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. Author manuscript; available in PMC: 2023 Oct 1.
Published in final edited form as: Exp Mol Pathol. 2022 Jul 4;128:104807. doi: 10.1016/j.yexmp.2022.104807

Sulfur Mustard Corneal Injury is Associated with Alterations in the Epithelial Basement Membrane and Stromal Extracellular Matrix

Laurie B Joseph a,*, Marion K Gordon a, Peihong Zhou a, Rita A Hahn a, Hamdi Lababidi a, Claire R Croutch b, Patrick J Sinko c, Diane E Heck d, Debra L Laskin a, Jeffrey D Laskin e
PMCID: PMC10044521  NIHMSID: NIHMS1871581  PMID: 35798063

Abstract

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8–17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45+ leukocytes and a decrease of keratocytes, along with areas of disorganization of collagen fibers. SM also disrupted the corneal basement membrane and altered the expression of perlecan, a heparan sulfate proteoglycan, and cellular fibronectin, an extracellular matrix glycoprotein. This was associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing. Tenascin-C, an extracellular matrix glycoprotein, was also upregulated in the stroma 14–28 d post SM, a finding consistent with its role in organizing structural components of the stroma necessary for corneal transparency. These data demonstrate that SM vapor causes persistent alterations in structural components of the cornea. Further characterization of SM-induced injury in rabbit cornea will be useful for the identification of targets for the development of ocular countermeasures.

Keywords: sulfur mustard, cornea, basement membrane, perlecan, tenacin-c

INTRODUCTION

Sulfur mustard (SM) is a highly toxic vesicant known to cause ocular injury including lacrimation, irritation, pain, and photophobia. This can progress to recurrent corneal ulceration and dry eye disease (Ghabili et al., 2010; Javadi et al., 2011; Kehe et al., 2009). Damage to the eye is dependent on SM exposure dose and duration, and time following exposure (Graham and Schoneboom, 2013; Jadidi et al., 2019; Kadar et al., 2009; McNutt et al., 2016). SM and the related vesicant nitrogen mustard (NM) target various ocular structures including the corneoscleral layer, the cornea, and the eyelids (Joseph et al., 2021; Panahi et al., 2017b; Uhde, 1946). Early effects of the vesicant on the cornea include extensive inflammation, edema and hazing, which is followed at later times by neovascularization (Amir et al., 2000; McNutt et al., 2012; Petrali et al., 2000; Tripathi et al., 2020). This is associated with epithelial cell necrosis, epithelial erosion, basement membrane damage, stromal deformation, and disruption of the corneal endothelium (Kadar et al., 2013a; Kadar et al., 2013b; McNutt et al., 2020). Damage to the basement membrane zone and epithelial layer is thought to be due, in part, to the action of metalloproteinases which degrade extracellular matrix proteins causing separation of the corneal epithelium from the basement membrane (Fini and Girard, 1990; Gordon et al., 2016; Matsubara et al., 1991).

In the present studies, we further characterized SM induced corneal injury in rabbits using a novel continuous vapor exposure model, which more closely reflects human exposure (Fuchs et al., 2021; Ghanei et al., 2010). We demonstrate that structural damage to the cornea in response to SM is correlated with changes in expression of corneal matrix proteins and matrix metalloproteinases. Moreover, these changes persist up to 28 days post SM exposure suggesting prolonged wound healing. Identification of macromolecules that are targeted by SM will aid in the development of therapeutics to mitigate tissue damage and improve wound repair.

MATERIALS AND METHODS

Animals and Exposures.

All SM exposures were performed as previously described (Goswami et al., 2021) at MRIGlobal, Kansas City, MO in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility following Institutional Animal Care and Use Committee (IACUC) guidelines. Animals (male New Zealand white rabbits,12 weeks of age, 2.5 – 4.0 kg, Charles River Laboratories, Kingston, NY) received humane care in compliance with an MRIGlobal IACUC approved protocol, and as outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Rabbits were treated with buprenorphine-SR (SQ) beginning 1 h before treatment with SM and up to 7–9 days post exposure, as required. Animals were anesthetized using ketamine+xylazine+acepromazine (IM) and then exposed to either air or SM vapors (at a flow rate of approximately 56mL/min) using continuous flow ocular goggles in a Chemical Surety hood. The left eye of the animal was treated as control (naïve, CTL) and the right eye was exposed to 420 μg/L SM at 37°C. After 8 min of SM vapor exposure, the corneas were flushed with saline and allowed to off-gas for 2 h. Rabbits were then returned to the general vivarium and evaluated daily for signs of injury. Corneal neovascularization, opacity/hazing, and thickness were assessed one day prior to exposure and on days 1, 3, 7, 21 and 28 post exposure as previously described (Gordon et al., 2010; Tewari-Singh et al., 2016). Rabbits were euthanized at 3, 14, and 28 days post exposure, the globes were excised, and corneas isolated with a 2–3 mm scleral rim. Corneas were then placed in OCT, frozen and shipped to Rutgers University for analysis.

Histology and immunohistochemistry.

Tissue sections (10 μm) were prepared using a Leica CM 1950 Cryostat (Leica Biosystems, Nussloch, Germany) and stained with hematoxylin and eosin (H&E) or Masson’s trichrome. Images were acquired at high resolution using an Olympus VS120 Virtual Microscopy System and analyzed using OlyVIA version 2.9 software (Center Valley, PA). Corneal epithelial-stromal separation was quantified as previously described (DeSantis-Rodrigues et al., 2021). Briefly, in histological sections, attached and detached areas of the cornea were measured. The percent detachment and remaining epithelial–stromal attachment preserved after SM exposure were calculated as follows:

1 − (width of total detachments) / (entire width of the cornea) × 100 = percent epithelial separation

For immunohistochemistry, tissue sections were fixed in ice cold 100% HPLC grade acetone (Thermo Fisher Scientific, Waltham, MA), air dried, and rehydrated in phosphate buffered saline (PBS). To remove endogenous peroxidase, tissue sections were incubated in Bloxall (Vector Labs, Burlingame, CA) for 15 min, rinsed with PBS and then incubated in horse serum (Gibco, Thermo Fisher Scientific). After 2 h, tissue sections were incubated overnight at 4°C with mouse monoclonal antibodies to aldehyde dehydrogenase 1A1 (ALHD1A1,1:40, Santa Cruz Biotechnologies, Dallas TX), CD 45 (1:200, Kingfisher Biotech Inc, St Paul, MN), cellular fibronectin (1:1000, cFN, Abcam, Cambridge, UK), keratin 3 (1:4000, Abcam), perlecan (1:4000, Abcam), tenasin C (TNC, 1:1000, Abcam), proliferating cell nuclear antigen (PCNA, 1:4000, MilliporeSigma, Burlington, MA), or control IgG (ProSci, Atlanta, GA). Tissue sections were washed and incubated at room temperature with biotinylated horse anti-mouse secondary antibody (Vector Labs, Burlingame, CA). Binding was visualized with DAB Peroxidase Substrate Kit (Vector Labs). Tissue sections were counterstained with hematoxylin and cover slipped with Permount (Thermo Fisher Scientific). Images were acquired at high resolution using an Olympus VS120 Virtual Microscopy System and analyzed with OlyVIA version 2.9 software. ALDH1A1 and CD45 cell density (cells/mm3) within the upper third of the stroma was determined using OlyVIA version 2.9 software.

For immunofluorescence studies, tissue sections were incubated with a mouse monoclonal antibody to ADAM17 (1:500, R&D Systems, Minneapolis, MN) or control IgG (ProSci). After overnight incubation at 4°C, sections were washed with PBS and incubated at room temperature for 1 h with donkey-anti-mouse Alexafluor488 (1:1000, Invitrogen, Carlsbad, CA). 4′,6-Diamidino-2-phenylindole (DAPI) was used to visualize nuclei. Fluorescence was visualized using a Zeiss fluorescent microscope with ProgRes Capture Pro software (Jenoptik, Jena, Germany).

Statistical Analyses.

Data are presented as the mean ± SE and were analyzed using Student’s-t test. Results are considered significant at p ≤ 0.05.

RESULTS

Clinical signs of SM ocular toxicity in the rabbit.

SM exposure resulted in a rapid increase in corneal opacity at 1–3 days, a response that persisted for at least 28 days (Fig. 1, Panels A and B). This was correlated with increases in corneal thickness (Fig. 1, panel B). During this process, corneal cloudiness partially obscured the delineation of the iris’ inner edge; an inflamed third eyelid/nictitating membrane also became evident (Fig. 1, panel A). SM also induced corneal neovascularization, which was apparent 7 days post exposure and increased continuously for at least 28 days (Fig. 1, Panel B). In contrast, control corneas exhibited a clear, glossy appearance with a crisp view of the underlying iris throughout the 28 day time course (Fig. 1, Panel A and B).

Figure 1:

Figure 1:

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Effects of SM vapor on rabbit cornea. Panel A: Rabbit eyes were treated with CTL or SM for 3 d, 14 d or 28 d as described in the Materials and Methods. Representative eyes are shown. Asterisk, third eyelid; arrowhead, neovascularzition. Panel B: Clinical profile of corneal injury. Rabbit corneas were treated with CTL or SM and corneal thickness, opacity and neovascularization assessed on days 0, 1, 3, 7, 14, 21 and 28. Each data point represents the mean ± SE (n = 4). *Significantly different (p<0.05) from CTL. Note opacification of the cornea 3–28 days post-SM. This was associated with increases in corneal thickness.

Changes in the cornea epithelium and stroma following SM exposure.

The cornea epithelium is composed of nonkeratinized stratified squamous epithelial cells, which include columnar basal cells anchored to the underlying basement membrane and suprabasal polyhedral wing cells and flattened superficial cells (Figs. 2A and 3). Keratin 3, a marker of corneal epithelial cells, was uniformly distributed throughout the epithelium of control corneas (Fig. 3). Thinning of the corneal epithelium was noted 3 days post-SM, with no change in keratin 3. At 14 days post-SM, keratin 3 expression decreased in squamous epithelial cells and columnar basal cells. The distribution and expression of keratin 3 within the corneal epithelial layer subsequently increased approaching control levels by 28 days post-SM. Consistent with wound healing, 14–28 days post SM, corneal epithelium thickness increased (Figs. 2A and 3). Areas of epithelial stromal separation or microblistering were evident 3 days post-SM which persisted for at least 28 days, although at this time, microblistering was reduced relative to 3 days (Figs. 2A and 2B). To further analyze changes in the thickness of the epithelium, we assessed expression of PCNA, a marker of cellular proliferation. In control corneas, PCNA was noted in small well-rounded nuclei within the multilayered stratified squamous epithelium (Fig.4A). PCNA was predominantly expressed in the suprabasal wing cells and flattened superficial cells. Treatment of corneas with SM resulted in a decrease in the number of PCNA positive cells in the epithelium at 3 and 14 days post-exposure (Fig. 4B); this was associated with increased numbers of superficial cells with flattened nuclei. At 28 days post-SM, there was an increase in the total number of epithelial cells expressing PCNA.

Figure 2:

Figure 2:

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SM vapor induces structural changes in the rabbit cornea. Panel A: Histological sections, prepared from control (CTL) corneas and corneas 3, 14 and 28 d post-SM, were stained with H&E. Note the degradation of the stromal epithelium on day 3. One representative section from 4 rabbits/exposure group is shown. Ep, epithelial cells; S, stroma; arrows, keratocyte. Panel B: Histological sections, prepared from control (CTL) corneas and corneas 3, 14, and 28 days post-SM, were stained with H&E and analyzed for epithelial-stromal separation in the central corneas. Each bar represents the mean ± SE (n = 4). *Significantly different (p≤0.05) from CTL.

Figure 3:

Figure 3:

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Effects of SM vapor on expression of keratin 3 in the rabbit cornea. Histological sections, prepared from control (CTL) corneas and corneas 3, 14, and 28 days post-SM, were stained with an antibody against keratin 3. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 4 rabbits/exposure group is shown. Ep, epithelium; S, stroma.

Figure 4:

Figure 4:

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Effects of SM vapor on expression of PCNA in the rabbit cornea. Panel A: Histological sections, prepared from control (CTL) corneas and corneas 3, 14, and 28 days post-SM, were stained with an antibody against PCNA. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 4 rabbits/exposure group is shown (n=4 rabbits/exposure). Ep, epithelium; S, stroma. Panel B:Control cornea epithelial cells and epithelial cells from corneas 3, 14, and 28 days post-SM expressing PCNA were enumerated (15–20 fields/cornea). Each bar represents the mean ± SE (n = 4). *Significantly different (p≤0.05) from CTL.

The stroma is composed of extracellular matrix proteins, primarily collagens. It forms a tightly packed lamellar structure with spindle shaped keratocytes (Figs. 2A, 5 and 6). Disorganization of the collagen fiber bundles, and edema were evident in the stroma following SM exposure; prominent areas with separation of lamellar structures were noted in the posterior stroma and to a lesser extent, in the anterior stroma (Fig. 5). SM caused a persistent decrease in the number of ALDH1A1 positive keratocytes in the edematous stroma (Fig. 6, left panel). The epithelial cell layer also expressed ALDH1A1, which decreased during the initial stage of damage (day 3) with increased expression observed 14–28 post SM-exposure (Fig 6, left panel and inset). SM also induced inflammation as indicated by an increase in the number of CD45+ leukocytes in the stroma (Fig. 6, right panel).

Figure 5:

Figure 5:

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Trichrome staining of rabbit conjunctiva following exposure to SM vapor. Histological sections, prepared from control (CTL) corneas and corneas 14 days post-SM, were stained with Masson’s trichrome containing hematoxylin, which stains nuclei dark blue/black, eosin which stains cytoplasm red, and aniline blue, which stains collagen I/III royal blue. One representative section 4 rabbits/exposure group is shown. Ep, epithelium; DM, Descemet’s membrane. Note the tightly packed lamellar structure in control corneal stroma. Areas of disorganized lamellar structures and edema were evident in the posterior stroma (arrow) and to a lesser extent in the anterior stroma (arrowhead) following SM exposure.

Figure 6:

Figure 6:

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Effect of SM vapor on epithelium, stromal keratocytes and leukocytes in rabbit cornea. Histological sections, prepared from control (CTL) corneas and corneas 3, 14, and 28 days post-SM, were stained with an antibody against ALDH1A1 (Left Panel) or CD45 (Right Panel). Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section from 4 rabbits/exposure group is shown. The upper third of the stroma of 4 rabbits/exposure group were measured. Left panel, Inset. ALDH1A1 epithelial expression in control cornea and corneas 3, 14, and 28 days post-SM were enumerated (15–20 fields/cornea). Each bar represents the mean ± SE (n = 4). *Significantly different (p≤0.05) from CTL. Arrows, keratocytes.

Effects of SM on the corneal basement membrane and stroma.

The matrix proteins, perlecan, and cellular fibronectin, a proteoglycan and a high molecular weight glycoprotein, respectively, are secreted by epithelial cells and stromal keratocytes (Barbariga et al., 2019; Torricelli et al., 2016; Wilson et al., 2020). Both proteins were highly expressed as a contiguous layer in control corneal basement membrane and in the stroma within the lamellar structures, and in keratocytes (Figs. 7 and 8). At 3 days post-SM, perlecan was upregulated in the basal lamina and extended into the stroma (Fig. 8). Expression levels of perlecan were reduced in the basement membrane and stroma 14 and 28 days post SM; a thickened diffuse band of perlecan was evident along the basement membrane (Fig. 7). SM caused a dramatic reduction in expression of cellular fibronectin at 3 days. Subsequently, levels began to increase. Low levels of cellular fibronectin were detected in the basement membrane with diffuse expression noted in the stroma after 14 days; by 28 days, the protein was identified in the basement membrane, throughout the stroma and in stromal keratocytes (Fig. 8).

Figure 7:

Figure 7:

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Effects of SM vapor on perlecan expression in the rabbit cornea. Histological sections, prepared from control (CTL) corneas and corneas 3, 14, and 28 days post-SM, were stained with an antibody against perlecan. Note changes in the distribution of perlecan in the basement membrane following treatment with SM. Antibody binding was visualized using a Vectastain Elite ABC kit. One representative section 4 rabbits/exposure group is shown. Ep, epithelium; S, stroma. Arrows, basal lamina.

Figure 8:

Figure 8:

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Effects of SM vapor on expression of cellular fibronectin in the rabbit cornea. Histological sections, prepared from control (CTL) corneas and corneas 3, 14, and 28 days post-SM, were stained with an antibody against cellular fibronectin. Antibody binding was visualized using a Vectastain Elite ABC kit. Note the decrease in cellular fibronectin following treatment with SM. One representative section from 4 rabbits/exposure group is shown. Ep, epithelium; S, stroma; arrowheads, basal lamina; arrows, keratocytes.

Earlier studies have shown that SM and nitrogen mustard are potent inducers of matrix metalloproteinase 9 (MMP9), an enzyme that degrades extracellular matrix (Goswami et al., 2019; Horwitz et al., 2014). ADAM17, a related metalloproteinase that degrades basement membrane proteins and heparan sulfate proteoglycans, is upregulated in the corneal epithelium following nitrogen mustard exposure (DeSantis-Rodrigues et al., 2016). Similarly, we found that ADAM17 was markedly upregulated in the corneal basement membrane 3 days post SM exposure; it was also interspersed between cells in the epithelium (Fig. 9). Increased expression of ADAM17 persisted for at least 28 days post-SM.

Figure 9:

Figure 9:

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Effects of SM vapor on expression of ADAM17 in the rabbit cornea. Histological sections, prepared from control (CTL) corneas and corneas 3, 14, and 28 days post-SM, were stained with an antibody against ADAM17. Antibody binding was visualized with donkey-anti-mouse Alexafluor488. Note the persistent increase in expression of ADAM17 in the corneal basement membrane following treatment with SM. One representative section from 4 rabbits/exposure group is shown. Ep, epithelium; S, stroma. Arrows, basement membrane zone.

Tenascin-C is an extracellular matrix glycoprotein synthesized by keratocytes and fibroblasts following injury (Saika et al., 2013; Tanaka et al., 2010). SM exposure was associated with a time related increase in tenascin-C expression throughout the stroma. Conversely, tenascin-C was not detected in epithelial cells from control or SM-treated corneas (Fig. 10).

Figure 10:

Figure 10:

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Effects of SM vapor on expression of tenascin-C in the rabbit cornea. Histological sections, prepared from control (CTL) corneas and corneas 3, 14, and 28 days post-SM, were stained with an antibody against tenascin-C. Antibody binding was visualized using a Vectastain Elite ABC kit. Note selective expression of tenacin-C in the corneal stroma. One representative section from 4 rabbits/exposure group is shown. Ep, epithelium; S, stroma.

Discussion

In both humans and animal models, SM causes sustained ocular injuries (Dachir et al., 2019; Dahl et al., 1985; Fuchs et al., 2021; Graham and Schoneboom, 2013; Ruff et al., 2013). To better understand damage in the cornea following SM exposure, we used a rabbit vapor exposure model. We found that SM readily induces an inflammatory response which includes edema, hazing and angiogenesis. Edema or swelling of the cornea following exposure to SM is largely the result of excessive buildup of fluids and disorganization of collagen fibrils (Farrell and McCally, 1976; Kamil and Mohan, 2021). When compared to the densely packed lamellae of the normal corneal stroma, separation of the lamellar layers was evident, most notably in the anterior area of the stroma. This is in line with earlier studies showing edema localized in the central cornea of rabbits exposed to SM (McNutt et al., 2012; Tripathi et al., 2020). Disruption of the lamellar structure, as well as the diameter and spacing of collagen fibrils, contributes to loss of corneal transparency during edema (Connon and Meek, 2004; Meek and Knupp, 2015). Other factors such as disruption of the extracellular matrix including proteoglycans, which participate in organizing corneal fibrillar collagens, as well as fibrotic scarring may also contribute to hazing (Fuchs et al., 2022; Hassell and Birk, 2010; Moller-Pedersen, 2004). Disruption of corneal stromal lamellar structures has been reported following corneal injury and in diseases such as bullous keratopathy, Fuchs’ endothelial dystrophy and herpetic keratitis, which are associated with corneal edema and opacification (Kenney et al., 2004).

Water and electrolyte/mineral balance are critical for maintaining corneal clarity (Cao et al., 2010). Under normal physiological conditions, the cornea is in a state of relative dehydration maintained by the barrier functions of the epithelial and endothelial layers (Santerre et al., 2020; Zhang et al., 2014). This state of relative dehydration or deturgescence of the corneal tissue, is required for normal vision (Srinivas, 2010). Alterations in the cornea (e.g., reduced epithelial adherence to the basement membrane and basement membrane abnormalities), and disruption of the corneal stroma contributes to the buildup of fluids and changes in electrolyte/mineral composition in the cornea following injury (von Fischern et al., 1998). Previous studies have reported damage to the corneal epithelium following exposure to SM including changes in epithelial thickness, numbers of proliferating and apoptotic cells, ulceration and microblistering, along with changes in the basement membrane zone (Kadar et al., 2011; Pajoohesh et al., 2017), and this may allow water penetration. SM has also been shown to cause a loss of corneal endothelial cells, a potential mechanism for increased permeability through the corneal endothelium (Kadar et al., 2013a; McNutt et al., 2013). It has been suggested that persistent corneal edema may be due to slow or limited repair of the corneal endothelium after SM exposure (Jafarinasab et al., 2010; McNutt et al., 2020).

The cornea is normally maintained in an avascular state, which is critical for visual acuity (Azar, 2006; Di Zazzo et al., 2021; Torricelli and Wilson, 2014). In humans, and in rodent and rabbit models, exposure to SM induces neovascularization (Baradaran-Rafii et al., 2011; Goswami et al., 2016a; Panahi et al., 2017a; Ruff et al., 2013). This is thought to be due, in part, to suppression of anti-angiogenic factors in the cornea such as endostatin, thyrosinase, semaphorin 3F, angiostatin and thrombospondin, and increases in pro-angiogenic factors including VEGF, FGF-2, TGF-α, MIF and IL-1β (Hadrian et al., 2021; Sabzevare et al., 2021). The importance of pro-angiogenic factors is evident from findings that interfering with VEGF in the cornea using bevacizumab, a humanized monoclonal anti-VEGF antibody or aflibercept, a fusion protein with high affinity for VEGF, suppress SM-induced neovascularization (Gore et al., 2021; Kadar et al., 2014). Changes in the composition of extracellular matrix proteins in the cornea are also important in the control of neovascularization (Pouw et al., 2021). For example, earlier studies have shown that both stromal fibronectin and tenascin-C are essential for neovascularization following corneal injury (Barbariga et al., 2019; Sumioka et al., 2013; Tuori et al., 1997).

The present studies show that both control and SM-treated corneas express keratin 3, a marker of corneal epithelial differentiation (Kao, 2020; Schermer et al., 1986). Keratin 3, in conjunction with its binding partner, keratin 12, form intermediate filaments which strengthen the corneal epithelium (Rodrigues et al., 1987). In control rabbit cornea, keratin 3 was largely expressed in the suprabasal layers, a finding consistent with a role for the keratin 3/keratin 12 pair in cell differentiation. Our findings are in line with earlier studies showing suprabasal expression of keratin 3 in epithelial cells of the human cornea and corneas in other vertebrate species (Chaloin-Dufau et al., 1993). Differentiation of corneal epithelial cells was evident as keratin 3 continued to be expressed 3 days post-SM. A marked decrease in keratin 3 was noted in the superficial layer of corneal epithelial cells, as well as in wing cells and basal cells 14 and 28 days following exposure to SM. These data suggest that the corneal epithelial wound healing process is incomplete. It should be noted that changes in expression of keratin 3/keratin 12, largely due to gene mutations, have been correlated with fragility of the corneal epithelium, disorganized and thickened epithelium, the formation of cysts and a thickened basement membrane keratin (Allen et al., 2016; Arin, 2009; Szaflik et al., 2008). We speculate that changes in keratin 3 following exposure to SM may contribute to epithelial cell and basement membrane zone dysfunction leading to microblistering.

Rabbit corneal keratocytes and epithelial cells are known to highly express ALDH1A1, a water-soluble antioxidant protein important in corneal transparency (Jester, 2008; Pei et al., 2006). Our data show that rabbit keratocytes and epithelial cells express ALDH1A1. A marked decrease in the number of keratocytes expressing ALDH1A1 was evident in the cornea 3 days post-SM, a response that persisted for at least 28 days. Conversely, in epithelial cells, ALDH1A1 increased 14 and 28 days post-SM. Recovery of ALD1A1 expressing epithelial cells is likely the result of rapid cellular proliferation during wound healing and the need for epithelial cells to detoxify cellular oxidants induced in tissues following injury (Stagos et al., 2010). Persistent decreases in ALDH1A1 expressing keratocytes is in line with our findings of reduced corneal transparency. Earlier studies in humans and rabbits reported decreases in numbers of corneal keratocytes following exposure to SM or nitrogen mustard (Goswami et al., 2021; Jafarinasab et al., 2010). Mechanisms underlying this response are not known. Changes in corneal hydration and/or electrolyte balance following SM injury may affect keratocyte turnover, a process that can reduce stromal keratocyte numbers. It should be noted that ALDH1A1 is important in detoxifying lipid peroxidation end products including 4-hydroxynonenal and malondialdehyde, which have been identified in vesicant treated cornea (Goswami et al., 2016b; Zheng et al., 2013). Fewer ALDH1A1 expressing keratocytes may lead to increased stromal oxidative stress, a process that could exacerbate tissue injury and delay wound healing (Pei et al., 2006; Stramer and Fini, 2004).

Our findings that CD45+ leukocytes accumulate in the corneal stroma following SM exposure is consistent with earlier studies showing vesicant-induced inflammatory cell infiltration into the cornea of rabbits (Goswami et al., 2021; Horwitz et al., 2018; Petrali et al., 2000). Leukocyte infiltration and activation are mediated by pro-inflammatory cytokines and chemokines including IL-6, MIP-1, TNFα and IL-8, which are known to accumulate in the cornea following SM injury (Ghazanfari et al., 2019; Horwitz et al., 2018; Singh et al., 2021). Release of cytotoxic mediators including reactive oxygen species and reactive nitrogen species by infiltrating macrophages and neutrophils are known to contribute to ocular injury (Milhorn et al., 2010). Macrophages are also important in wound repair and dysregulation of these cells can impair this process. This is supported by findings that anti-inflammatory agents that suppress leukocyte infiltration including corticosteroids and non-steroidal anti-inflammatory agents blunt vesicant-induced ocular damage (Amir et al., 2000; Gore et al., 2021; Goswami et al., 2022; Tewari-Singh et al., 2012).

In earlier studies, our laboratory and others have shown that the matrix metalloproteinase, MMP9, is upregulated in the cornea by mustards including SM, nitrogen mustard and the half-mustard, 2-chloroethyl ethyl sulfide (Anumolu et al., 2010; Gordon et al., 2016; Horwitz et al., 2014). Activation of MMP9 can lead to degradation of the extracellular matrix, disrupting cell-cell and cell-matrix attachments, which may result in epithelial-stromal separation (Fini and Girard, 1990; Tewari-Singh et al., 2012). Another matrix metalloproteinase upregulated in the corneal epithelium and basement membrane by SM is ADAM17, which cleaves the hemidesmosomal component, collagen XVII, and other basement membrane components including perlecan as well as mediators of cell adhesion (DeSantis-Rodrigues et al., 2016). Inhibitors of MMP9 and ADAM17 have been shown to suppress SM-induced corneal injury in a rabbit model demonstrating the importance of these enzymes in toxicity (DeSantis-Rodrigues et al., 2021; Horwitz et al., 2014).

We found that perlecan and cellular fibronectin are expressed in the rabbit corneal epithelial basement membrane. By interacting with laminins and collagens, these molecules are thought to function as scaffolds in basement membrane organization (Medeiros et al., 2018; Wilson et al., 2020). Both matrix proteins were tightly associated with the corneal epithelial basement membrane. Over time following SM exposure, perlecan expression decreased with diffuse expression from the epithelial basement membrane zone extending into the stroma. This is possibly due to increased matrix metalloproteinase expression following SM injury. In contrast, early SM-induced decreases in cellular fibronectin were followed by increased expression in the epithelial basement membrane zone and stroma during wound repair 14 and 28 days post exposure. Earlier studies have shown that tenascin-C acts as a provisional extracellular matrix glycoprotein in the stroma following injury and is important in triggering wound repair (Sumioka et al., 2013; Yamanaka et al., 2013). These data are consistent with our findings of increased tenascin-C in the stroma following SM-injury where it likely functions to stimulate keratocyte migration, growth and differentiation (Schmidinger et al., 2003; Sumioka et al., 2021). Taken together, these data indicate persistent damage to the epithelial basement membrane zone and stroma of the rabbit cornea after SM-exposure.

In summary, the present studies identify novel aspects of the injury and wound healing process in rabbit cornea following exposure to SM. These include changes in the corneal epithelium and stroma and associated modifications of epithelial basement membrane components. Potential mechanistic targets for the development of countermeasures against vesicant injury in the cornea include extracellular matrix proteins and matrix metalloproteinases, which are essential for epithelial basement membrane and stromal integrity. Proper functioning of these proteins may in part suppress corneal edema, hazing and neovascularization, thereby contributing to the restoration of normal vision. Other aspects of corneal injury including the inflammatory response and control of keratocyte growth and differentiation may also be targets for therapeutic intervention.

Acknowledgements

This work was supported by the U.S. Department of Health and Human Services, National Institutes of Health under grants AR055073 and ES005022.

Footnotes

Declaration of Competing Interest

The authors have no conflict of interest for the subject matter of this paper.

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