Acute corneal injury in rabbits following nitrogen mustard ocular exposure

Abstract

Sulfur mustard (SM), a potent vesicating chemical warfare agent, and its analog nitrogen mustard (NM), are both strong bi-functional alkylating agents. Eyes, skin, and the respiratory system are the main targets of SM and NM exposure; however, ocular tissue is most sensitive, resulting in severe ocular injury. The mechanism of ocular injury from vesicating agents' exposure is not completely understood. To understand the injury mechanism from exposure to vesicating agents, NM has been previously employed in our toxicity studies on primary human corneal epithelial cells and ex vivo rabbit cornea organ culture model. In the current study, corneal toxicity from NM ocular exposure (1%) was analyzed for up to 28 days post-exposure in New Zealand White male rabbits to develop an acute corneal injury model. NM exposure led to conjunctival and eyelid swelling within a few hours after exposure, in addition to significant corneal opacity and ulceration. An increase in total corneal thickness and epithelial degradation was observed starting at day 3 post-NM exposure, which was maximal at day 14 post-exposure and did not resolve until 28 days post-exposure. There was an NM-induced increase in the number of blood vessels and inflammatory cells, and a decrease in keratocytes in the corneal stroma. NM exposure resulted in increased expression levels of cyclooxygenase-2, Interleukin-8, vascular endothelial growth factor and Matrix Metalloproteinase 9 indicating their involvement in NM-induced corneal injury. These clinical, biological, and molecular markers could be useful for the evaluation of acute corneal injury and to screen for therapies against NM- and SM-induced ocular injury.

Keywords: Corneal injury; Inflammation; Mustard; Nitrogen mustard; Sulfur mustard; Vesicant.

Figure 1.

Clinical progression of ocular injury following NM exposure. Right eye of New Zealand white rabbits was exposed to NM (1%) for 5min and the left eye was exposed to safine and ciinical progression of injury was observed for 28 days post-exposure as detailed in the “Materials and methods” section. Representative pictures showing injury to the rabbit eyes and the progression of ocular injury following NM exposure (A). Representative Slit lamp pictures showing corneal opacity (B), and corneal ulceration (C) following NM exposure. Green arrows, scarring or clouding of cornea or corneal opacity; red arrows, corneal ulceration; fluorescein stain taken up by exposed stroma, due to the disruption of epithelial layer, appears green. Quantification of corneal-stromal injury and corneal ulceration (D). Control (blue line) represents both corneal-stromal injury and corneal ulceration. Data presented are mean ± SEM (n=3–6). +, p<0.05 compared to the control group (corneal stromal injury); *, p<0.05 compared to the control group (corneal ulceration).

Figure 2.

Effect of NM exposure on corneal thickness (A), epithelial degradation (B), and epithelial-stromal separation (C). Right eye of New Zealand white rabbits was exposed to NM (1%) for 5min and the left eye was exposed to saline. The rabbits were euthanized at days 3, 14, and 28 post NM-exposure, the eyes were dissected, and the corneal tissue was fixed for histopathologic evaluation. The total corneal thickness, epithelial degradation, and epithelial-stromal separation in the hematoxylin and eosin (H&E) stained corneal sections was measured detailed under the “Materials and Methods” section. Representative images showing corneal thickness and bar diagram showing quantitative data from the measurement of the corneal thickness (A) in control and NM exposed corneal sections. Representative images showing epithelial-degradation and bar diagram showing quantification of the epithelial-degradation (B) in control and NM exposed corneal sections. Representative images showing epithelial-stromal separation and bar diagram showing quantification of the epithelial-stromal separation (C; no control bars indicate that there was no epithelial-stromal separation in control corneas). Representative control images are from 3-day post NM exposure. Data presented are mean ± SEM (n=3). *, p<0.05 compared to the control group, e, epithelium; s, stroma; red arrows, epithelial-stromal separation; red size bar (corneal thickness), 200 μm; black size bar (epithelial degradation and epithelial-stromal separation), 50μm.

Figure 3.

NM exposure causes a decrease in the number of keratocyte (A), and an increase in the number of inflammatory cells (B) and blood vessels (C) in the stroma of rabbit cornea. Right eye of New Zealand white rabbits was exposed to NM (1%) for 5min and the left eye was exposed to saline. The rabbits were euthanized at days 3, 14, and 28 post NM-exposure, the eyes were dissected, and the corneal tissue was fixed for histopathologic evaluation. The corneal sections were H&E stained and number of keratocytes, inflammatory cells, and blood vessels were quantified as detailed under the “Materials and Methods” section. Representative images showing a decrease in keratocytes and bar diagram showing the quantification of keratocytes in the stroma (A). Representative images showing an increase in the inflammatory cells in the corneal stroma following NM exposure and bar diagram showing the quantification of inflammatory cells (B; no control bars at day 3 indicate that there was no significant inflammatory cell infiltration observed in the control corneas) detailed under the material and methods section. Representative images showing an increase in number of blood vessels in the corneal stroma and bar diagram showing the quantification of blood vessels (C; no control bars at day 3 and day 14 indicate that blood vessels in the control corneas were not observed at these time points). Representative control images are from 3-day post NM exposure. Data presented are mean ± SEM (n=3); *, p<0.05 compared to the control group, e, epithelium; s, stroma; red arrows, keratocytes/inflammatory cells/blood vessels; size bar, 50μm

Figure 4.

NM exposure causes an increase in the expression of COX-2 (A), MMP-9 (B), and VEGF (C) expression in rabbit cornea. Right eye of New Zealand white rabbits was exposed to NM (1%) for 5min and the left eye was exposed to saline. The rabbits were euthanized at days 3, 14, and 28 post-NM exposure, the eyes were dissected, and were either fixed for immunohistochemistry (IHC) evaluation or frozen for cytokine array analysis. The corneal sections were IHC stained for COX-2, MMP-9, and VEGF expression as detailed under the “Materials and Methods” section. Representative images showing an increase in COX-2 expression (A) following NM exposure. IL-8 levels in corneal lysates at 3-day post-NM exposure (B). Representative pics for MMP-9 staining (C) and MMP-9 levels in corneal lysates at 3-day post NM-exposure (D). Representative images showing VEGF expression (E) and bar diagram showing quantification of the staining (F; no control bars at day 14 indicate that there was no significant VEGF staining observed in control corneas) following NM exposure. Representative control images are from 3-day post NM exposure. Data presented are mean ± SEM (n=3). *, p<0.05 compared to the control group, e, epithelium; s, stroma; size bar, 50μm.