Abstract
Purpose :
Vesicant agents, such as nitrogen mustard (NM), have been used in biological warfare and are known to cause severe ocular injury. The mechanisms of vesicant-induced ocular injuries, both acute and chronic, are not well-understood, and there is a lack of effective treatments.
Methods :
Porcine corneas were exposed to NM by placing sterile Whatman #3 filter discs (5 mm) wetted with media containing either 0 (the control) or 5 mg/ml NM onto the central cornea. The disks were allowed to stay on the corneas for 0, 5, 10, and 15 min. After exposure, the corneas were rinsed extensively and transferred to a new 35-mm dish with 2 ml fresh medium for 24 and 48 h. Corneas were collected; H&E, TUNEL, and Immunohistochemistry with antibodies against cleaved caspase3, p-RIPK3, and p-MLKL, were performed. The epithelial defects of NM-exposed corneas were assessed with fluorescence staining at 24 and 48 h.
Results :
Epithelium, stroma, and endothelial layers were all intact in the control and with 5 minutes of exposure to the cornea (MEC). With 10 MEC, injury extends into the stroma. With 15 MEC, the entire cornea was affected, with no identifiable endothelial cells. There is an increase in both apoptosis and necroptosis, illustrated by tunnel staining, cleaved casp3, p-RIPK3 and p-MLKL. In rat corneas, exposure to 5 mg/ml NM for 10 min resulted in damage to the cornea at 24 h, manifesting as erosion of the epithelium at 48 h. A significant stromal injury was also observed. Inhibition of RIPK3 with GSK872 resulted in wound closure without any detectable epithelial defects in the cultured pig corneas.
Conclusions :
NM induces exposure time- and dosage-dependent damage to the cornea that manifests with the time post-NM exposure. NM-caused epithelial defects can be attenuated by targeting necroptosis, suggesting that necroptosis might be a potential target(s) for medical countermeasures against chemical ocular toxicity.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.