Fig. 4.

Rev-erbα^−/−eyes are more sensitive to chemical-induced oxidative stress injury. (A) Reactive oxygen species (ROS) production was assessed in 3-, 6-, and 12-month-old Rev-erbα^+/+ and Rev-erbα^−/− RPE cells by using a quantitative cellular ROS assay kit measuring 2’,7’ –dichlorofluorescin diacetate (DCFDA), a fluorogenic dye indicative of hydroxyl, peroxyl and other ROS activity inside the cells. (B) Representative images of immunostaining of 8-hydroxy-2' -deoxyguanosine (8-OHdG) in 6- and 12-month-old Rev-erbα^+/+ and Rev-erbα^−/− retinas. Scale bar: 10 μm. (C) Three representative fundus images of Rev-erbα^+/+ and Rev-erbα^−/− mice and associated OCT images at 14 days post-NaIO₃ injection (20 mg/kg body weight, a low dose to evaluate mouse sensitivity to RPE damage). (D) Quantification of RPE atrophy (loss of pigment) in fundus images at 3, 7, and 14 days post-NaIO₃ injection. n = 7–8 mice/group. (E) RPE/choroid flat mounts from Rev-erbα^+/+ and Rev-erbα^−/− mice were stained with β-Catenin (red) and DAPI (blue) at 14 days post-NaIO₃ injection (upper panel). Severe RPE loss was observed at peripheral regions of Rev-erbα^−/− eyes in the RPE/choroid flat mounts, as well as hematoxylin and eosin (H&E) staining of eye cross-sections (lower panel). H&E staining also shows focal loss of outer segments and a discontinued RPE layer in Rev-erbα^−/− retinas at 14 days post-NaIO₃ injection. Scale bar: flat mounts, 50 μm; H&E staining, 10 μm. (F) Representative images of TUNEL staining (magenta) in Rev-erbα^+/+ and Rev-erbα^−/− eye cross-sections (peripheral regions) co-stained with DAPI (blue) at 14 days post-NaIO₃ injection. Arrows indicate TUNEL positive RPE cells. Scale bar: 10 μm. (G) Quantification of the TUNEL positive RPE cells and photoreceptors. n = 15 images/genotype. (H) Primary RPE cells isolated from 8-week-old Rev-erbα^−/− mice showed decreased cell viability after paraquat (PQ, 0.5 mM, 4 h)-induced oxidative damage compared to Rev-erbα^+/+ RPE cells. n = 6/group. (I) Oxygen consumption rate (OCR) of Rev-erbα^+/+ and Rev-erbα^−/− primary RPE cells were analyzed after PQ treatment (0.5 mM, 2 h) using Seahorse XFe96 extracellular flux analyzer. Rev-erbα^−/− RPE cells showed significantly decreased maximal respiratory capacity under oxidative stress compared with Rev-erbα^+/+ RPE cells. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Error bars indicate SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)