Retinal Vascular Permeability Assay

Retinal vascular permeability assay was performed using 6 to 8 weeks old C57BL/6 mice as described previously.11^–13 All the animals were provided by and experimental protocols were reviewed and approved by The Institution of Animal Medical Research of Sichuan Provincial People's Hospital in accordance with the ARVO STATEMENT for the use of animals in ophthalmic and vision research. For intravitreal injection of IL-1β (or BSA control; 2 µl of 100 ng/µl) and compound (1 µl of 200 ng/µl) or DMSO control (1 µl of 100% DMSO), the mice were sedated by 4% chloral hydrate (8 µl/g, intraperitoneal), then carefully injected with the mixture of IL-1β (or BSA control) with compound (or DMSO control) from the side of the eye into the vitreous without touching the retina. Evans blue (45 mg/kg; Sigma Aldrich, USA) was injected via the tail vein at 2 hours post intravitreal injection. The mice were sacrificed by CO₂ 10 hours after receiving Evans blue. Mouse eyes and retina were collected for Evans blue quantification. The Evans blue that penetrated into retina was extracted using formamide (Sigma Aldrich) for 20 hours at 70°C. The absorbance of extracts was measured at 620 nm and 740 nm after clarification by centrifugation (13,000 rpm, 5 minutes). Data are presented as mean ± SEM.