Repairing Effects And Mechanisms Of RAAV2-Neuritin On The Injured Optic Nerve Of Adult Rat

Objective:As a part of central nervous system,the optic nerve has become a privileged site in the therapy of injury and regeneration because of its special anatomical and physiological characteristics.It is very significant to study the regeneration of optic nerve injury,not only to restore the therapy of optic nerve,but also to the treatment and functional recovery of the disease and injury in the central nervous systerm.Clinical treatments of optic nerve injury include drug treament,optic canal decompression surgery,electro-acupuncture and so on.Optic nerve injury induces apoptosis of retinal ganglion cells(RGCs),abortion of axon regeneration and low potentinal of intrinsic regeneration.The main methods for promoting optic nerve regeneration: peripheral nerves transplantation,cells transplantation,adding neurotrophic factors and gene therapy.As a focus of recent researches,the gene therapy could be became one of the effective methods to repair the injured optic nerve.Adeno-associated virus 2 is a suitable vector for the retina in the eye,and Nrn1 has unique neurotrophic effect.Our present study used the models of optic nerve crush in adult rats,and Nrn1 was a target molecule in the gene therapy which may have effect on RGCs survival and axons regeneration.Methods:(1)Plasmid of rAAV2-Nrn1-EGFP and rAAV2-Nrn1-EGFP was constructed and the titer of the virus was determined.(2)Intravitreal injection and rats model of the optic nerve injury: rats were intraritreally accepted rAAV2-EGFP,rAAV2-Nrn1-EGFP or normal saline.Four weeks later,a Yasargil aneurysm clip was used to crush the optic nerve for 9 seconds probably 2 mm behind the eyeball,retinas of all rats were taken for observation after two weeks.(3)The immunohistochemical staining were used to determine the cell types and percentage of transfection.(4)Real-time PCR and Western blot were used to detect the expression of mRNA and protein of Nrn1 in the retina.(5)The effect of Nrn1 overexpression on RGCs after optic nerve injury: HEstaining,immunohistochemical staining,CTB anterograde tracing and TUNEL staining.(6)The effect of Nrn1 overexpression on axon survival and regeneration after optic nerve injury: RITC anterograde tracing and transmission electron microscopy.(7)The effect of Nrn1 overexpression on visual function after optic nerve injury: pupillary light reflex and flash visual evoked potentials.(8)Western blot wasused to detect the expression of neurotrophic factors related signal pathway proteins: pAkt1,pAkt2,pErk1/2 and pStat3.Further more,Western blot wasused to detectmolecules involved in cell apoptosis: Bcl-2 and Bax,Caspase 3 and Cleaved Caspase 3.Results:(1)rAAV2-EGFP and rAAV2-Nrn1-EGFP was constructed,the titer of rAAV2-EGFP was 2.24×1012 vg/ml and titer of rAAV2-Nrn1-EGFP was 2.34×1012 vg/ml.(2)rAAV2-Nrn1-EGFP was injected into the vitreal body for 4 weeks,and most of retinal cells and nerve fibers expressed green fluorescence;the result of immunofluorescence showed that the majority of RGCs with a percentage of 70% were transfected by rAAV2-EGFP,but astrocytes were hardly transfected.(3)The expression of Nrn1 was over in the retina,the expression of Nrn1 mRNA and proteinupregulated 19-flod and 2.3-fold respectively in the rAAV2-Nrn1-EGFP group compared with the rAAV2-EGFP group;the expression of Nrn1 mRNA and protein upregulated 6-flod and 2-fold respectively in the ONC/rAAV2-Nrn1-EGFP group compared with the ONC/rAAV2-EGFP.(4)The effect of Nrn1 overexpression on RGCs survival,optic nerve regeneration and visual function restoration: the result of HE staining showed that the numbers of cells survival was increased in the RGCs layer in the ONC/rAAV2-Nrn1-EGFP group.Gamma synuclein immunohistochemical staining,CTB anterograde tracing and TUNEL staining results showed that Nrn1 overepression increased the number of survival RGCs.The RITC anterograde tracing results showed that Nrn1 overepression increased regenerated axons.Transmission electron microscopy results showed that most of the myelin layers keep tightness.Pupillary light refiex results showed that the ratio of pupil coustriction reduced significantly.Flash visual evoked potentials results showed that the latency period of P wave was reduced,and the amplitude was increased.(5)Western blot results showed that overexpression of Nrn1 could upregulate the expression of the pAkt1 and pStat3 proteins.Western blot detected that overexpression of Nrn1 decreased the expression of Bax,wheres increased the expression of Bcl-2,and overexpression of Nrn1 decreased the expression of Caspase 3 and Cleaved Caspase 3.Conclusions:(1)The most of RGCs with a percentage of 70% were transfected by rAAV2-EGFP.(2)Nrn1 overexpression in the retina after intravitreal injection of rAAV2-Nrn1-EGFP.(3)Nrn1 overexpression in the retina can promote the survival of RGCs,stimulate the axonal regeneration,and improve the visual function.This effect is accomplished through activating Akt1 and Stat3 pathways and then inhibiting mitochondrial apoptotic pathway.