Abstract
Purpose :
Regional differences in the mouse retinal pigment epithelium (RPE) are under characterized, leaving a knowledge gap in how these differences may contribute to regional degeneration in retinal disease models. We created a single-cell resolution map of RPE subpopulations based on cell area and studied their susceptibility to oxidative stress.
Methods :
Two-month-old C57BL/6J mice (three males and three females) were used to generate the morphometric RPE maps. Four additional mice were injected with 20 mg/kg of NaIO3 into the tail vein one time to test RPE sensitivity to oxidative stress. The mice were euthanized 24 hours after the injections. Eyeballs were enucleated, fixed in 4% PFA for five minutes, cut once in the cornea, fixed in 4% PFA for 10 more minutes, and then placed in 1x PBS. The cornea and lens were removed, and the eye cup was cut into four quadrants before removing the retina. RPE flatmounts were then fixed in 4% PFA for 1 hour at room temperature and transferred to PBS.
The RPE flatmounts incubated in a blocking solution for 1 hour at room temperature. RPE cell borders were stained with an anti-ZO1-AF594 antibody overnight at room temperature. Tiled 2D projection images of the entire RPE monolayer were acquired with an Axioscan 7 (Zeiss) and analyzed with REShAPE.
Using a linear mixed effects model and Tukey HSD test, we assessed the differences between RPE subpopulations.
Results :
We found three statistically significant concentric subpopulations of RPE cells based on cell area. We named them P1 to P3 starting from the center. P1 cells were the largest (420.27 mm2), followed by P3 (350.33 mm2) and P2 (308.05 mm2). We found that cells became more elongated and less hexagonal from P1 to P3 (Aspect ratio = 1.21 in P1 and 1.38 in P3; Hexagonality = 9.18 in P1 and 8.62 in P3). Additionally, P1 and P2 were more sensitive to oxidative stress induced by sodium iodate. The average atrophy extended 1.9 mm from the center of the flatmount, encompassing P1 and P2.
Conclusions :
Based on cell area, there are three concentric RPE subpopulations in mice, with the center-most subpopulation being the largest followed by the outer-most subpopulation. The two subpopulations in the center are more susceptible to oxidative stress compared to the outermost subpopulation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.