Abstract
Purpose :
Guttae in Descemets membrane, the basement membrane of the corneal endothelium, are the fundamental clinical finding diagnostic of Fuchs endothelial corneal dystrophy (FECD). However, there are no in vitro models to study guttae. The purpose of this study was to develop a model to generate guttae in vitro. We hypothesized that methods to stress bovine corneal endothelial cells (BCEnCs), which deposit substantial extracellular matrix (ECM) in culture, would result in guttae formation.
Methods :
Primary BCEnCs were cultured without passage in DMEM with 10% FBS, 1% antibiotic/antimycotic, at 37°C, 2.5% O2, 5% CO2 ([O2]2.5). Sets of cultures (n=3) were seeded to allow paired experiments with treatment and control groups. Following confluence, cells were exposed to the following conditions: continuous oxygen stress with atmospheric O2 (room air, 5% CO2; [O2]A) or [O2]2.5; 10 μM menadione added to cultures 3x/week at [O2]A, and ultraviolet-A (UV-A) stress with weekly exposure to 2.5 J/cm2 for 4 weeks at [O2]A. Cultures were observed by phase contrast microscopy for changes in ECM and guttae-like mounds of ECM were counted. Guttae densities between culture conditions were compared with paired t-tests. Decellularized ECM was studied by scanning electron microscopy and compared to decellularized Descemets membrane from patients with FECD.
Results :
BCEnCs were resilient in culture and monolayers appeared intact except for transient disruptions to cell morphology with exposures to menadione and UV-A. Following 10-20 weeks in culture, guttae-like protrusions in the ECM were noted under all culture conditions. There were no significant differences in guttae density for menadione (mean ± SD, guttae/cm2; 0 μM: 9.5 ± 3.1; 10 μM: 13.3 ± 3.9; p = 0.212) or UV-A stress (0 J/cm2: 23.5 ± 19.9; 10 J/cm2: 34.4 ± 31.5; p = 0.320). However, significantly higher guttae density was observed with [O2]2.5 (33.7 ± 9.5) compared to [O2]A (7.4 ± 0.9; p = 0.049) conditions. With scanning electron microscopy, the shapes of guttae appeared similar between the decelluarlized BCEnC cultures and FECD specimens (Figure).
Conclusions :
BCEnCs in culture generate guttae-like protrusions of ECM that are enhanced under [O2]2.5 conditions. This is a novel in vitro model to study the role of external stressors in the development and progression of guttae.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.