Abstract
Purpose :
The objective of this project is to establish a cell model to mimic chemical damaging processes in corneal cells and compare the cell model to the previously established animal model of corneal wound healing at cellular level.
Methods :
Human telomerase-immortalized corneal epithelial (hTCEpi) cells were used as the in-vitro model of the human corneal epithelium layer and human foreskin fibroblasts (HFF-1) served as the model of the human corneal stroma layer. Alkaline treatment was applied for 1 minute on the cells respectively, and they were cultured at different time points of 6 hours, 1, 4, 7 and 14 days. Cells were stained with 8 different immunofluorescent (IF) antibodies to check cell viability, proliferation, inflammation and repair. Confocal microscopy and ImageJ software were used to count the cells collected at each time point. RT-PCR was used in the same cell samples to look for different genes that correlated with cell viability, proliferation, inflammation, and repair. Finally, the hTCEpi and HFF-1 cell outcomes were compared, as well as in-vitro data to the previously established in-vivo data.
Results :
The IF data reveals dynamic changes in cellular markers over time after alkali treatment. Stromal cells exhibit a significant increase in Ki67 concentration (mean ± SD: Day 7, 85.36 ± 11.32) and ZEB1 concentration (Day 14, 97.84 ± 6.57). NFkB levels vary considerably (Day 0.25, 169.64 ± 68.94), and distinct patterns emerge in COL IV (Day 7, 14.75 ± 7.47), SMA-a (Day 14, 66.81 ± 29.77), VEGF (Day 14, 173.58 ± 101.08), IL-6 (Day 7, 99.77 ± 32.82), and TNFa (no measurable concentrations across all days). Epithelial cells display a peak in Ki67 concentration (Day 0, 59.02 ± 14.76) and a delayed rise in ZEB1 (Day 14, 184.96 ± 54.12). NFkB levels remain stable, COL IV increases at Day 7 (1.79 ± 3.57), SMA-a decreases at Day 7 (29.14 ± 24.32), and VEGF peaks at Day 14 (82.18 ± 26.53). RT-PCR cell model results mimicked the in-vivo model ZEB1 results, but had limitations in mimicking the inflammatory markers IL-1b and TGFB2.
Conclusions :
The cell model mimics cell responses to chemical burn in animal corneas. Despite its limitations, there are advantages to utilizing it in the lab setting because it has no ethical issues, it saves time and money, and it is easy to control. Establishing this cell model and dissecting pathways in the cells responding to wound healing will assist drug discoveries in treating corneal wound by alkaline burn.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.