Background Retinal flatmount of oxygen-induced retinopathy (OIR) animal models is a useful tool in the study of ischemic retinopathy.The retinas of OIR of rat or mouse pups were small and thick and difficult in operating of conventional preparation and quantitative analysis of retinal flatmounts.Objective This study was to explore an easy and stable operating method of retinal flatmount combined with immunofluorescence staining in rodent.Methods Forty ＜6-hour-old SD rat pups were randomly assigned to OIR model group and normal control group.The pups were raised with nursing mothers in hyperoxia environment (80％) and normal oxygen environment (21％) alternately at a 24-hour interval for 14 days in the OIR model group,and the pups were raised in the room air for 14 days in the normal control group.The eyeballs of the rats were extracted to isolate the retinas intactly.The retinas were stained with glutamine synthetase (GS)-isolectin B4 firstly and then expanded into flatmounted and cut into 4 petals radially.Adobe Photoshop CS3 imaging analysis system was used to match the pictures into entire retinal vascular images and analyzed under the fluorescence microscope.The pixel values of retinal avascular areas and the entire retina were quantified by this system.The percentage of avascular areas to the entire retina was calculated to analyze the severity of non-perfusion areas.The use and care of the animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results An intact and smooth retinal flatmount could be obtained by firstly staining method.Ora serrata structure was seen surrounding the whole retina.Strong green fluorescence was exhibited in retinal vessel net with clearly visible vascular branches;while the background fluorescence was weaker.Fully developed blood vessels were displayed in the retinas of the normal control group.Non-capillary areas around the central optic disk and large peripheral avascular areas could be seen in the retinal flatmounts of OIR models.Conclusions The preparation of retinal flatmount is easy and feasible by first immunofluorescence staining for retinal vessels followed by radially cutting of retina.This method of retinal flatmount can ensure the integrity of retinal vascular system and it is available for the observation and evaluation of retinal vascular structure in OIR models.%背景 氧诱导视网膜病变(OIR)动物模型的视网膜铺片是缺血性视网膜病变研究的有用工具.OIR大鼠或小鼠的幼鼠视网膜面积小且厚,采用传统方法先剪开视网膜再进行铺片实际操作难度较大,影响了对实验结果的定量分析. 目的 探讨一种易操作、稳定性好的鼠视网膜铺片联合免疫荧光染色技术.方法采用随机数字表法将40只出生后＜6 h的SD大鼠随机分为OIR模型组和正常对照组,OIR模型组幼鼠与母鼠一起以24 h的时间间隔交替在体积分数80％高氧环境或21％常氧环境中喂养14d,正常对照组大鼠在常氧环境中喂养.于喂养至14 d时摘除眼球并剥离视网膜,对完整的视网膜先行谷氨酰胺合成酶(GS)-isolectin B4染色,然后展平视网膜铺片并呈放射状切为4瓣,并于荧光显微镜下拍照,应用Adobe PhotoshopCS3图像分析软件系统进行图像拼接处理,获得全视网膜血管图像,采用软件测量每张完整视网膜铺片图像中无血管区及整个视网膜的像素值,最后计算无血管区的像素百分比进行无灌注区严重程度分析. 结果采用先行视网膜血管免疫荧光染色再放射状切开视网膜的方法可见视网膜结构完整,展开的视网膜铺片平整,视网膜全周均可见锯齿缘结构.视网膜血管呈强的绿色荧光,血管分支清晰可见,而视网膜的背景绿色荧光较弱.正常对照组大鼠视网膜铺片显示其血管基本发育完全,OIR模型组大鼠视网膜铺片可显示中央视盘旁无毛细血管区及周边大片无血管区形成. 结论 采用先行视网膜血管免疫荧光染色再放射状切开视网膜的方法制备视网膜铺片简便、易行,较传统的视网膜铺片法更容易确保视网膜结构的完整性,有利于OIR模型视网膜血管形态学的观察和分析.
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