Induction and Evaluation of EAU

EAU was induced in B6 mice by subcutaneous injection at 6 spots at the tail base and on the flank with an emulsion containing 200 μg of the human interphotoreceptor retinoid-binding protein (IRBP) peptide (IRBP_1-20) (Sigma-Aldrich) in phosphate-buffered saline (PBS) and complete Freund’s adjuvant (CFA) (Difco, Detroit, MI) and intraperitoneal (i.p.) injection of 300 ng of pertussis toxin. The mice were then randomly grouped and injected i.p. with oxATP in PBS (300 μg/mouse, twice a week) or with PBS alone (vehicle), starting 1 day post-immunization. Mice were examined for clinical signs of EAU three times a week until the end of the experiment (day 30 post-immunization) by indirect fundoscopy, in which the pupils were dilated using 1.25% phenylephrine hydrochloride ophthalmic solutions and 0.5% tropicamide. The fundoscopic grading of disease was performed using the scoring system reported previously [29]. Histopathological evaluation was performed on eye sections on day 21 post-immunization, because the actively induced EAU in mouse is a monophasic disease that peaks at 20–25 days post-immunization. For details of related tissue fixation, embedding and slicing, see previous report [38].