Abstract
Purpose :
Aqueous tear deficiency (ATD) is characterized by increased evaporation of the ocular surface and decreased tear volume and accompanied by multiple ocular surface pathologic changes. This study aims to establish a dry eye model in vitro to mimic ATD pathologic changes and providing a new strategy for drug screening.
Methods :
For establishing the dry eye model, the corneal limbal epithelial cells and the conjunctival vault epithelial cells of SD rats co-cultured with NIH3T3 cell line in cell culture inserts respectively to form colonies. The colonies were submerged for the first 8 days, and air-exposed culture thereafter until the day 14, while the control group remained submerged. The macroscopic characteristic was observed by crystal violet staining. The colony epithelial stratification was observed in transverse section. The ultrastructure of the epithelium was observed by electron microscope. The normal and abnormal differentiation markers of corneal and conjunctival epithelium were detected by the qRT-PCR, Western blot assay, IF staining. The inflammatory cytokines were detected by qRT-PCR. Addition of 0.001% fluorometholone to the lower medium of insert on day 8 (corneal epithelium cell colonies) to test drug sensitivity of the model and to repeat the portion of the markers on day 14.
Results :
The corneal and conjunctival colonies which were air-exposed from day 8 to day 14 showed obvious stratification and multilateral structure vanished accompanied by a decrease in colony size. Electron microscopy showed that the microvilli became thinner and less tightly connected compared with submerged group. The tight junction marker ZO-1 showed reduced expression range. In both corneal epithelium and conjunctival colonies, the expression of abnormal marker Sprr1b and K10 increased, and the proliferation marker Ki67 decreased in air-exposed culture group. The inflammatory cytokines IL-1β, TNF-α, S100A9 and MMP9 increased after air-exposed. Addition of 0.001% fluorometholone to the lower medium of insert in corneal epithelium cell colony culture can inhibit the inflammatory cytokines activation and keep the well tight junction. The abnormal differentiation reduced and the size of colonies became larger.
Conclusions :
The in vitro dry eye model, based on ocular surface cell colony air-exposed culture, can mimic ocular surface pathologic changes of ATD and possesses sensitivity for drug screening.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.