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Sulfur mustard (SM) ocular exposure severely damages the cornea and causes vision impairment. At present, no specific therapy exists to mitigate SM-induced corneal injury and vision loss. This study performed transcriptome profiling of naïve, SM-damaged, and SM-undamaged rabbit corneas using RNA-seq analysis and bioinformatic tools to gain a better mechanistic understanding and develop SM-specific medical countermeasures. The mRNA profiles of rabbit corneas 4 weeks post SM vapor exposure were generated using Illumina-NextSeq deep sequencing (Gene Expression Omnibus accession # GSE127708). The RNA sequences of naïve (n = 4), SM-damaged (n = 5), and SM-undamaged (n = 5) corneas were subjected to differential expression (DE) analysis after quality control profiling with FastQC. DE analysis was performed using HISAT2, StringTie, and DESeq2. The log2(FC)±2 and adjusted p˂0.05 were chosen to identify the most relevant genes. A total of 5930 differentially expressed genes (DEGs) (upregulated: 3196, downregulated: 2734) were found in SM-damaged corneas compared to naïve corneas, whereas SM-undamaged corneas showed 1884 DEGs (upregulated: 1029, downregulated: 855) compared to naïve corneas. DE profiling of SM-damaged corneas to SM-undamaged corneas revealed 985 genes (upregulated: 308, downregulated: 677). The DE profiles were subsequently subjected to signaling pathway enrichment, and protein‒protein interactions (PPIs) were analyzed. Pathway enrichment was performed for the genes associated with cellular apoptosis, death, adhesion, migration, differentiation, proliferation, extracellular matrix, and tumor necrosis factor production. To identify novel targets, we narrowed the pathway analysis to upregulated and downregulated genes associated with cell proliferation and differentiation, and PPI networks were developed. Furthermore, protein targets associated with cell differentiation and proliferation that may play vital roles in corneal fibrosis and wound healing post SM injury were identified.

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Exposure to mustard agents, such as sulfur mustard (SM) and nitrogen mustard (NM), often results in ocular surface damage. This can lead to the emergence of various corneal disorders that are collectively referred to as mustard gas keratopathy (MGK). In this study, we aimed to develop a mouse model of MGK by using ocular NM exposure, and describe the subsequent structural changes analyzed across the different layers of the cornea. A 3 µL solution of 0.25 mg/mL or 5 mg/mL NM was applied to the center of the cornea via a 2-mm filter paper for 5 min. Mice were evaluated prior to and after exposure on days 1, 3, 7, 14, and 28 for 4 weeks using slit lamp examination with fluorescein staining. Anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM) tracked changes in the epithelium, stroma, and endothelium of the cornea. Histologic evaluation was used to examine corneal cross-sections collected at the completion of follow-up. Following exposure, mice experienced central corneal epithelial erosion and thinning, accompanied by a decreased number of nerve branches in the subbasal plexus and increased activated keratocytes in the stroma in both dosages. The epithelium was recovered by day 3 in the low dose group, followed by exacerbated punctuate erosions alongside persistent corneal edema that arose and continued onward to four weeks post-exposure. The high dose group showed persistent epitheliopathy throughout the study. The endothelial cell density was reduced, more prominent in the high dose group, early after NM exposure, which persisted until the end of follow-up, along with increased polymegethism and pleomorphism. Microstructural changes in the central cornea at 4 weeks post-exposure included dysmorphic basal epithelial cells and reduced epithelial thickness, and in the limbal cornea included decreased cellular layers. We present a mouse model of MGK using NM that successfully replicates ocular injury caused by SM in humans who have been exposed to mustard gas.

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Sulfur mustard gas (SM) is a vesicating and alkylating agent used as a chemical weapon in many mass-casualty incidents since World War I. Ocular injuries were reported in >90% of exposed victims. The mechanisms underlying SM-induced blindness remain elusive. This study tested the hypothesis that SM-induced corneal fibrosis occurs due to the generation of myofibroblasts from resident fibroblasts via the SMAD2/3 signaling pathway in rabbit eyes in vivo and primary human corneal fibroblasts (hCSFs) isolated from donor corneas in vitro. Fifty-four New Zealand White Rabbits were divided into three groups (Naïve, Vehicle, SM-Vapor treated). The SM-Vapor group was exposed to SM at 200 mg-min/m3 for 8 min at the MRI Global facility. Rabbit corneas were collected on day 3, day 7, and day 14 for immunohistochemistry, RNA, and protein lysates. SM caused a significant increase in SMAD2/3, pSMAD, and ɑSMA expression on day 3, day 7, and day 14 in rabbit corneas. For mechanistic studies, hCSFs were treated with nitrogen mustard (NM) or NM + SIS3 (SMAD3-specific inhibitor) and collected at 30 m, 8 h, 24 h, 48 h, and 72 h. NM significantly increased TGFß, pSMAD3, and SMAD2/3 levels. On the contrary, inhibition of SMAD2/3 signaling by SIS3 treatment significantly reduced SMAD2/3, pSMAD3, and ɑSMA expression in hCSFs. We conclude that SMAD2/3 signaling appears to play a vital role in myofibroblast formation in the cornea following mustard gas exposure.

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Exposure to mustard agents, such as sulfur mustard (SM) and nitrogen mustard (NM), often results in ocular surface damage. This can lead to the emergence of various corneal disorders that are collectively referred to as mustard gas keratopathy (MGK). In this study, we aimed to develop a mouse model of MGK by using ocular NM exposure, and describe the subsequent structural changes analyzed across the different layers of the cornea. A 3 µL solution of 0.25 mg/mL NM was applied to the center of the cornea via a 2-mm filter paper for 5 min. Mice were evaluated prior to and after exposure on days 1 and 3, and weekly for 4 weeks using slit lamp examination with fluorescein staining. Anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM) tracked changes in the epithelium, stroma, and endothelium of the cornea. Histologic evaluation and immunostaining were used to examine corneal cross-sections collected at the completion of follow-up. A biphasic ocular injury was observed in mice exposed to NM, most prominent in the corneal epithelium and anterior stroma. Following exposure, mice experienced central corneal epithelial erosions and thinning, accompanied by a decreased number of nerve branches in the subbasal plexus and increased activated keratocytes in the stroma. The epithelium was recovered by day 3, followed by exacerbated punctuate erosions alongside persistent stromal edema that arose and continued onward to four weeks post-exposure. The endothelial cell density was reduced on the first day after NM exposure, which persisted until the end of follow-up, along with increased polymegethism and pleomorphism. Microstructural changes in the central cornea at this time included dysmorphic basal epithelial cells, and in the limbal cornea included decreased cellular layers and p63+ area, along with increased DNA oxidization. We present a mouse model of MGK using NM that successfully replicates ocular injury caused by SM in humans who have been exposed to mustard gas. Our research suggests DNA oxidation contributes to the long-term effects of nitrogen mustard on limbal stem cells.

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Sulfur mustard (SM) remains a highly dangerous chemical weapon capable of producing mass casualties through liquid or vapor exposure. The cornea is highly sensitive to SM toxicity and exposure to low vapor doses can cause incapacitating acute injuries. At higher doses, corneas fail to fully heal and subsequently develop a constellation of symptoms known as mustard gas keratopathy (MGK) that causes reduced quality of life and impaired or lost vision. Despite a century of research, there are no specific treatments for acute or persistent ocular SM injuries. Here I summarize toxicological, clinical and pathophysiological mechanisms of SM vapor injury in the cornea, describe a preclinical model of ocular SM vapor exposure for reproducible therapeutic studies, and propose new approaches to improve evaluation of therapeutic effects. I also describe recent findings illustrating the delayed development of a transient but severe recurrent corneal lesion that, in turn, triggers the emergence of secondary keratopathies characteristic of the chronic form of MGK. Development of this recurrent lesion is SM dose-dependent, although the severity of the recurrent lesion appears SM dose-independent. Similar recurrent lesions have been reported in multiple species, including humans. Given the mechanistic relationship between the recurrent lesion and chronic, secondary keratopathies, I hypothesize that preventing the development of the recurrent lesion represents a novel and potentially valuable therapeutic approach for treatment of severe corneal SM injuries. Although ocular exposure to SM vapor continues to be a challenging therapeutic target, establishing consistent and reproducible models of corneal injury that enhance mechanistic and pathophysiological understanding will help satisfy regulatory requirements and accelerate the development of effective therapies.

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An array of corneal pathologies collectively called mustard gas keratopathy (MGK) resulting from ocular exposure to sulfur mustard (SM) gas are the most prevalent chemical warfare injury. MGK involves chronic ocular discomfort that results in vision impairment. The etiology of MGK remains unclear and poorly understood primarily due to a lack of scientific data regarding structural and cellular changes in different layers of the cornea altered by mustard vapor exposure in vivo. The goals of this study were to (a) characterize time-dependent changes in different layers of corneal epithelium, stroma, and endothelium in live animals in situ by employing state-of-the-art multimodal clinical ophthalmic imaging techniques and (b) determine if SM-induced acute changes in corneal cells could be rescued by a topical eye drop (TED) treatment using in an established rabbit in vivo model. Forty-five New Zealand White Rabbit eyes were divided into four groups (Naïve, TED, SM, and SM + TED). Only one eye was exposed to SM (200 mg-min/m3 for 8 min), and each group had three time points with six eyes each (Table-1). TED was topically applied twice a day for seven days. Clinical eye examinations and imaging were performed in live rabbits with stereo, Slit-lamp, HRT-RCM3, and Spectralis microscopy system. Fantes grading, fluorescein staining, Schirmer's tests, and applanation tonometry were conducted to measure corneal haze, ocular surface aberrations, tears, and intraocular pressure respectively. H&E and PSR staining were used for histopathological cellular changes in the cornea. In vivo confocal and OCT imaging revealed significant changes in structural and morphological appearance of corneal epithelium, stroma, and endothelium in vivo in SM-exposed rabbit corneas in a time-dependent manner compared to naïve cornea. Also, SM-exposed eyes showed loss of corneal transparency characterized by increased stromal thickness and light-scattering myofibroblasts or activated keratocytes, representing haze formation in the cornea. Neither naive nor TED-alone treated eyes showed any structural, cellular, and functional abnormalities. Topical TED treatment significantly reduced SM-induced abnormalities in primary corneal layers. We conclude that structural and cellular changes in primary corneal layers are early pathological events contributing to MGK in vivo, and efficient targeting of them with suitable agents has the potential to mitigate SM ocular injury.

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Sulfur mustard (SM) is a lethal chemical agent that affects many organs, particularly the eyes, respiratory system and skin. Even asymptomatic patients with documented SM vapor exposure may develop organ disorder many years later. Patients with even minor signs in the acute stage may experience late complications that necessitate surgery. Early decontamination and conservative measures could help the patients and decrease the complications. Despite decades of research, there is still no effective treatment for either acute or long-term SM-induced ocular complications. Even after multiple medications and surgical procedures, the majority of patients continue to have symptoms. For dry eye, punctual occlusion, autologous eye drops, and aggressive lubrication are used; for persistent epithelial defects (PED), tarsorrhaphy, amniotic membrane transplant, and stem cell transplantation are used; for total limbal stem cell deficiency (LSCD), living-related conjunctivolimbal allograft and keratolimbal allograft are used; for corneal vascularization, steroids, non-steroidal anti-inflammatory drugs, and anti-vascular endothelial growth factor prescribed; and for corneal opacities, corneal transplantation is done. Platelet rich plasma and topical drops containing stem cell transplantation for LSCD, photodynamic therapy paired with subconjunctival or topical anti-vascular endothelial growth factors for corneal vascularization, topical curcumin and topical ciclosporin-A for dry eye, and orbital fat-derived stem cells for PED are all alternative treatments that can be suggested. Despite the experimental and clinical research on the complications of SM exposure over the past decades, there is still no effective treatment for eye complications. However, supportive medical and surgical management has been applied with relatively good outcome.

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Sulfur mustard (SM) is a chemical warfare agent that has been used throughout recent history and remains a threat today. Exposed soldiers and civilians experience a variety of symptoms primarily in the respiratory system, skin, and eyes. The ocular tissues are highly sensitive to damage by SM and undergo unique manifestations of acute, chronic, and delayed complications that can persist for months and years after exposure. The mechanisms of this unique mustard gas keratopathy are still not fully understood and animal models for the study of this disease are discussed. Recent advances in mechanisms of injury are included in this review. Ophthalmic manifestations of SM injury including persistent epithelial defects, limbal stem cell deficiency, corneal neovascularization, dry eye, and corneal opacification have been reported. A wide variety of medical and surgical therapies have been studied and are reviewed here along with potential future therapies.

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Sulfur mustard (SM) is a lipid soluble alkylating agent that causes genotoxic injury. The eye is highly sensitive to SM toxicity and exposures exceeding 400 mg min/m3 can elicit irreversible corneal pathophysiologies. Development of medical countermeasures for ocular SM exposure has been hindered by a limited understanding of dose-dependent effects of SM on corneal injury. Here, clinical, histological and ultrastructural analyses were used to characterize the effects of SM dose on corneal injury progression. Corneas were evaluated for up to 20 wk following exposure to saturated SM vapor for 30-150 s, which corresponds to 300-1,500 mg min/m3. In acute studies, a ceiling effect on corneal edema developed at doses associated with full-thickness corneal lesions, implicating endothelial toxicity in corneal swelling. Recurrent edematous lesions (RELs) transiently emerged after 2 wk in a dose-dependent fashion, followed by the development of secondary corneal pathophysiologies such as neovascularization, stromal scarring and endothelial abnormalities. RELs appeared in 96 % of corneas exposed for ≥ 90 s, 52 % of corneas exposed for 60 s and 0 % of corneas exposed for 30 s. While REL latency was variable in corneas exposed for 60 s, REL emergence was synchronized at exposures ≥ 90 s. Corneas did not exhibit more than one REL, suggesting RELs are part of a programmed pathophysiological response to severe alkylating lesions. In post-mortem studies at 12 wk, corneal edema was positively correlated to severity of endothelial pathologies, consistent with previous findings that endothelial toxicity influences long-term outcomes. These results provide novel insight into long-term corneal pathophysiological responses to acute toxicity and identify exposure conditions suitable for therapeutic testing.

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BACKGROUND: Degenerative biomechanical factors and immunologic processes with effect on collagen and corneal reparative process are known as the main cause of ocular surface dysfunction in mustard gas keratopathy (MGK) and may cause changes in the corneal biomechanical values. Therefore, we evaluate corneal biomechanical properties of these patients. METHODS AND MATERIALS: This case-control study includes 61 chemical warfare victims with MGK. After omission according to our exclusion criteria, 88 eyes of patients with MGK were enrolled as the case group and also a group of 88 normal eyes, which were matched regarding their age and sex in the control group, were enrolled. Measurements of corneal biomechanical properties which reported by ORA and Corvis ST (CST) devices were compared. The SPSS software version 23.0 was used in the statistical analysis. For comparisons between groups, if the data had a normal distribution, were analyzed by Student's t-test and ANOVA, and if the data didn't have a normal distribution, Mann-Whitney U test, and Kruskal-Wallis were used. Furthermore, to identify a relationship between two groups of data Spearman's rank Correlation test was used. P value < 0.05 were considered statistically significant. RESULTS: In the MGK group, A1 length (A1L), A1 velocity (A1V), A2 velocity (A2V), deformation amplitude (DA) and peak distance (PD) were higher than the control group (P < 0.001). However, the corneal hysteresis (CH) (P = 0.003), corneal resistant factor (CRF), non-corrected IOP (IOPnct), corrected IOP based on corneal thickness (IOPpachy), and central corneal thickness (CCT) were lower than the control group (P < 0.001). The visual acuity according to the LogMAR scale and severity of MGK was positively associated with IOPpachy and negatively associated with CH, CRF, CCT and highest concavity radius (Radius). CONCLUSION: Measurement of corneal biomechanical properties may be, have a useful role in the classification, monitoring or diagnosis of MGK.

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Sulfur mustard (SM) is a strong blistering, highly reactive, lipophilic chemical war agent that causes injury in different organs including the skin, eyes, and respiratory tract. The Eyes are especially susceptible to the consequences of SM poisoning because of the aqueous and mucosal nature of conjunctiva and cornea. DNA alkylation and depletion of glutathione, are the most important mechanisms of SM action in the eye injuries. Acute clinical symptoms are including decrease in visual acuity, dryness, photophobia, blepharospasm, conjunctivitis, and complaints of foreign body sensation and soreness that gradually progress to severe ocular pain. Corneal abrasions, ulcerations, vesication, and perforations are common corneal consequences in SM injured victims. Appearance of chronic symptoms has been reported as chronic inflammation of the corneal and conjunctival vasculature, ischemia, lipid and cholesterol deposition, scarring in cornea, corneal thinning, opacification and perforation of the cornea, limbal stem cell deficiency (LSCD), and neovascularization. Different medical and surgical protocols have been documented in the management of SM-induced ocular injuries, including preservative-free artificial tears, topical steroids and antibiotic, mydriatic, antiglaucoma drops, therapeutic contact lenses, dark glasses and punctal plugs/cauterization, N-acetylcysteine, tarsorrhaphy, amniotic membrane transplantation, stem cell transplantation, and corneal transplantation. New drugs such as resolvin E1, topical form of essential fatty acids, thymosin ß4, 43 amino-acid polypeptides, topical form of curcumin, newly formulated artificial tears, diquafosol, rebamipide, tretinoin, and oral uridineseems to be beneficial in the management of ocular lesion associated with sulfur mustard poisoning. Further studies are needed to approve these drugs in SM victims. J. Cell. Biochem. 118: 3549-3560, 2017. © 2017 Wiley Periodicals, Inc.

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Sulfur mustard (SM; mustard gas) is a classic chemical warfare agent that has been used in several wars and is still a potential threat especially in the Middle-East region. Victims experience acute symptoms in airexposed organs including skin, respiratory tract and the eyes. Survivors of the acute stage might develop chronic or delayed-onset complications in the exposed organs. The exact mechanism(s) of SM-induced tissue damage is still unknown, however DNA alkylation and oxidative damage are the most relevant mechanisms. Eye is the most sensitive organ to the SM vapor and ocular symptoms usually precede other manifestations. Ocular findings including blepharitis, dry eye disease, corneal vascularization, persistent epithelial defects, limbal ischemia, limbal stem cell deficiency, corneal thinning, corneal opacity and corneal innervation abnormalities have been reported several years after SM exposure. In this review, mechanisms of acute and chronic/delayed ocular manifestations of SM and their current management and potential future therapies have been discussed. We have also included recent advances in amniotic membrane transplantation, cultivated stem cell transplantation and anti-angiogenic therapies which might be considered as therapeutic options in SM-induced ocular damage in the future.

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PURPOSE: Sulfur mustard (SM) is a highly reactive vesicant that causes severe ocular injuries. Following exposure to moderate or high doses, a subset of victims develops a chronic injury known as mustard gas keratopathy (MGK) involving a keratitis of unknown etiopathogenesis with secondary keratopathies such as persistent epithelial lesions, corneal neovascularization, and progressive corneal degeneration. This study was designed to determine whether SM exposure evokes acute endothelial toxicity and to determine whether endothelial pathologies were specifically observed in MGK corneas as opposed to healed corneas. METHODS: Corneas of New Zealand white rabbits were exposed to SM vapor, and the corneal endothelium was evaluated at 1 day and 8 weeks using scanning electron microscopy (SEM), transmission electron microscopy (TEM), in vivo confocal microscopy (IVM), and fluorescent microscopy. Barrier function was measured by uptake of a fluorescent dye injected into the anterior chamber. RESULTS: A centripetal endothelial injury at 1 day was observed by SEM, TEM, IVM, and fluorescent microscopy. In vivo confocal microscopy revealed additional cytotoxicity between 1 and 13 days. In contrast to healed corneas, which appeared similar to sham-exposed naive eyes at 8 weeks, MGK corneas exhibited significant evidence of continued pathological changes in the endothelium. CONCLUSIONS: Endothelial toxicity occurs at the right time and with the appropriate pathophysiology to contribute to MGK. Based on these findings, we propose a model that explains the relationships among SM dose, the biphasic progression, and the various clinical trajectories of corneal SM injury and that provides a mechanism for temporal variations in MGK onset. Finally, we discuss the implications for the management of SM casualties.

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