Nitrogen mustard causes progressive tissue injury, severe DNA damage, and necroptosis in the cornea

The eye is sensitive and vulnerable to vesicants such as sulfur and nitrogen mustard (NM). To assess the adverse effects of NM exposure on the ocular surface, we used porcine and rat corneas and NM-wetted filtrate paper disks to assess NM's ex vivo and in vivo effects on corneal health. In cultured porcine corneas (N = 5), 5 mg/ml NM caused exposure time-dependent damage, which progressed from 1 to 2 days post-NM exposure (dpe), with increased programmed cell death, particularly necroptosis at 2 dpe. In rat corneas (N = 4), 5 mg/mL NM caused corneal opacification, punctate keratitis, pupil anisocoria, and decreased corneal sensitivity. At the tissue level at 1-dpe, epithelial cells exhibited a leading edge, the front row of epithelial cells that actively migrate to cover the wound area, with apical layers being c-Casp3 positive and basal and/or wing cells being p-RIPK3 positive. Most corneal cells were γH2AX-positive, and stromal cells were TUNEL-positive but c-Casp3-or p-RIPK3-negative. At 2-dpe, only a few cells were c-Casp3 positive, while most cells on the apical side of the cornea were p-RIPK3 positive. These changes were associated with basement membrane breakdown and tight junction loss in NM-exposed rat corneas. Finally, in cultured porcine corneas, the inhibition of necroptosis resulted in the prevention of corneal tissue destruction caused by NM. Our data suggest that the severity of NM-induced corneal injuries may be linked to increased necroptosis, and targeting necroptosis may reduce or prevent corneal deterioration caused by NM exposure.