Modification and application of Evans blue-perfused retinal wholemount method in blood-retinal barrier evaluation of diabetic rats

Background Retina fixed flat-mount perfused by Evans blue (EB) is a common method for the evaluation of blood-retinal barrier (BRB). However, previous method is inconvenient for some laboratories because the retinal specimen can not be observed by gereral microscope rather than confocal laser scanning microscope after the fixation. Objective This study was to modify the preparing way of flat-mounted retina in order to obtain transparent specimen for the observation of rat retinal vessels and the evaluation of leakage under the ordinary fluorescence microscope. Methods Forty male SD rats were divided into the control group, diabetes mellitus (DM) 1-month group, DM 3-month group and DM 6-month group according to the random number table. Streptozotocinum (STZ) of 2% dissolved in 0.05 mmol/L sodium citrate-hydrochloric acid buffer was intraperitoneally injected in SD rats to establish DM models, and the equal volume of solvent was injected in the same way in the control rats. One month, three months and six months after injection, EB of 30 g/L was injected via rat femoral vein in the dose of 45 mg/kg. Fifteen minutes after injection of EB, the rats were sacrificed and the retinas were isolated and cut radially to prepare the flat-mounted retinas in PBS immediately and then were dried till the specimens were transparent. The specimens were examined under the fluorescence microscope. The percentage of EB leakage was quantitatively calculated by IPP 6.0 software. All procedures were performed following approval of the institutional animal care and use committee of Tianjin Medical University. Results The retina morphology was normal in the control group, and EB filled the vessels, exhibiting the red fluorescence under the fluorescence microscope. Compared with the control group, retinal background fluorescence was enhanced slightly in the DM 1-month group, and focal leakage of the EB from capillaries and focal dilated vessels were found in the DM 3-month group, further, vascular caliber inequality, retinal hypoperfusion area and a larger number of hyperfluorescence areas were seen in the DM 6-month group. The percentage of leakage area was (0.05±0.02)%, (0.27±0.06)%, (1.17±0.18)% and (4.77±0.66)% in the control group, DM 1-month group, DM 3-month group and DM 6-month group, respectively, showing a significant difference among the four groups (F=795.800, P<0.001), and the leakage area was obviously larger in the DM 3-month group and DM 6-month group than that in the control group (q'=10.338, q'=43.475, both at P<0.001). Conclusions Modified EB-perfused retinal wholemount method is easy and helpful for clear visualization of retinal vessel leakage induced by BRB breakdown in the diabetic rats under the common fluorescence microscope. Key words: Retinal wholemount; Diabetic retinopathy; Disease model, animal; Rats, Sprague-Dawley; Blood-retinal barrier; Evans blue