Role of Nrf2 signaling pathway in chloropicrin-induced cornea injury

Abstract

Purpose : Chloropicrin (CP), a choking agent, has been used during World War I as a chemical weapon and its alleged use in the recent Russian-Ukraine conflict has also been reported. CP is currently a popular pesticide and fumigating agent; thereby easing its acquisition for terrorism. Although CP exposure most frequently results in cornea injury, there are no targeted treatments. In vitro studies indicate that CP exposure induces oxidative stress and inflammation, and that targeting both pathways can better counteract CP-induced damage. Nuclear factor-erythroid factor-2-related factor2 (Nrf2) pathway regulates the cellular expression of both anti-oxidative and -inflammatory genes. Using a relevant mouse in vivo injury model, we tested the hypothesis that Nrf2 plays a protective role in CP-induced corneal injury and its genetic-loss of function results in exacerbated corneal injury.

Methods : The left eye of male 8-10 weeks old wild type (WT) and Nrf2 knockout (KO) Balb/C mice was exposed to CP (~0.7652ppb for 1min) and the right eye served as control. Clinical assessments were carried out at multiple timepoints post exposure. Mice were euthanized at day 1- and 7- post exposure. Tissue injury using fixed ocular tissue was assessed using hematoxylin & eosin staining. Myeloperoxidase (MPO) for neutrophil infiltration and gene expression of proinflammatory cytokines and angiogenesis markers was assessed using immunohistochemistry (IHC-P) and qPCR, respectively. Statistical analysis (p<0.05; n=3-10/group) of our results was done using one-way ANOVA with multiple comparisons where appropriate.

Results : Our studies shows that CP causes an early, more severe, and significant increase in gross corneal lesions in Nrf2 KO mice compared to WT mice. Analysis showed a significant degradation of corneal epithelial layer and increased stromal thickness in both mice groups on day 1- and 7- post exposure. However, regeneration of epithelial layer was reduced, and neutrophils infiltration was increased in Nrf2 KO mice compared to WT mice. Furthermore, qPCR showed an increase in vascular endothelial growth factor, and a significant increase in tumor necrosis factor-alpha and interleukin-6, at day 7-post exposure in Nrf2 KO mice compared to WT mice.

Conclusions : These novel findings support our hypothesis that Nrf2 signaling pathway plays a protective role in CP-induced corneal injury, and this pathway could be a therapeutic target.

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