Effects of mustard gas exposure to the corneal basement membrane and epithelial-stromal attachment to human cornea ex vivo

Abstract

Purpose : Sulfur mustard (SM) exposure to the eye causes many keratopathies and vision loss. The damage to various corneal components and underlying mechanisms are still largely unknown. This study investigated the effects of SM on the corneal epithelial basement membrane and epithelial-stromal attachment using a human corneal ex vivo organ culture model.

Methods : An ex vivo human cornea organ culture model available in the lab was used. Thirty donor healthy human corneas suitable for research were purchased from various Eye Banks. Corneas were placed into a well of a standard 6-well tissue culture dishes on a sterile customized corneal conformer to maintain in culture for long durations. Each well was fed with MEM medium having 10% FBS (4.5-5.5ml), allowing corneal endothelium to continually bathed in medium with an air-lift environment. The axial cornea air-exposed received one drop of medium every 8h to prevent corneal desiccation. Cultures were maintained in a humidified 5% CO₂ incubator at 37°C for 2-4 weeks in +/- SM analog (200μM of Nitrogen Mustard). An eight mm circular filter disc was used for SM exposure to the cornea. Standard H&E staining, real-time quantitative RT-PCR (qRT-PCR), western blotting, and Immunofluorescence (IFC) techniques were used to study cellular and molecular parameters.

Results : The vehicle-treated human cornea revealed normal corneal epithelium, epithelial basement membrane, epithelial-stromal association, stromal arrangement, and endothelium in H&E staining. Conversely, SM exposure to cornea demonstrated varying levels of damage to the corneal epithelium, epithelial basement membrane, epithelial-stromal association, stromal arrangement, and endothelium under similar culture conditions in H&E staining. The IFC and qRT-PCR data showed a significant change in the level of integrin β4 (p<0.001), Collagen XVII (p<0.01), and Col IV (p<0.001) compared to vehicle-treated control. Tested proteins play a crucial role in the maintenance of normal corneal architecture and function. The Western blot analyses validated findings of the IHC and qRT-PCR.

Conclusions : Mustard gas exposure to the human eye compromises corneal basement membrane and epithelial-stromal attachment crucial for maintaining refraction.

This is a 2021 ARVO Annual Meeting abstract.