视神经脊髓炎相关性视神经炎大鼠模型的构建与评价  

Construction and Evaluation of a Rat Model of Neuromyelitis Optica-associated Optic Neuritis

作　　者：韩梦雨 马传政 邓婷婷 金明[1] HAN Mengyu;MA Chuanzheng;DENG Tingting;JIN Ming(China-Japan Friendship Hospital,Beijing 100029,China)

机构地区：[1]中日友好医院,北京100029 [2]北京中医药大学,北京100029 [3]中日友好临床医学研究所,北京100029

出　　处：《中国中医眼科杂志》2024年第10期908-916,共9页China Journal of Chinese Ophthalmology

基　　金：国家自然科学基金项目(82174440);中央高水平医院临床科研业务费(ZRJY2021-QM24);中日友好医院“菁英计划”人才培育工程(ZRJY2021-QM24)。

摘　　要：目的 以上结膜入路法构建视神经脊髓炎相关性视神经炎(NMO-ON)大鼠模型,从活体与病理、视神经及脊髓、脑等多维度评价该模型。方法 将SD雄性大鼠30只随机分为对照组(CG)、模型组(MG),各15只。2组大鼠上结膜入路暴露视神经后,在球后2 mm处分别将健康对照人群血清和AQP4免疫球蛋白G(AQP4-IgG)阳性NMO患者血清注射至蛛网膜下腔,常规饲养14 d。术后第3天免疫荧光法检测视神经人AQP4-IgG表达。术前1 d、术后第7、14天分别行闪光视觉诱发电位(F-VEP)及瞳孔对光反射检测;术后第14天取2组大鼠视网膜、视神经、脑及脊髓组织制片,苏木精-伊红(HE)染色、勒克斯坚牢蓝(LFB)髓鞘染色观察大鼠组织形态,透射电镜观察大鼠视神经超微结构。免疫荧光法检测视神经水通道蛋白4(AQP4)、胶质纤维酸性蛋白(GFAP)、神经丝蛋白L(NFL)、簇分化抗原68(CD68)表达。结果 (1)AQP4-IgG:MG组大鼠视神经中AQP4-IgG强表达,AQP4和GFAP在AQP4-IgG沉积区域丢失;CG组则为阴性表达。(2)F-VEP:第14天MG组N1-P1振幅低于CG组,差异有统计学意义(t=96.639,P=0.000);2组第14天N1、P1波值比较,差异无统计学意义(P>0.05)。(3)瞳孔对光反射实验:第14天MG组大鼠曝光前后瞳孔收缩的百分比低于CG组,差异有统计学意义(t=81.022,P=0.000)。(4)组织形态:MG组大鼠视神经肿胀,轴突数量减少,微管微丝大量溶解,伴有不规则空洞化,其间见大量炎症细胞浸润,髓鞘严重分层、结构不完整。视网膜神经节细胞层细胞水肿明显,数量减少,且大小不一,核仁不清晰。脊髓与脑组织形态未见明显异常。(5)NFL、CD68:MG组大鼠视神经NFL表达低于CG组,CD68表达高于CG组,差异均有统计学意义(t_(NFL)=28.103、t_(CD68)=385.335,均P=0.000)。结论通过在大鼠视神经蛛网膜下腔显微注射人类AQP4-IgG阳性血清可成功构建NMO-ON大鼠模型,该模型显示了NMO-ON的特征性病理变化,包括星形胶质细胞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

关 键 词：视神经脊髓炎 视神经炎 上结膜入路 动物模型 

分 类 号：R774.61[医药卫生—眼科]