To Establish Mice Model Of Experimental Autoimmune Optic Neuritis And Study Protective Effects Of NGF On Neurons

Objective:1,To establish animal model of experimental autoimmune optic neuritis induced by petide myelin oligodendrocyte glycoprotein(MOG_35-55).2,To explore incidence rate of optic neuritis and visul dysfunction in the mice model of experimental optic neuritis by monitoring f-VEP and f-ERG combined with pathological techniques.3,To investigate protective effects of NGF against RGCs and neurons damage on optic neuritis in mice.Methods:1,C57BL/6 mice were induced to optic neuritis and EAE by injecting subcutaneously MOG_35-55 peptide in CFA.F-VEP and f-ERG tests were performed in experimental group and control group at different time points after immunization.Optic neuritis were initially diagnosed by recording f-VEP.2,At days 0,7,14,20,30,consecutive sections of optic nerve were stained with hymatoxylin/eosin,Luxol fast blue and Bielschowsky silver staining to assess inflammation,demyelination and axonal pathology,and used to observe ultrastructural changes of optic nerve with electron microscopy in EAE mice and control group mice.Apoptosis of RGCs was measured by TUNEL technique and apoptotic index were calculated.3,40 EAE model mice were randomly divided into 2 groups:NGF-treated group(NGF+EAE) and control group(BSS+EAE).Nerve growth factor was intravitreal injected to the eyes in NGF-treated group and balanced satls solution was intravitreal injected to the eyes in control group on days 4,10 post immunization.We recorded the f-VEP and f-ERG response at days 0,7 and 14 in two groups.After examination of visual electrophysiology,histological evaluation was performed.Consecutive sections of optic nerve were stained with hymatoxylin/eosin,Luxol fast blue and Bielschowsky silver impregnation to assess inflammation,demyelination,and axonal pathology. Apoptosis of RGCs was measured by TUNEL technique.Results:1.In the experiment,C57BL/6 mice were induced to optic neuritis and EAE by injecting subcutaneously MOG35-55 peptide in CFA,f-VEP changed in 60% eyes and the incidence of optic neuritis was 57.5%by pathological diagnosis in EAE model group.Both of eyes had pathological changes in most mice of model group.2.The results indicated that the recording of f-VEP and f-ERG in EAE mice did not differ from control ones before immunization.At days 7 post immunization, amplitudes and latency of P-wave in f-VEP and b-wave in f-ERG changed in the EAE group.At days 14 post immunization,the recordings of f-VEP and f-ERG in EAE mice were different significantly from that of the control mice.During our observation,until days 20 and days 30 post immunization,the results of VEP and ERG were slightly serious and not improved.3.At days 7 post immunization,the optic nerve in sections were not different from control mice by general HE staining and Bielschowsky silver staining in EAE mice,and confused myelin but no loss was found by LFB staining.The optic nerve showed the myelin was broken,layered and separated from axon by electron microphotographs in EAE mice.The apoptosis of RGCs was not found obviously and not different from control group.At days 14 post immunization,there were inflammation,demyelination,axonal lesion in different level in model group.The optic nerve in EAE mice by electron microphotographs showed serious and layered demyelination,and axonal damage.The apoptotic index of RGCs was significantly higher than that in control mice.During our observation,until days 20 and days 30 post immunization,the pathological results of optic nerve were slightly serious and not improved.The apoptotic index of RGCs was gradually highten according to the course of optic neuritis.4.The clinical score and weight in NGF-treated group(NGF+EAE) and control group(BSS+EAE)did not changed.At days 7 post immunization,the results of f-VEP and f-ERG was not different between two groups(P＞0.05).The area of demyelination didn't decrease in NGF-treated group both at days 7 and days 14 after immunization.Amplitudes and latency of b-wave in ERG changed significantly and the VEP results did not differ from control ones at days 14 after immunization(P＜0.05).At the same time,we observed the apoptotic index of RGCs in NGF-treated group was significantly lower than that in control group by TUNEL assay(P＜0.05).Conclusions:1.The EAE mice induced by MOG35-55 peptide was stable and had high incidence of optic neuritis,which might be an ideal model to study ON. We observed apoptosis of RGCs and damage of axon accompanied with serious demyelination of optic nerve.2.f-VEP can be used to diagnose optic neuritis initially and assess the severity of disease,which can be been index of screening and drug therapy of optic nertitis.3.NGF has no significant effects on the NGF-treated group mice such as clinical score and the degree of demyelination.It couldn't decrease the incidence of disease,but treatment of NGF showed a protective effects on survival and function of RGCs.