2.6. Measurement of Retinal Vascular Permeability

Retinal vascular permeability was analysed by measuring Evans blue-albumin complex leakage from retinal vessels, as described previously [28]. Briefly, Evans blue dye was dissolved in normal saline (45 mg/mL). Then, under deep anaesthesia, the dye (45 mg/kg) was injected into the jugular vein of each rat. Blood (200 μL) was withdrawn from the iliac artery 2 min after Evans blue injection and then every 30 min for up to 120 min. After the dye had circulated for 120 min, the chest cavity was opened, and the left ventricle was cannulated. Each rat was perfused with 0.05 M citrate buffer, pH 3.5 (37°C), for 2 min at 66 mL/min to clear the dye. Immediately after perfusion, the eyes were enucleated, and the retinas were carefully dissected under an operating microscope. Evans blue in the retina and blood samples was detected as described previously [29]. Retinal vascular permeability was calculated using the following equation, and the results were expressed as μL plasma/g retina dry weight/h: retinal vascular permeability = Evans blue (μg)/retina dry weight (g)/time-averaged Evans blue concentration (μg)/plasma (μL) × circulation time (h).