorbital margin exposure of the optic nerves was used to establish adult SD rats' optic nerve inhausted injury model.Then the purified OECs were injected into the optic nerve sheaths,and CNTF were injected into the vitreous body simultaneously.According to different methods of treatment, the experimental rats were divided into control group,CNTF group,OECs group and OECs+CNTF combined group.At 4W postoperatively,cholera toxin B subunit(CTB) anterograde tracing technique and fluorescence(FG) diotinylated dextran amine(BDA) retrograde tracing technique were adopted to evaluate the effects of OECs and CNTF on the survival of retinal ganglion cells(RGCs) and the regeneration of optic nerve axons.By calculating the number of survival neurons of the same vision field and analyzing the length and density of neurons by analysis of variance and multivariate linear regression analysis,the protective effect of OECs and CNTF on the RGCs and the axons were evaluated.Then the effects of the single use or combined use of OECs and CNTF on the optic nerves jury reparation and regeneration were compared to verify their synergistic effect.[Results]1.Primary cultured OECs with high purityPrimary cultures of OECs were from 2.5 month old rat.Purification of OECs was developed with cytosine arabinoside(Ara-C)treatment and differential adhesion method.OECs were stimulated to proliferate by BPE or forskolin.The purity of OECs was determined according to immunostaining positively for p75.The result demonstrated that total purification time of OECs derived from adult rats was 10d with more than 95%purity,and cell viability decreased about 12-14 days after purification.Adult OECs culture was selected for our experiment because of the high purity.2.Neuronal protection effects of CNTF and OECs after optic nerve injuryEstablish adult SD rat optic nerve inhausted breakdown injury model,to be going to imbed cultured and purified in vitro OECs into optic nerve inhausted breakdown spot below tunica vaginalis,meanwhile corporis vitre inject CNTF。According to therapeutic tool diversity,divided experiment rat into 4 groups:control group,CNTF group,OEC group and CNTF+OEC group.At the end of 8 weeks,by calculating RGCs' survival number,neuronal ecphyma length and density,to carry out analysis of variance and multiple linear regression analysis,evaluate OECs.The number of the rats'RGCs in the control group significantly decreased compared with the normal group.The survived RGCs in each treated groups were significantly more than the control group,and the neuron axon length and density were also higher than the control group.The survived RGCs of OECs group was more than CNTF group, proved that OECs was more effective on the protection of the optic nerve.3.Transplantation of OECs genetically modified to secrete human CNTF promote optic nerve injury repairwhen observing and statisticsing above-mentioned each optic nerve damage criterion,we found that CNTF and OECs associated application group has strongest repair effect,which has higher contribution on optic nerve injury than CNTF group or OECs single used group.That hint CNTF and OECs have synergistic effect on the treatment of optic nerve injury and repair. [Conclusions]The experiment manifest local applied exogenous OECs and corporis vitre injected CNTF homoenergetic abatement behind the eyeball optic nerve inhausted injury originated retrogression neuron damage.The OECs' protective effects are better than the CNTF's.Especially,the OECs and CNTF combined group were significantly better than the single use groups on optic nerves reparation and regeneration,because the two have a synergistic effect.