MUSTARD GAS EXPOSURE AFFECTS LIMBAL STEM CELLS IN RABBIT CORNEA IN VIVO AND HUMAN CORNEA EX VIVO

Abstract

Purpose : Sulfur mustard gas (SM) has been used frequently as a chemical weapon by numerous countries in many wars and most recently in the Syrian conflict. We investigated impact of SM exposure on the corneal limbal stem cells using an in vivo rabbit and an ex vivo human cornea organ models of mustard gas keratopathy (MGK).

Methods : Naïve and SM-injured (200 mg/m3 for 8 min) corneas of New Zealand White Rabbits were used. Healthy donor human corneas were exposed to vehicle or nitrogen mustard (NM; 200uM), a SM surrogate, in an organ culture setting for 24h, 48h, or 72h. Slit lamp, confocal, and optical coherence tomography were used for clinical evaluations and H&E, immunostaining, qRT-PCR, and fluorescence microscopy techniques were used for molecular and histopathology studies. The ABCG2 (stem cell marker), ABCG5 (limbal stem cells), K12 (limbal epithelial cells), and p63 (S phase proliferation) expression in -/+ SM/NM exposed corneas were examined changes in corneal limbal stem cells in vivo and ex vivo corneas.

Results : SM-exposed rabbit eyes showed recurrent corneal epithelial erosions, inflammation, haze, and neovascularization, ocular pain, and increased tearing compared to naive. In Vivo confocal microscopy in live rabbits showed decreased basal limbal epithelial cell density. Post-euthanasia, H&E staining of rabbit corneal sections showed irregular morphology and/or cell organization in limbal stem cell region and decreased nuclear staining in limbal crypts. NM-exposed human cornea developed haze/opacity and swelling in a time dependent manner in an organ culture setting while vehicle-exposed cornea remained normal. The H&E histology of NM-exposed human corneas revealed compromised cellular density and disorganization of basal limbal epithelial stem cells in limbal crypts compared to vehicle-treated cornea.. qRT-PCR and immunofluorescence exhibited notably altered ABCG2, ABCG5, K12, and p63 expression in the limbal crypts in NM-exposed human corneas compared to vehicle-treated cornea. Quantification and statistical analyses are underway.

Conclusions : Mustard gas exposure damages corneal limbal stem cells in rabbits in vivo and human cornea ex vivo. In depth additional studies are warranted.

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