慢性光照联合氢醌诱导小鼠年龄相关性黄斑变性模型

A novel murine model of age-related macular degeneration induced by combined chronic exposure to light and hydroquinone

目的采用慢性光照联合氢醌喂食的方法诱导建立小鼠年龄相关性黄斑变性(AMD)模型,观察其病理特征、超微结构及视网膜功能改变,为AMD发病机制及防治研究提供新模型。方法4月龄C57BL/6小鼠20只随机分为模型组和正常组,每组10只。模型组小鼠被喂以基于基础纯净合成的含8 g/(kg·bw)氢醌的饮食,同时每日接受12 h、强度2500 lx的光照。正常组小鼠被喂以不含氢醌的同配方饮食,正常光照明,饲养3.5个月。采用视网膜电图(ERG)、光镜及电子显微镜技术观察两组小鼠视网膜功能和结构改变,TUNEL方法观察视网膜细胞凋亡情况,免疫荧光法检测视网膜、脉络膜血管内皮生长因子(VEGF)及CD31的表达和分布。结果ERG检测结果显示模型组小鼠视网膜功能较正常组下降;光镜观察显示模型组小鼠视网膜色素上皮层呈现萎缩样改变,Bruch膜结构破坏,可见新生血管样组织细胞长入,感光细胞计数模型组小鼠为(164.67±34.37),正常组为(243.33±15.23),模型组感光细胞数较正常组显著减少,差异有统计学意义(t=-9.77,P<0.05);透射电子显微镜显示,模型组视网膜的感光细胞的膜盘结构松散变形,部分碎裂,RPE细胞微绒毛变短,Bruch膜不规则增厚,外胶原层见层状沉积物,内皮细胞突入Bruch膜内。TUNEL结果显示正常组几乎没有凋亡细胞,模型组视网膜色素上皮细胞(RPE)层及感光细胞层出现了大量凋亡细胞,感光细胞凋亡率(43±2.73)%,两组差异有统计学意义(P<0.01)。免疫荧光结果显示,正常组外丛状层、神经纤维层可见散在VEGF弱阳性表达,模型组除上述细胞外,在RPE细胞层亦可见强阳性染色。正常组视网膜各层细胞未见明确CD31阳性染色,模型组RPE细胞层可见较强CD31表达,提示RPE层新生血管发生。结论慢性光照联合氢醌饲料喂养的小鼠非常接近人AMD的发病进程及特点,可为进一步探讨AMD发病机制及防治研究提供可靠的动物模型。

Objective To develop a murine model of age-related macular degeneration(AMD),using combined chronic exposure to light and hydroquinone,and to characterize the pathological and ultrastructural changes,and retinal function,thus to provide a more suitable model for AMD pathogenesis and treatment research.Methods Twenty 4-month-old C57 BL/6 mice were randomly divided into a model group and a normal group,with 10 mice in each.The mice in the model group were fed a diet containing 8 g/(kg·bw)hydroquinone and received 12 h light exposure daily with an intensity of 2500 lx.Mice in the normal group were fed with the same diet without hydroquinone,and were exposed to conventional illumination.After 3.5 months,light and electron microscopy and electroretinograms(ERGs)were used to detect structural and functional changes in the retina.Photoreceptor apoptosis was evaluated with TdT-mediated dUTP nick-end labeling(TUNEL)staining,and vascular endothelial growth factor(VEGF)and cluster of differentiation 31(CD31)expression and distribution were detected by immunofluorescence.Results ERGs showed that retinal function of the mice in the model group was lower than that of mice in the normal group.Light microscopy showed atrophic changes in the retinal pigment epithelium(RPE)layer in the model group,and a reduced number of photoreceptor cells(164.67±34.37 versus 243.33±15.23 in the normal group).There were significantly fewer photoreceptor cells in the model group than in the normal group(t=-9.77,P<0.05).Transmission electron microscopy showed that the photoreceptor outer segments of the mice in the model group were loose,deformed and partially fragmented.In addition,the RPE-cell microvilli were shortened;the Bruch’s membrane became irregularly thickened;and endothelial cells from the choroidal capillary basement membrane penetrated into the Bruch’s membrane.Apoptosis was rarely found in the normal group.TUNEL staining showed that there were many positive cells in the RPE and photoreceptor layers in the model group.The apoptosis rate was(43±2.73)%(P<0.01).Immunofluorescence showed marked VEGF-positive staining in the RPE layer in the model group,whereas no specific staining was found in the normal group.Immunofluorescence of CD31 showed scattered positive staining in the outer plexiform layer and ganglion cell layer in the normal group,whereas it could also be found in the RPE layer in the model group,which indicated the development of neovascularization.Conclusions The retinas of mice treated with combined hydroquinone and chronic light damage closely mimic the development and characteristics of human AMD,this may provide a reliable animal model for further studies of AMD pathogenesis and management.