Application of modified retinal flatmount combined with immunofluorescence staining in oxygen-induced retinopathy model

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. Key words: Retinal vessels/pathology; Oxygen/toxicity; Animals, newborn; Rats, Sprague-Dawley; Retinopathy, oxygen-induced; Retinal flatmount