Construction and Evaluation of a Rat Model of Neuromyelitis Optica-associated Op-tic Neuritis
OBJECTIVE To construct a rat model of neuromyelitis optica-associated optic neuritis(NMO-ON)using a superior conjunctival approach and to evaluate the model from various dimensions,including in vivo and pathological assessments of the optic nerve,spinal cord,and brain.METHODS A total of 30 male SD rats were randomly divided into control group(CG)and model group(MG),with 15 rats in each group.After exposing the optic nerve via a superior conjunctival approach,healthy human serum was injected into the subarachnoid space 2 mm behind the globe in the CG,while aquaporin 4 immunoglobulin G(AQP4-IgG)-positive serum from NMO patients was injected in the MG.The rats were then maintained under standard conditions for 14 days.On the third day post-operation,immunofluorescence was used to detect AQP4-IgG expression in the optic nerve.Flash visual evoked potential(F-VEP)and pupillary light reflex tests were performed one day before the surgery and on the 7th and 14th days post-surgery.On the 14th day post-surgery,retinal,optic nerve,brain,and spinal cord tissues were collected from both groups.Histological assessments using hematoxylin-eosin(HE)and Luxol fast blue(LFB)staining were conducted to observe tissue morphology,while transmission electron microscopy was used to examine the ultrastructure of the optic nerve.Immunofluorescence was employed to detect the expression of aquaporin 4(AQP4),glial fibrillary acidic protein(GFAP),neurofilament light chain(NFL),and cluster of differentiation 68(CD68)in the optic nerve.RESULTS(1)AQP4-IgG:Strong expression of AQP4-IgG was observed in the optic nerve of the MG group,with loss of AQP4 and GFAP in the areas of AQP4-IgG deposition;The CG group showed negative expression.(2)F-VEP:On the 14th day post-surgery,the N1-P1 amplitude in the MG group was significantly lower than in the CG group(t=96.639,P=0.000);However,there was no significant difference in the N1 and P1 wave values between the two groups(P>0.05).(3)Pupillary light reflex test:The percentage of pupillary constriction before and after exposure in the MG group was significantly lower than in the CG group on the 14th day post-surgery(t=81.022,P=0.000).(4)Tissue morphology:The MG group showed optic nerve swelling,reduced axon number,extensive microtubule and microfilament dissolution with irregular cavitation,and substantial inflammatory cell infiltration,as well as severe demyelination with disorganized structure.Retinal ganglion cell layer showed significant edema,decreased cell number with varying sizes,and unclear nucleoli.No significant abnormalities were observed in the spinal cord and brain tissue morphology.(5)NFL and CD68:NFL expression in the optic nerve was lower,and CD68 expression was higher in the MG group compared to the CG group,with both differences being statistically significant(tNFL=28.103,tCD68=385.335,both P=0.000).CONCLUSIONS An NMO-ON rat model can be successfully established by microinjecting AQP4-IgG-positive human serum into the subarachnoid space of the optic nerve.This model exhibits characteristic pathological changes of NMO-ON,including astrocyte destruction,inflammatory infiltration,axonal injury,and demyelination.
neuromyelitis opticaoptic neuritissuperior conjunctival approachanimal model
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