Oxidative Stress-Induced Crystallin Aggregation

Animal experiments were approved by the Institutional Animal Care and Use Committee of the Sixth Affiliated Hospital of Sun Yat-sen University. The experimental procedures with animals complied with ARRIVE guidelines and were performed in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Four-week-old male C57BL/6 J mice were purchased from Gempharmatech-GD (Guangdong, China). The eyeballs of the mice were removed and the lenses were carefully dissected after sacrifice with CO₂ inhalation. Dissected lenses were placed in a 10-cm dish containing 20 ml Medium 199 (M4530, Sigma-Aldrich), and incubated at 37 °C in a 5% CO₂ atmosphere for 12 h. Then, three transparent lenses were transferred into a 6-cm dish and incubated with 8 ml Medium 199 containing 10 mU/ml glucose oxidase (GO, G7141, Sigma-Aldrich)^{34 (link)}, which continuously generated oxidative stress and induced crystallin aggregation. The morphological and crystallin protein changes of the lenses were analyzed at 0 and 24 h after GO treatment.