Chlorine gas toxicity to the mouse cornea in vivo and human cornea in vitro

Abstract

Purpose : Chlorine (Cl₂) is widely used in household cleaning. Also, it is a warfare agent and used during world wars. Cl₂ ocular exposure damages cornea and causes vision impairment. This study investigated the toxicological effects of Cl₂ on mouse cornea in vivo and optimized conditions for an in vitro model of Cl₂ toxicity to human cornea.

Methods : Eyes of C56BL/6 mice were exposed to Cl₂ gas (100ppm for 20min) by placing mouse in a leakproof custom gas-exposure chamber equipped with gas-sensor inside chemical hood. Treated eyes were subjected to clinical microscopic (slit-lamp, confocal, spectralis, and specular) examinations and fluorescein eye test in live animals at regular intervals. Primary human corneal stromal fibroblasts (hCSF) isolated from healthy cadaver corneas were used for in vitro model development. Cultures, at 70% confluence, were exposed to Cl₂ (0-1ppm) using sodium hypochlorite for 0-72h in culture plates sealed with a commercial aluminum sealing foil preventing gas leak. Cultures were maintained for a desired times in a standard humidified CO₂ incubator at 37°C. Phase-contrast microscope, fluorescence microscope, MTT, Trypan blue, and Click-iT^™ EdU proliferation assays analyzed changes in phenotype and functional parameters.

Results : Cl₂ exposure led to mild ocular inflammation, corneal epithelium erosion/defect, and sparse corneal haze in mouse eyes in vivo in a time dependent manner after initial exposure. Age-matched naive eyes exposed to air/vehicle showed no clinical symptoms. hCSF exposed to Cl₂ exposure showed a dose- and time-dependent decrease in cellular growth, viability, and proliferation (p<0.01 to 0.0001). Cl₂ doses of 0.05ppm for 72h and 0.08ppm for 6-8h appear optimal for in vitro model of Cl₂ toxicity profiling experiments. Ongoing H&E and immunofluorescence supported in vivo clinical eye examinations. The quantitative molecular studies are pending.

Conclusions : Chlorine exposure to eye jeopardizes corneal integrity in vivo and in vitro. Ongoing additional animal and culture studies will yield useful mechanistic information.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.