Roles And Mechanism Of PirB In The Regeneration After Optic Nerve Injury

Background:The repair and regeneration of the injured central nervous system(CNS)has always been a research hotspot and medical challenge.The optic nerve is a unique CNS component that is difficult to regenerate after damage.Active treatment methods for disease remain scarce,resulting in visual dysfunction or even blindness.Myelin-associated inhibitors(MAIs),found in large amounts in the microenvironment,cause regeneration difficulty after optic nerve damage.Myelin sheaths of the optic nerve usually surround optic nerve axons and are exposed to a large number of MAIs after optic nerve damage.Then,MAIs specifically bind to receptors on optic nerve axons,resulting in an unstable cytoskeleton of growth cone-plate pseudopods and filopodia,thereby hindering optic nerve regeneration.Among the MAIs,Nogo-A,myelin-associated glycoprotein(MAG),and oligodendrocyte-myelin glycoprotein(OMgp)play major roles in the inhibition of neuronal axon regeneration.In the CNS,the above three MAIs have two known co-receptors,NgR and PirB.The presence of MAIs can result in the collapse of neuronal growth cones after binding to NgR to cause cell actin depolymerization,but myelin-associated inhibition of NgR in CNS regeneration is very limited.Indeed,knocking down NgR alone does not significantly reduce the inhibitory effects of MAIs on neuronal axon growth?However,the inhibitory effects of PirB on nerve regeneration after binding to MAIs are controversial too.We hypothesize that PirB may also play an important role in the inhibiting axon growth after CNS injury.This study,the interference virus were used to inhibit the expression of PirB in vitro and in vivo respectively,to observe the axonal regeneration of RGCs and optic nerve.As a result,it will providing a new basis for the use of PirB as a target molecule for promoting optic nerve regeneration and to provide experimental basis for the development of gene drugs for the treatment of central nerve injury.Objective:To clarify the role and possible molecular mechanism of PirB in the repair and regeneration after optic nerve injury.Methods:1.To establish the model of optic nerve clamp damage in SD rats,the expression of PirB in retina were observed by immunofluorescence staining on the 1,3,7day after injury,and the mRNA and protein levels of PirB in retina were detected by q PCR and Western blotting on the 1,3,7,14 d after injury.2.Primary RGC cells were cultured in vitro.MAG,Nogo-A and OMgp were used to inhibit the growth of RGC axons respectively.AD shRNA PirB was used to interfere with the expression of RGC PirB.The length of RGC axons were measured by immunofluorescence staining.3.Primary müller cells were cultured in vitro,and the expression of PirB was interfered by AD shRNA PirB.The PirB interference efficiency by virus in Müller cells were verified by immunofluorescence staining,q-PCR and Western blotting.P-Stat3,P-SHP1,SHP1,SHP2,P-SHP2,NGF,CNTF and BDNF expression changes in Müller cells were detected by Western blotting;then müller cells were co-cultured with injured RGC,and RGC axon regeneration were detected by immunofluorescence;finally,siRNA CNTF was used to interfere with the CNTF expression of Müller cells,and RGC axon regeneration was detected by immunofluorescence staining too.4.The effect of PirB were verified in animal experiments further.AAV shRNA PirB was injected into the vitreous cavity of SD rats for 21 days,and the interference efficiency of AAV shRNA PirB were detected by Western blotting;the model of optic nerve clamped damage were made by SD rats that interfered with AAV shRNA PirB into the vitreous cavity,the survival rates of RGC were investigated by CTB labeled at 7,14,28 day after the optic nerve clamped damage,respectively;the expression of GAP43 in optic nerve were observed by immunofluorescence staining at 7,14,28 day after the ioptic nerve clamped damage;the recovery of optic nerve function at 28 day after the optic nerve clamped damage were investigated by FVEP.Results:1.Under normal circumstances,PirB is mainly expressed in GCL layer of retina;and the fluorescence of PirB in retina were activated from GCL layer to ONL layer after optic nerve clamped damage;and the activation of PirB in Müller cells is time-dependent,the PirB fluorescence expression is gradually enhanced with the prolongation of clamped damage time.The mRNA and protein levels of retina PirB were significantly higher than blank and sham groups after optic nerve clamped damage.2.AD shRNA PirB can effectively knock down the PirB expression of RGC cells in vitro,but directly knock down the PirB expression of RGC cells has no significant effect on the growth of axons inhibited by MAG,Nogo-A and OMgp.3.The expression of PirB in Müller cells was decreased by AD shRNA PirB,which could promote the expression of P-SHP1,P-STAT3 and CNTF in Müller cells.The RGC cells which co-cultured with Müller cells could significantly promote the regeneration of RGC axons after injury,and this effect could be reversed by si RNA CNTF.4.Knocking down the expression of retina PirB can promote the increase of GAP43 expression in optic nerve after clamped damage,but it has no obvious protective effect on the survival of RGC cells that there was no significant difference in the RGC survival rate at 7,14,28 d after the optic nerve clamped damage between AAV shRANA and AAV sh RANA PirB group;knockdown retina PirB did not significantly improve the visual function of N2,N1-P1 and N2-P2 at 28 day after clamped damage.Conclusion:1.The expression of PirB in RGC and Müller cells are all increased after optic nerve injury,suggesting that PirB plays an important role in the pathological changes of optic nerve injury.2.PirB in Müller cells may be more important to promote axon growth than RGC PirB.The mechanism may be related to promote the CNTF expression through pirb-shp1-jak / stat3-cntf pathway.3.Inhibition of PirB expression in retina can significantly promote axonal regeneration after optic nerve clamp injury,but it does not have RGC protective effect,and does not show significant improvement in visual function recovery in this study,suggesting that PirB only promotes optic nerve regeneration but does not protect RGC survival,which leads to its limited therapeutic effect.