Cataract Induction and NLRP3 Inhibition

Sixty male SD rats underwent lens isolation and organ culturing as previously described [42 (link), 43 (link)]. Briefly, the rats were humanely killed with a carbon dioxide (CO₂) euthanasia apparatus. Their eyes were dissected posteriorly, and lenses were extracted and cultured in M-199 medium (M5017, Sigma-Aldrich, St. Louis, USA) containing 5% fetal bovine serum (FBS, Gibco, Grand Island USA) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B). The cultured lenses were treated with increasing doses (200 to 1000 μM) of soluble UA (U2625, Sigma-Aldrich) to determine the effective dose for inducing cataract formation. Subsequent experiments used the established model to test the effects of 400 μM MCC950 (CSN18163, CSNpharm, Chicago, USA), a selective NLRP3 antagonist [44 (link)]. The lenses were cultured at 37 °C in 5% CO₂ for a week with daily lens opacity assessments (Supplementary Table S1 and Fig. S1). “Clear lens survival” was defined as an opacity grade ≤1 under microscopy. Additionally, lenses were collected at 12, 24, or 48 h to measure NLRP3/caspase-1/IL-1β signaling. Eventually, all lenses underwent Western blot and histological analysis.