Abstract
Purpose :
Nitrogen mustard (NM) is a cytotoxic and alkylating agent with no antidote. As a potent vesicant, it causes debilitating eye injury in the anterior segment. However, its effect on the retina is unknown despite evidence of retinal deficits in humans exposed to the agent. Here, we studied NM’s pathological effects in murine cornea and retina. We hypothesized that NM will cause degeneration of corneal Schwann cells (cSCs) and axons, and induce reactive gliosis and citrullination in injured mice retinas.
Methods :
Adult C57LB/6 wild-type (WT) mice and PLP-eGFP transgenic (TG) mice were used (Bargagna-Mohan et al., J. Neurosci. Res., 2020). After sedation, 5 μL of 0.2% or 1.0% NM was applied for 5 min in the right eye to induce injury, and flushed with 5 mL of sterile saline. The left eye was used as control. Mice were euthanized at 5 and 14 days post-injury. TG mice eyes were dissected for immunohistochemical (IHC) analyses of whole mount corneas (WMCs) and cryosectioned posterior eye cups (PECs). Whole eyes of WT mice were cryosectioned for IHC analyses. WMCs were stained for axonal β3-tubulin and imaged for eGFP of cSCs, while PEC tissues were co-stained for GFAP and citrullination (F95 antibody). Epifluorescence microscopy was used for imaging.
Results :
We found that 0.2% and 1.0% NM caused corneal injury and regression of stromal cSCs and axons, with no notable changes in the uninjured cornea. These findings were evident in the central and peripheral zones of the cornea, and were observed at both 5 and 14 days post-injury. In the injured retina, a global increase in reactive Muller glia with ample staining for GFAP was observed. Notably, the F95 antibody revealed increased citrullination in Muller glia at both 5 and 14 days post-injury time points at the 1.0% NM dose. Although the 0.2% NM dose caused corneal injury, retinal gliosis and increased citrullination were not observed. The cornea and retina responses to NM is consistent with our lab’s findings from previous models of corneal injury.
Conclusions :
Our initial results suggest that NM causes loss of corneal nerves leading to reduction in overall density, and increases Muller cell gliosis and citrullination of GFAP filaments in murine retinas. Our novel in vivo NM corneal injury model potentially supports real-life exposure and provides the first evidence that NM-induced retinal gliosis and hypercitrullination may be involved in retinal degeneration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.