Time-dependent in situ structural and cellular aberrations in rabbit cornea in vivo after mustard gas exposure

Abstract

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.

1. Introduction

Sulfur mustard (Bis (2-chloroethyl) Sulfide; SM), a devastating vesicating chemical, has been deployed in multiple combats and often against civilians since World War I. On exposure to the body, SM rapidly penetrates mucous membranes and causes debilitating wounds, mainly to the skin, lungs, and eyes, significantly compromising the quality of life for decades (Emad and Rczaian, 1999; Greenberg MI et al., 2016; Kilic et al., 2018; RENSHAW, 1947; Wattana and Bey, 2009). Ocular exposure to SM causes many incapacitating and painful acute and chronic corneal injuries that are not easy to treat and require lengthy rehabilitation. In addition, SM causes low morbidity to many organs and mortality. SM was described as the “king of battlefield gases” because it caused more chemical casualties than all the other agents combined, including chlorine, phosgene, and cyanogen chloride. (Borak and Sidell, 1992; Milhorn et al., 2010).

The eyes are the most vulnerable tissue to SM injury. SM causes dose- and time-dependent damage to ocular tissues (Safarinejad et al., 2001; Solberg et al., 1997). Ocular injuries are reported in 90% or more of victims exposed to SM with symptoms lasting months to years after the initial exposure (Ghasemi et al., 2009; Safarinejad et al., 2001; Safi et al., 2017; Solberg et al., 1997). SM is a potent alkylating agent that upon contact with the eye causes severe corneal injury, pain, and acute vision loss within hours (CDC, 2018; Javadi et al., 2005; McNutt et al., 2012b, 2012a; Milhorn et al., 2010; Rowell et al., 2009). The ocular damage and clinical symptoms depend on the amount and duration of SM exposure to the eye. A low-duration (mild) SM exposure tends to heal fully over time, but high-duration SM exposure causes a grade II to IV ocular pathologies called mustard gas keratopathy (MGK). MGK is a delayed pathology characterized by severe ocular pain, inflammation, recurrent corneal epithelial erosions, ulcerations, epithelial-stromal separation, haze/fibrosis, and neovascularization (McNutt et al., 2012b, 2012a; Milhorn et al., 2010; Rowell et al., 2009; Safarinejad et al., 2001; Solberg et al., 1997). MGK can be a progressive corneal degeneration with delayed onset or chronic manifestations involving multiple mechanisms (Javadi et al., 2005; Rowell et al., 2009).

The cornea is a unique tissue comprising three cellular layers: epithelium, stroma, and endothelium (Mohan et al., 2022). The corneal epithelium is the outer superficial layer made of 5–7 layers of non-keratinized cells. The corneal stroma contributes 85–90% of corneal thickness and primarily comprises collagens and quiescent keratocytes. The distinctive collagen fibrils arrangement and characteristic organization are essential in maintaining corneal shape, size, and optical properties. At the same time, its unique hexagonal pattern in the cross-section is critical for minimizing light scattering and preserving optical property (Meek, 2009; Meek and Knupp, 2015). The endothelium is a single monolayer of cells at the innermost portion of the cornea that maintains corneal hydration (Kamil and Mohan, 2021). The primary function of the cornea is to protect the eye and render light refraction (vision) (Mohan et al., 2020). Several studies, including ours, have demonstrated damage to corneal cells post-SM exposure. Our recent pilot rabbit study suggests acute injury to the corneal epithelium, breakage in the epithelial-stroma barrier, damage to keratocytes, and endothelium post-SM exposure in vivo (Tripathi et al., 2020).

Mild SM vapor exposure symptoms are manageable with variable patient outcomes (Murray and Volans, 1991). The standard of care for MGK includes amphoteric rinsing, topical steroids, a concurrent topical steroid with non-steroidal anti-inflammatory drugs (NSAIDs), Zinc Desferrioxamine, and a combination of Dexamethasone and Silibinin (Etemad et al., 2019; Gore et al., 2021). Nevertheless, recurrence of clinical signs after therapy withdrawal remains a significant challenge (Amir et al., 2000; Gordon et al., 2010; Kadar et al., 2009). However, most formulations rely heavily on a steroidal component to manage SM vapor toxicity. In addition, long-term steroid use in patients causes many side effects, including glaucoma (Phulke et al., 2017). Recently, a pilot study has reported the potential of non-steroidal topical eye drop (TED) to clinically mitigate acute SM vapor toxicity (Tripathi et al., 2020). The TED formulation contained 4 FDA-approved drugs, Ketorolac, SAHA (a histone deacetylase inhibitor), Enalapril, and Vitamin C (Tripathi et al., 2020). Ketorolac has been approved for ocular use by the FDA. This study examined the acute ocular toxicity of mustard gas vapor and the therapeutic effects of Topical Eye Drops (TED) for 14 days. Additionally, advanced methods of clinical images were performed to collect in situ from cellular layers of the cornea following SM vapor toxicity and TED treatment in live rabbits. The imaging techniques allowed us to examine corneal pathological changes in the same rabbits over time. Furthermore, this is the first study to quantify ultrastructural changes in the corneal stroma collagen architecture post-SM vapor exposure.

Accumulating literature reveals that the cornea post-SM injury undergoes unique manifestations of acute, chronic, and delayed complications that can prolong and persist from months to several years (Fuchs et al., 2021; McNutt et al., 2013). However, the lack of scientific data about structural and cellular changes in corneal cells in vivo after SM exposure and knowledge of mechanisms related to MGK are significant knowledge gaps in the literature. Recent reviews have provided preliminary knowledge regarding various animal models, clinical symptoms seen in human patients, and perceived mechanisms of MGK, but not much information on cell-specific alterations occurring after SM ocular exposure (Fuchs et al., 2021; Gore et al., 2021). Therefore, the primary objectives of this study were to collect time-dependent structural and cellular data revealing changes in different layers of corneal epithelium, stroma, and endothelium in live animals in situ with state-of-the-art multimodal clinical eye imaging techniques and to determine if SM-specific changes in various corneal cells could be rescued by TED using an established rabbit in vivo model.

2. Materials and methods

2.1. Animals

The Institutional Animal Care and Use Committees of the University of Missouri, Columbia, MO, and MRI Global, Kansas City, MO have approved the study. Rabbits were treated following the ARVO Statement for the Use of Animals in Ophthalmology and Vision Research. For this study, 45 male New Zealand White rabbits purchased from Charles Rivers Laboratories were used. All rabbits were between 2.5 and 4.0 kg in good health, with no ocular defects during the study. Upon delivery, animals were inspected for signs of ill-health and quarantined in the animal facility for more than two weeks under the supervision of a Veterinarian. All rabbits were fed certified feed and water ad libitum. The rabbits were housed in environmentally controlled rooms at a temperature between 16 °C and 22 °C and relative humidity of 50% ± 20% with a 12-h light/dark cycle per day.

2.2. Experimental treatment groups and sulfur mustard exposure

Animals were randomly divided into four groups. Each group has three timepoints (day-3, day-7, and day-14) and six eyes were used in the study, as shown in Table 1, to study changes in corneal cells and the effectiveness of TED in rescuing MGK. Group-I (Naive) was untreated eyes; Group-II (TED) eyes had TED treatment alone twice a day for seven days; Group-III (SM) eyes had SM vapor exposure only; and Group-IV (SM + TED) eyes had SM vapor exposure and beginning 2 h post-SM vapor exposure TED treatment twice a day for seven days.

Table 1.

Research design showing details of treatment in animal of various groups, sample size, and clinical eye imaging timepoints.

S.N.	Groups	Number of Rabbits	Used eyes	Eye imaging timepoints
1	Naïve	9	OS (n = 9) and OD (n = 9) had no treatment	3 (day-3, day-7, day-14)
2	SM	18	OD (n = 18) received SM and OS received no treatment	3 (day-3, day-7, day-14)
3	SM + TED	18	OD (n = 18) received SM + TED	3 (day-3, day-7, day-14)
4	TED	0	OS (n = 18) (contralateral eyes of “SM + TED” group) received TED only and no SM	3 (day-3, day-7, day-14)
Total used	4 groups	45 rabbits	72 eyes	3 timepoints

The SM vapor and other procedures in rabbits were initially performed at MRI Global Inc, Kansas City, MO, on animals under general anesthesia administered via ketamine (100 mg/kg) and xylazine (5 mg/kg) intramuscular injection. In addition, the slow-release buprenorphine HCl (0.05–0.1 mg/kg) was administered 30–60 min prior to anesthesia for pain management. Once the rabbit was anesthetized, ocular vapor goggles were secured around the animal’s head for SM vapor exposure inside the hood line. The animals were placed on top of an absorptive pad and positioned on a shelf unit within the chemical hood for SM exposure. All animals received a vapor exposure of SM to the whole eye, including the eyelids and conjunctiva at a target concentration of 200 mg-min/m³ for 8 min. Animals remained on the shelf unit throughout the exposure until a sufficient wash-out of the SM in the goggles was achieved. After the target SM vapor exposure was completed, the goggles were removed. Next, all eyes were rinsed copiously with balanced salt solution (BSS) to decontaminate the exposed area in the eyes. Animals were allowed to recover from anesthesia in the chemical fume hood for 2 h or more if needed. In addition to buprenorphine administration, the lights in the animal rooms were dimmed or turned off to aid in ocular pain or sensitivity for a time determined by the attending veterinarian. Once the animal’s eyes had been washed, the rabbits were transferred to a clean room at the MRI Global vivarium upon approval by the attending veterinarian. Twenty-four hours after initial procedures, animals were transported to the University of Missouri, Columbia by an approved animal carrier company under veterinarian supervision. The BSS was purchased from Fisher, and TED was formulated by the Micro Lab Ltd, Bangalore, India, under GLP lab for use in animal research. The final concentrations of four drugs in TED were 25 μM of SAHA, 25 μM of enalapril, 0.5% of Ketorolac, and 10% of Vitamin-C. TED was given topically by holding the rabbit’s head slightly upwards towards the ceiling for about 5 s.

2.3. Clinical eye examinations, multimodal ophthalmic imaging, and ocular health evaluations

Clinical eye evaluation in live rabbits pre- and post-procedure was performed on day-3, day-7, and day-14 under general anesthesia with a Slit-lamp microscope (Kowa, SL-15 portable slit-lamp, Torrance, CA). This clinical microscope was fitted with a high-definition digital imaging system to record/access ocular health and corneal haze (Kowa, portable VK- 2 Ver. 5.50) (Gupta et al., 2018). Additionally, corneal health was examined with a stereomicroscope (Leica MZ16F, Leica Microsystems Inc., Buffalo Grove, IL) equipped with a digital camera system (SpotCam RT KE, Diagnostic Instruments Inc., Sterling Heights, MI) to record the levels of corneal/ocular damage (Gupta et al., 2018). All in vivo clinical examinations were performed by at least two independent investigators in a masked manner (Author initials: NS, RT, PB, SG, or EG) following protocol (Tripathi et al., 2020). Eyes were kept adequately hydrated during the procedure with BSS to prevent corneal desiccation.

Corneal epithelial defects were measured with a commercial ophthalmic fluorescein-stain (Altafluor Benox, Sigma Pharmaceuticals, North Liberty, IA) as mentioned earlier (Gupta et al., 2018; Mohan et al., 2011). The epithelial defects were assessed under a cobalt light blue filter and recorded under a green fluorescence filter using a stereomicroscope equipped with an image-capturing system (Leica MZ16F, Leica Microsystems Inc.) and with a digital camera (SpotCam RT KE, Diagnostic Instruments Inc.) (Gupta et al., 2018; Mohan et al., 2011). The clinical findings were documented by photography and assessed by a minimum of two independent observers in a masked manner (NS, RT, PB, SG, or EG). In addition, the size of the corneal epithelial defect in rabbit eyes was digitally computed by counting pixels in images taken with a stereomicroscope at 7.1x magnification using Photoshop software (Adobe, San Jose, CA) following the procedure reported earlier (Mohan et al., 2003).

2.4. Intraocular pressure and tear flow measurements

The changes in intraocular pressure (IOP) were measured with a handheld tonometer (TonoPen AVIA Tonometer, Scottsdale, AZ) while animals were under general anesthesia following published protocol (Tripathi et al., 2020). Additionally, Schirmer Tear Test (STT) Strips (Fisher Scientific, Pittsburgh, PA) were used to quantify tear volume at three timepoints in live animals.

2.5. Optical coherence tomography (OCT) imaging in live animals

OCT was conducted using the Heidelberg SPECTRALIS microscopy system equipped with an anterior segment module (ASM) to review structural changes in corneal cells in situ. This state-of-the-art imaging system allowed us to collect high-resolution images of cornea, sclera, and anterior chamber. The lateral scan images were taken at 16 mm with two different scan patterns: single and raster. There were predefined scan patterns depending on the application (cornea, anterior chamber angle, and sclera). The Heidelberg Noise Reduction and the TruTrack Active Eye Tracking allow for enhanced detailed images and precise alignment, respectively. The corneal layers can be seen in detail with the SPECTRALIS ASM, aiding in the measurement of corneal thickness. A single image was taken at the central cornea and raster images were taken to capture the whole cornea. Images used for central corneal thickness and gross anatomy analysis were the average of serial images taken from raster scans of each animal.

2.6. In vivo live scanning corneal confocal microscopy

The HRT III with Rostock Corneal Module (HRT3-RCM) was used for in vivo live scanning confocal. The HRT III compared and quantified time-depended morphological modifications in different layers of the corneal epithelium, various regions of the stroma (anterior, middle, and posterior), and endothelium in situ in live animals. GenTeal (Novartis, FortWorth, TX) water-based gel was applied to prevent the rabbit cornea from drying and as a coupling media. For quantification purposes, six images were randomly selected from each layer. Quantification is based on the number of cells, cell death, and presence of debris in the HRT3-RCM camera field of view (400 × 400 μm). Single monolayer images of basal epithelium were taken for cell-to-cell quality comparison. Additionally, high-detail images detailing sub-differentiation of cell layers were present. Serial images were taken from the central cornea at 20 μm intervals directly below the epithelium until the endothelium became present on days 3, 7, and 14. Stromal morphologic categories were identified based on patterns of cell quiescence, cell death, cell activation, and cell repopulation. Single endothelial non-overlapping images (25 manually selected cells per field) were taken for endothelial cell analysis (HRT3-RCM software Heidelberg Eye Explorer version 1.7.1.0). Total cell density counts were performed in triplicate for each eye and averaged. Cells were counted in a predetermined square box area of the same size for each cornea using the Heidelberg Eye Explorer software. Cells crossing the borders of the boxed selected area were only counted on the left and lower sides of the box to avoid over or underestimations of cell count and were expressed in cells per square millimeters. Endothelial cell hexagonality and heterogenicity were analyzed using specular imaging software (CellChek).

2.7. Corneal tissue collection

At selected termination points, animals were humanely euthanized on day-3, day-7, and day-14 after all final clinical examinations were completed. Animals were placed under general anesthesia prior to intravenous pentobarbital (SomaSol, Euthanasia-III Solution, Henry Schein, Dublin, OH) (150 mg/kg) administration for euthanasia. The corneas were harvested using sharp dissection scissors, and collected corneas were placed in 15x15 × 5 mm molds (Fisher Scientific, Pittsburgh, PA) filled with optical cutting temperature (OCT) compound (Sakura Finite, Torrance, CA, USA) (Mohan et al., 2003, 2011). The molds were immediately snap-frozen in a liquid nitrogen container immersed in a cryo-cup containing 2-methyl butane and blocks were stored at −80 °C until further use. Serial corneal sections (8-μm thick) were prepared using a cryostat (HM525 NX UV; Microm GmbH, Walldorf, Germany) and placed on glass microscope slides (Superfrost Plus; Fisher Scientific) and used for histology studies.

2.8. Histopathological examinations

Hematoxylin & Eosin (H&E) and Picrosirus Red (PSR) staining were performed for histopathological examinations following standard technique (Gupta et al., 2018). Briefly, cryo-frozen corneal sections were incubated for 15–20 min at room temperature and then washed in PBS for 10 min and dipped in hematoxylin for 5 min, followed by a rinse in running tap water, 1 dip in 1% acid-alcohol, and 10–15 dips in 0.3% ammonia water. Next, the slides were incubated in 95% alcohol followed by eosin. The tissues were then dehydrated in absolute alcohol, cleared in CitriSolv solution (Decon Laboratories, King of Prussia, PA), and finally mounted in cytoseal (Richard- Allan Scientific, Kalamazoo, MI).

H&E-stained tissue images were captured with a bright-field microscope (Leica) equipped with a digital camera and imaging software (SpotCamRT KE; Diagnostic Instruments, Sterling Heights, MI, USA). One slide had four serial tissue sections. Six images were taken from each tissue section. Images were assessed for corneal thickness and epithelial-stroma separation by verifying consistent horological changes in the four serial sections.

PSR staining was performed by the Veterinary Medical Diagnostic Laboratory at the University of Missouri, Columbia, MO, as described earlier (Tandon et al., 2012). The images of H&E and PSR stained tissues were captured with a bright-field microscope (Leica) equipped with a digital camera and imaging software (SpotCamRT KE; Diagnostic Instruments, Sterling Heights, MI, USA). The level of red staining was digitally measured by counting pixels of red color in three randomly selected non-overlapping regions at 100X magnification using RStudio software.

2.9. Transmission electron microscopy (TEM)

Immediately after euthanasia, a small portion of the cornea was sliced and placed in EM primary fixative (2% paraformaldehyde, 2% glutaraldehyde in 100 mM sodium cacodylate buffer; pH 7.35) provided by the University of Missouri TEM Core facility, which processed tissue and TEM imaging. Next, corneas were transferred into acetone, infiltrated with Epon resin (250 W for 3 min), polymerized at 60 °C overnight, and cut transversely with an ultramicrotome to obtain 2 μm thick sections for histologic examination. The cornea was trimmed to a length of 500 μm and cut with a diamond knife to yield 75-nm sections for transmission EM. Images were acquired with a JEOL JEM 1400 transmission electron microscope (JEOL USA Inc., Peabody, MA, USA) at 80 kV on a Gatan Ultrascan 1000 CCD at 50000x magnification. Automated quantification of collagen fibrils of TEM images was conducted using an image processing and analysis software developed in collaboration with Filiz Buyank (Sinha et al., 2021). In brief, an algorithm is made of three main modules, (i) fibril detection, (ii) shape analysis and cluster decomposition, and (iii) size and interfibrillar distance analysis. A multiscale Hessian matrix was used to detect collagen fibrils. Hessian matrix (Equation (1)) described the second-order structure of local intensity variations around each point of the image L (x, y) and eigenvalues ƛ_1,2 (Equation (2)) of the Hessian matrix used to detect blob-like or ridge-like structures. Hessian matrix was computed by convolving the image with derivatives of the Gaussian kernel, where scale s represented the standard deviation of the Gaussian kernel and controlled the radius of the detected structures. A coarse fibril mask was produced by computing the Hessian matrix and thresholding ƛ₁ (Hessian) as below (Equation (3)):

$$Hessia{n}_{\sigma }\left(X,Y\right)=\left[\begin{array}{cc}{L}_{xx}\left(X,Y\right)& L_{xy}\left(X,Y\right)\\ L_{xy}\left(X,Y\right)& L_{yy}\left(X,Y\right)\end{array}\right]$$ (1)

$${\lambda }_{1,2}=\frac{1}{2}\left(L_{xx}+L_{yy}\pm \sqrt{{\left(L_{xx}+L_{yy}\right)}^2+{\left(2L_{xy}\right)}^2}\right)$$ (2)

$$Mas{k}_{Fibril}\left(X,Y\right)=\{\begin{array}{ll}1& \lambda \left(Hessian\left(x,y\right)\right)<\epsilon \\ 0& otherwise\end{array}$$ (3)

The quantification of morphology and spatial organization of fibrils requires accurate identification and localization of individual fibrils. Hessian-based detection efficiently segmented regions occupied by fibrils from the background but failed to separate some neighboring fibrils and merge into clusters. To identify individual fibrils, shape analysis and cluster decomposition modules were developed based on previously reported modules (Ersoy et al., 2012; Sun et al., 2014). Specifically, the first connected component labeling was applied to the detection mask and disconnected blobs. Then, to each detected blob B_i, an ellipse E_i was fitted. Blob and ellipse areas area (B_i), area (E_i), and ratio r = area (E_i) − area (B_i))/area (B_i) was then computed. Using size and shape indices and their means and variances over the image, each detected blob B_i was classified into one of the three classes (spurious detection, single fibril, or fibril cluster). Blobs classified as spurious detection were removed; blobs classified as single fibril were kept intact.

The marker-controlled watershed transformation was used to decompose fibril clusters into individual fibrils (Vincent et al., 1991). Regional maxima of distance transform were used as markers. H-maxima transform was applied to the distance transform before detecting regional maxima to suppress spurious regional maxima and prevent over-segmentation (Soille, 1999). The module computed fibril morphology and spacing parameters, such as fibril radius, area fraction, and interfibrillar distance. From the refined segmentation, fibril centroids were computed. Delaunay triangulation & vertex coloring were applied to the centroids, and a colored neighborhood graph was generated, as previously described (Ersoy et al., 2012; Nath et al., 2006). In the neighborhood graph, nodes corresponded to individual fibrils, and edges linked immediate neighbors. Two neighbors were identified for each node in this neighborhood graph, specifically the nearest neighbor (red edges) and the farthest immediate neighbor (blue edges). A second graph (nearest/farthest neighborhood graph) was constructed, using only these specific links. Fibril-to-fibril interactions were assessed, and the spatial organization of collagen fibrils was described using these two neighborhood graphs. Various descriptors from these graphs, such as mean and standard deviation were then calculated as described previously (Gronkiewicz et al., 2016).

2.10. Statistical analysis

The GraphPad Prism 9.2 (GraphPad Software, La Jolla, CA, USA) software was used for statistical analysis. Each experiment was conducted independently in triplicate and the values were expressed as mean ± SEM. For statistical analysis, the student’s t-test and two-way analysis of variance (ANOVA) with Bonferroni post-hoc test was used. The value of p ≤ 0.05 was considered significant. The sample size was determined using the G*Power (3.1.9.4 software, www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3) priori power analysis method to achieve α = 0.05; power ≥0.9.

3. Results

3.1. Structural and morphological changes in different layers/regions of the cornea by SM

3.1.1. Changes in corneal epithelium after SM injury and mitigation by TED

In vivo live scanning confocal microscopy via Heidelberg HRT3-RCM was used to examine changes in the corneal epithelium. Naïve corneas have a healthy basal epithelium in which cells are densely populated with minimal gaps and minimal hyper-reflective areas between epithelial cells on day-3, (Fig. 1A; 130.42 ± 9.45), day-7 (Fig. 1E; 132.3 ± 7.26), and day-14 (Fig. 1I; 132.38 ± 6.41). SM exposure to the cornea causes severe injury to the epithelial cells. A significant decrease in epithelial cells density can be seen on day-3 (Fig. 1C; 48.42 ± 5.45; p < 0.0001), day-7 (Fig. 1G; 60.42 ± 6.86; p < 0.0001), and day-14 (Fig. 1K; 96.83 ± 7.63; p < 0.0001). On day-3 post SM exposure, a snow-like pattern of scattered hyper-reflective round dots with an obscure pattern and large accumulated aggregates can be seen (Fig. 1C). A highly reflective border around basal epithelial cells can be seen on day-7 (Fig. 1G) and day-14 (Fig. 1K). TED treatment to SM exposed corneas had significant mitigation of epithelial cell density loss on day-3 (Fig. 1D; 120.83 ± 13.59; p < 0.0001), showing snow-like spots on day-3, day-7 (Fig. 1H) had significant epithelial cell density recovery (129.73 ± 19.24; p < 0.0001) with a highly reflective border, and an almost normal basal cell layer on day-14 (Fig. 1L). A significant loss of epithelial cell density was mitigated by TED on day-14 (139.31 ± 9.67; p < 0.0001) compared to SM only group. Topical application of TED alone did not cause any visible changes in basal epithelial cells on day-3 (Fig. 1B; 131.36 ± 6.80), day-7 (Fig. 1F; 132.25 ± 8.47), and day-14 (Fig. 1J; 133.56 ± 8.32).

Fig. 1.

HRT3-RCM images of corneal epithelium exhibiting SM vapor-induced damage to the corneal epithelium on day-3 (C), day-7 (G), and day-14 (K). Topical TED drops significantly mitigated SM-induced corneal toxicity in vivo and restored epithelium damage on day-3 (D), day-7 (H), and day-14 (L) compared to SM only group. TED only drops group on day-3 (B), day-7 (F), and day-14 (J) did not cause corneal epithelium damage as compared to naïve at day-3 (A), day-7 (E), and day-14 (I). White arrowhead depicts damage the hyper-reflective border formed due to SM damage. White arrow depicts snow-like aggregates found on basal epithelium. n = 6/group/time point.

3.1.2. Changes in anterior, mid, and posterior stroma after SM injury and mitigation by TED

A multimodal approach was used to examine the corneal stroma in vivo and post-euthanasia tissue histology. In vivo techniques include optical coherence tomography via Heidelberg Spectralis and In vivo live scanning confocal microscopy via Heidelberg HRT3-RCM.

Spectralis imaging of naïve corneas showed a healthy confluent stroma. SM-exposed corneas led to a corneal stroma with an increased thickness in the central and peripheral regions on day-3 (Fig. 2C), day-7 (Fig. 2G), and day-14 (Fig. 2K). The corneal thickness gradually decreased from day-3 to day-14 but remained thicker than the corresponding naïve control. The topical application of TED to SM-exposed corneas showed a reduction in corneal thickness on day-3 (Fig. 2D), day-7 (Fig. 2H), and day-14 (Fig. 2L) compared to SM exposed corneas. Corneal thickness was reduced using TED on day-14 but is still recovered to naïve. The topical application of TED only did not cause a change in the corneal stroma on day-3 (Fig. 2B), day-7 (Fig. 2F), and day-14 (Fig. 2J) compared to the naïve on day-3 (Fig. 2A), day-7 (Fig. 2E), and day-14 (Fig. 2I).

Fig. 2.

Spectralis images exhibiting SM vapor-induced corneal thickness to the corneal stroma on day-3 (C), day-7 (G), and day-14 (K). TED drops potently mitigated SM-induced corneal toxicity in vivo by restoring corneal stromal thickness at day-3 (D), day-7 (H), and day-14 (L) compared to SM only group. TED topical drops only group on day-3 (B), day-7 (F), and day-14 (J) did not change corneal thickness as compared to naïve at day-3 (A), day-7 (E), and day-14 (I). Spectralis images were taken on a horizontal plane across the center of the cornea. n = 6/group/time point. Scale bar = 50 μm.

HRT3-RCM imaging was used to determine the quality of stromal cells and the transparency of the stroma. In a naïve cornea, normal stromal cells are present on day-3 (Fig. 3A; 70.94 ± 3.12), day-7 (Fig. 3E; 71.83 ± 3.57), and day-14 (Fig. 3I; 70.42 ± 2.95). SM exposure resulted in the loss of corneal transparency due to stress fiber formation and significant loss of stromal cell density on day-3 (Fig. 3C; 23.92 ± 5.54; p < 0.0001), day-7 (Fig. 3G; 30.33 ± 4.51; p < 0.0001), and day-14 (Fig. 3K; 54.47 ± 5.70; p < 0.0001) compared to corresponding naïve group. Additionally, SM-exposed corneas showed zones of anuclear hyperreflective cell remnants (cell debris) and the formation of spindles on day-3 (Fig. 3C), day-7 (Fig. 3G), and day-14 (Fig. 3K) using in vivo confocal microscopy. The presence of stress fibers and spindles in the stroma is associated with opacity/haziness in the cornea (Lim et al., 2013). Corneas treated with TED after SM exposure showed progressively rapid improvement in stromal quality. A significant mitigation of stromal cell density loss was seen on day-3 (Fig. 3D; 45.89 ± 2.89; p < 0.0001), day-7 (Fig. 3H; 52.22 ± 3.29; p < 0.0001), and day-14 (Fig. 3L; 69.97 ± 2.49; p < 0.0001) compared to corresponding SM group. Day-3 (Fig. 3D) imaging showed zones of anuclear hyper-reflective cell remnants (cell debris) and the formation of spindles like SM injury. However, on day-7 (Fig. 3H), cells begin to repopulate the stroma, and on day-14 (Fig. 3L), a noteworthy improvement can be seen. Topical application of TED only did not cause changes in the corneal stromal cell density on day-3 (Fig. 3B; 70.81 ± 2.82), day-7 (Fig. 3F; 70.67 ± 3.14), and day-14 (Fig. 3J; 69.94 ± 2.53).

Fig. 3.

HRT3-RCM images of corneal stroma exhibiting SM vapor-induced loss of keratocyte density in the corneal stroma on day-3 (C), day-7 (G), and day-14 (K). Topical TED drops significantly mitigated SM-induced keratocyte density in vivo on day-3 (D), day-7 (H), and day-14 (L) compared to SM only group. TED treatment after SM exposure reduced the amount of stress fibers making cornea more transparent compared to SM only group. TED drops caused no loss of corneal stromal keratocytes density on day-3 (B), day-7(F), and day-14 (J) as compared to naïve on day-3 (A), day-7 (E), and day-14 (I). Images were taken at 200 μm ± 20 μm depth in the anterior stroma. n = 6/group/time point.

3.1.3. Changes in corneal endothelium after SM injury and mitigation by TED

HRT3-RCM imaging was used to determine the size and hexagonality of endothelial cells. In a naïve cornea, endothelial cells are hexagonal and organized in a honeycomb pattern seen on day-3 (Fig. 4A; 511.22 ± 27.16), day-7 (Fig. 4E; 512.22 ± 21.08), and day-14 (Fig. 4I; 514.25 ± 21.04). Corneas exposed to SM vapor caused severe injury to the corneal endothelium. A significant loss of endothelial cell density was seen on day-3 (Fig. 4C; 349.64 ± 23.27; p < 0.0001), day-7 (Fig. 4G; 370.58 ± 18.73; p < 0.0001), and day-14 (Fig. 4K; 320.17 ± 11.70; p < 0.0001) compared to naïve groups. On day-3, SM exposed corneas (Fig. 4C) had endothelial cell nuclei present; however, cells have undergone excessive polymegathism (enlargement in size) and pleomorphism (change in the number of facets and shape). Endothelial cells show similar deformation on day-7 (Fig. 4G) and day-14 (Fig. 4K). The topical application of TED after sulfur mustard injury significantly mitigated the damage to corneal endothelium on day-3 (Figs. 4D and 397.31 ± 33.26; p < 0.0001), day-7 (Figs. 4H and 405.50 ± 17.68; p < 0.0001), and day-14 (Fig. 4L; 412 ± 43.98; p < 0.0001). Polymegathism and pleomorphism are still present in the SM + TED group compared to naïve corneas; however, the severity of endothelial damage was mitigated, as seen by the maintenance of the honeycomb-like pattern for at least two weeks. The use of Topical TED only did not cause changes in the corneal endothelium on day-3 (Fig. 4B; 514.25 ± 21.55), day-7 (Fig. 4F; 511.47 ± 22.64), and day-14 (Fig. 4J; 524.75 ± 33.24).

Fig. 4.

HRT3-RCM images exhibiting SM vapor-induced loss of morphology and cell density in corneal endothelium on day-3 (C), day-7 (G), and day-14 (K). Topical TED drops partially restored SM-induced loss of morphology (hexagonal shape) and mitigated loss of cell density in the corneal endothelium in vivo on day-3 (D), day-7 (H), and day-14 (L) compared to SM only group. TED only drops group did not cause any change in corneal endothelium on day-3 (B), day-7 (F), and day-14 (J) as compared to naïve on day-3 (A), day-7 (E), and day-14 (I). n = 6/group/time point.

3.2. Clinically relevant changes in ocular and corneal health by SM

3.2.1. Development of corneal haze after SM injury and mitigation by TED

Stereo and slit-lamp bio-microscopy was used for gross in vivo observation of rabbit corneas. Stereo bio-microscopy showed SM vapor exposure to rabbit eyes caused grade III alkali corneal injury based on the Roper-Hall scale (Roper-Hall, 1965). Corneal haze was assessed using a Fantes score. SM exposure caused haze that persisted on day-3 (Fig. 5C; 2.3 ± 0.5), day-7 (Fig. 5G; 2.7 ± 0.5), and day-14 (Fig. 5K; 2.8 ± 0.5). The degree of haze was mitigated through the use of TED on day-3 (Fig. 5D; 1.3 ± 0.5; p < 0.01), day-7 (Fig. 5H; 1.5 ± 0.5; p < 0.01), and almost absent by day-14 (Fig. 5L; 1.5 ± 0.5; p < 0.01). Naïve and TED only groups showed healthy eyes with a Fantes score of 0.0 ± 0.0 on day-3 (Fig. 5A and F), day-7 (Fig. 5E and F), and day-14 (Fig. 5I and J).

Fig. 5.

Biomicroscopy images describing corneal opacity. Corneal haze was graded based on Fantes score. SM-vapor caused severe corneal haze on day-3 (C), day-7 (G), and day-14 (K) compared to naïve group. TED drops significantly mitigated SM-induced haze over-time on day-3 (D), day-7 (H), and day-14 (L) compared to SM only group. TED topical drops only on day-3 (B), day-7(F), and day-14 (J) did not cause any haze compared to naïve on day-3 (A), day-7 (E), and day-14 (I). n = 6/group/time point. Scale bar = 2.0 mm.

Slit-Lamp analysis confirmed haze and grade III corneal injury in the SM exposure group on day-3 (Fig. 6C), day-7 (Fig. 6G), and day-14 (Fig. 6K). Narrow beam slit lamp evaluation showed cornea opaqueness due to the absence of parallelepiped lines on day-3 (Fig. 6C) and day-7 (Fig. 6G). Additionally, corneal haze/edema was seen at all three-time points. Corneal edema was characterized by the blueish color seen in the slit-lamp beam. On the other hand, topical TED treatment to SM exposed corneas reduced corneal opacity and edema on day-3 (Fig. 6D) and day-7 (Fig. 6H) to grade II. Moreover, TED treatment showed cornea injury reduced to grade I on day-14 (Fig. 6L). Narrow beam slit lamp evaluation showed recovery of corneal transparency due to parallelepiped lines on day-7 and day-14. Additionally, a visible reduction in cornea haze was seen at all time points compared to the SM exposure-only group. TED dosing to the non-exposure contralateral eye did not cause any ocular opacity or edema on day-3 (Fig. 6B), day-7 (Fig. 6F), and day-14 (Fig. 6J) as compared to naïve day-3 (Fig. 6A), day-7 (Fig. 6E), and day-14 (Fig. 6I).

Fig. 6.

Slit-lamp narrow beam images exhibiting SM vapor-induced changes in the cornea on day-3 (C), day-7 (G), and day-14 (K) compared to naïve group. Corneal edema is present in SM exposure groups on day-3 (C), day-7 (G), and day-14 (K) based on the blueish color of the slit lamp beam. Furthermore, corneal opacity was seen by the loss of the second beam of light on the pupil on day-3 (C) and day-7 (G). Corneal edema and opacity are present in TED treatment after SM exposure on day-3 (D). On day-7 (H) corneal edema is present but transparency is partially restored supported by the presence of second beam from the slit-lamp on the pupil. Day-14 has minimal corneal edema, and the transparency was restored. Topically applied TED drops only group did not cause corneal damage on day-3 (B), day-7 (F), and day-14 (J) as compared to naïve on day-3 (A), day-7 (E), and day-14 (I). n = 6/group/time point. Scale bar = 2.0 mm.

3.2.2. Detection of corneal epithelial erosion after SM injury and mitigation by TED

Epithelial defects were observed using fluorescein staining. A positive fluorescein stain is associated with defects in the epithelial-stroma barrier (Banayan et al., 2018). Observed values of percentage positive fluorescein staining on day-3, day-7, and day-14 for the four test groups: naïve, TED, SM, and SM + TED are in Table 2. Fluorescein staining of naïve corneas resulted in no stain on day-3 (Fig. 7A), day-7 (Fig. 7E), and day-14 (Fig. 7I). A significant increase in fluorescein-positive area can be seen in the SM exposed group on day-3 (Figs. 7C and 83.53 ± 5.03; p < 0.0001), day-7 (Figs. 7G and 57.08 ± 8.57; p < 0.0001), and day-14 (Figs. 7K and 15.00 ± 7.65; p < 0.0001) compared to naïve. The topical application of TED after SM exposure has a significantly reduced area of fluorescein-positive stain on day-3 (Figs. 7D and 59.31 ± 8.55; p < 0.0001), day-7 (Fig. 7H; 46.53 ± 8.93; p < 0.0001), and day-14 (Fig. 7H; 0.00 ± 0.00; p < 0.0001) compared to the SM exposed group. The application of TED only resulted in no fluorescein stain on day-3 (Fig. 7C), day-7 (Fig. 7G), and day-14 (Fig. K), demonstrating that TED did not cause any damage to the corneal epithelial-stroma barrier.

Table 2.

Clinical measurements of corneal haze, central corneal thickness, tear volume, and intraocular pressure in study groups.

Group	Naïve			TED			SM			SM + TED
Study Time	Day-3	Day-7	Day-14	Day-3	Day-7	Day-14	Day-3	Day-7	Day-14	Day-3	Day-7	Day-14
Haze level (Fantas score)	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	2.3 ± 0.5 ****	2.7 ± 0.5 ****	2.8 ± 0.5 ****	1.3 ± 0.5 **** $$	1.5 ± 0.5 **** $$	1.5 ± 0.5 **** $
STT	7.7±6.0	8.0±4.3	7.3±2.2	12.3±9.6	6.7±2.3	15.0±5.7	13.5±8.2	14.7±7.9	21.0±7.7	14.8±6.8	17.8±7.2	17.0±7.1 **** $
IOP	12.3±2.3	14.5±3.1	10.5±3.4	13.5±3.1	13.5±3.7	12.5±3.5	11.5±7.2	14.8±8.3	32.3±14.1	9.7±3.6	14.3±3.2	11.5±0.7

Significant changes between SM vs Naïve or TED: Significant changes between SM vs SM + TED.

^****p < 0.001

^$$p < 0.01.

^***p = 0.001

^$p = 0.05.

Legend: SM = sulfur mustard, TED = topical eyedrops, STT = Schirmer’s Tear Test, IOP = intraocular pressure, n = 6 for all groups and timepoints.

Fig. 7.

Fluorescein dye test showing the progression of corneal epithelial damage and recovery in rabbit eyes. The degree of epithelial damage was graded based on percentage of positive fluorescein staining. SM vapors severely damaged corneal epithelium on day-3 (C), day-7 (G), and day-14 (K) compared to naïve group. TED drops application after SM exposure effectively mitigated the damage on day-3 (D), day-7 (H) and day-14 (L) compared to SM group. TED only group on day-3 (B), day-7(F), and day-14 (J) did not cause damage to corneal epithelium as compared to naïve on day-3 (A), day-7 (E), and day-14 (I). n = 6/group/time point. Scale bar = 2.0 mm.

3.2.3. Appearance of other common MGK symptoms after SM injury and mitigation by TED

Table 2 depicts the observed values of haze, IOP (interocular pressure), and STT (Schirmer’s tear test) on day-3, day-7, and day-14 for the four test groups: naïve, TED, SM, and SM + TED (data on Table 2). Corneal haze was scored based on the Fantes score. A significant increase in the haze was found in the SM exposure group on day-3 (2.3 ± 0.5; p < 0.001), day-7 (2.7 ± 0.5; p < 0.001), and day-14 (2.8 ± 0.5; p < 0.001) compared to the naïve group. The topical application of TED after SM exposure led to a significant reduction on day-3 (1.3 ± 0.5; p < 0.01), day-7 (1.5 ± 0.5; p < 0.01), and day-14 (1.5 ± 0.5; p < 0.05) compared to SM only group. No significant changes were found in the group receiving topical TED only on day-3 (0 ± 0), day-7 (0 ± 0), and day-14 (0 ± 0). No significant change in IOP was detected between four groups (Naïve, TED, SM, and SM + TED) on day-3 (12.3 ± 2.3; 13.5 ± 3.1; 11.5 ± 7.2; 9.7 ± 3.6), day-7 (14.5 ± 3.1; 13.5 ± 3.7; 14.8 ± 8.3; 14.3 ± 3.2), or day-14 (10.5 ± 3.4; 12.5 ± 3.5; 32.3 ± 14.1; 11.5 ± 0.7) (data on Table 2). Shimmer tear test showed no significance in Naïve vs. SM on day-3 (7.7 ± 6.0; 13.5 ± 8.2) and day-7 (8.0 ± 4.3; 14.7 ± 7.9). There was a significant increase on day-14 (7.3 ± 2.2; 21.0 ± 7.7; p < 0.001). There was no significance in SM vs. SM with TED treatment on day-3 (13.5 ± 8.2; 14.8 ± 6.8), day-7 (14.7 ± 7.9; 17.8 ± 7.2), and day-14 (21.0 ± 7.7; 17.0 ± 7.1), and no significant change in Naïve vs. TED alone on day-3 (7.7 ± 6.0; 12.3 ± 9.6), day-7 (8.0 ± 4.3; 6.7 ± 2.3), and day-14 (7.3 ± 2.2; 15.0 ± 5.7) (data on Table 2). Thus, clinical observations demonstrate that the topical application of TED effectively mitigates SM-related corneal injury and safe to use.

3.3. Histopathology showing gross anatomical changes in the cornea after SM injury

3.3.1. Changes in corneal epithelium after SM injury and mitigation by TED

H&E staining was used to assess morphologic changes in the corneal epithelium-stroma barrier. SM vapor exposure caused epithelial detachment on day-3 (Fig. 8C) and day-7 (Fig. 8G). On day-14 (Fig. 8K), the epithelium was recovered. The use of TED after SM exposure (SM + TED) showed epithelial detachment on day-3 (Fig. 8E). On day-7 (Fig. 8H) and day-14 (Fig. 8L), the corneal epithelium was attached to the stroma. The use of TED helped to recover the epithelial-stromal border. Naïve and TED-only did not show morphologic changes on day-3 (Fig. 8A and B), day-7 (Fig. 8E, I), or day-14 (Fig. 8G, K).

Fig. 8.

H&E stained images showing SM vapor caused loss of epithelium-stroma integrity on day-3 (C) and day-7 (G). On day-14 (K) SM injured corneas had restored epithelial-stromal barrier. Black arrowhead points to damage on the epithelial-stromal barrier. TED topical drops effectively restored SM-induced epithelium-stromal integrity over time on day-3 (D), day-7 (H), and day-14 (L). TED topical drops alone group on day-3 (B), day-7 (F), and day-14 (J) did not compromise epithelium-stroma integrity as compared to naïve on day-3 (A), day-7 (E), and day-14 (I). n = 6/group/time point. Scale bar = 100 μm.

3.3.2. Changes in corneal stroma after SM injury and mitigation by TED

H&E staining shows the health of rabbit cornea. SM vapor causes increased stroma spacing on day-3 (Fig. 8C), day-7 (Fig. 8G), and day-14 (Fig. 8K) compared to corresponding naïve groups. TED treatment after SM exposure had less stromal separation on day-3 (Fig. 8D) and day-7 (Fig. 8H) compared to the SM group. On day-14 (Fig. 8L), TED treatment after SM exposure had minimal stromal spacing compared to SM group. TED-only groups do not have morphological changes on day-3 (Fig. 8B), day-7 (Fig. 8F), or day-14 (Fig. 8J) as compared to naïve day-3 (Fig. 8A), day-7 (Fig. 8E), and day-14 (Fig. 8I).

Picrosirius Red stain showing collagen I and collagen III levels in rabbit cornea. TED-only groups show normal collagen I (yellow) and III (red) stains indicating a healthy stroma on day-3 (Fig. 9B; 9.39 ± 0.5), day-7 (Fig. 9E; 9.53 ± 0.61), and day-14 (Fig. 9J; 10.17 ± 1.59) as compared to naïve day-3 (Fig. 9A; 9.61 ± 0.76), day-7 (Fig. 9E; 9.71 ± 1.21), and day-14 (Fig. 9I; 10.03 ± 1.59). SM vapor significantly increased collagen III staining (red) on day-3 (Figs. 9C and 81.14 ± 3.25; p < 0.0001), day-7 (Fig. 9G; 83.39 ± 1.34; p < 0.0001), and day-14 (Fig. 9K; 83.33 ± 2.92; p < 0.0001), indicating haze/fibrosis. SM + TED group had significantly less red staining compared to SM only group on day-3 (Figs. 9D and 61.14 ± 3.25; p < 0.0001), day-7 (Figs. 9H and 63.39 ± 1.49; p < 0.0001), and day-14 (Fig. 9L; 63.33 ± 1.34; p < 0.0001) but to a lower intensity compared to the SM group indicating TED is mitigating SM induced corneal haze/fibrosis.

Fig. 9.

Picrosirius Red stain showing collagen I (yellow) and collagen III (red) levels in rabbit cornea showing SM vapor-induced loss of collagen I and accumulation of collagen III on day-3 (C), day-7(G), and day-14 (K) as compared to naïve groups. TED drops significantly mitigated collagen III production on day-3 (D), day-7(H), and day-14 (L) as compared to SM group. TED drops alone on day-3 (B), day-7(F), and day-14 (J) did not cause any loss of collagen I as compared to naïve group on day-3 (A), day-7 (E), and day-14 (I). n = 6/group/time point. Scale bar = 100 μm.

3.4. Modifications in characteristic corneal stromal fibrillogenesis by SM and mitigation by TED

Transmission electron microscopy was conducted 14 days following SM vapor exposure, SM vapor + TED treatment, TED only, or naïve. Images of transverse sections were taken at 50,000x magnification (Fig. 10 original TEM). Quantitative analysis of stroma transverse sections did not show a change in the size (Fig. 10 ellipse) or inter-fibril distance (IFD) of stromal collagens after adding TED (Fig. 10). SM vapor exposure shows a trend in which collagen size has been decreased and maximum, and minimum IFD have increased. Though the use of TED after SM exposure did not show a change compared to the naïve eye in maximum and minimum IFD, a variation in collagen size distribution was still present. Quantification and graphical representation of the collagen area, maximum IFD, and minimum IFD are shown in Fig. 11.

Fig. 10.

TEM images of corneal tissue sections showing pattern of collagen fibrils in cross-sections at 50,000 × magnification. Ellipses are digitally processed TEM image showing segmentation for individual collagen fibrils, and further fitted with individual bead like structure. Ellipses were used to quantify collagen fibril size. Fibril graph was digitally processed from TEM images showing minimal (red lines) and maximal (blue lines) inter fibril distances (IFD) between neighboring collagen fibrils. SM exposure caused increased IFD, while TED treatment restored collagen fibrils arrangement on day-14. TED only treatment group did not affect collagen fibril arrangement and was similar to naïve on day-14. n = 6/group/time point.

Fig. 11.

Comparative analysis of fibril size, maximum inter-fibril distance (IFD), and minimum inter-fibril distance in naïve, TED, SM, and SM + TED. A trending change in fibril size and IFD was observed to SM exposure. Please mention how many images were processed.

4. Discussion

Sulfur mustard (SM) exposure to the eye causes a severe corneal pathology called mustard gas keratopathy (MGK). MGK is well described and documented in human subjects (Javadi et al., 2005; Rowell et al., 2009; Safarinejad et al., 2001; Solberg et al., 1997). In human subjects, pathological symptoms appear after several hours of SM exposure that include severe ocular pain, photophobia, excessive tear production, eyelid swelling, blepharospasms, and blurred vision (Javadi et al., 2005; McNutt et al., 2012b, 2012a; Milhorn et al., 2010; Rowell et al., 2009; Safarinejad et al., 2001; Solberg et al., 1997). To the best of our knowledge, there are limited studies examining the structural changes in the cornea following SM exposure in live animals in situ. Therefore, this study used a state-of-the-art live animal multimodal clinical eye imaging system to study time-dependent structural and cellular changes in different corneal cells in vivo after SM exposure. Additionally, this study tested if TED could prevent or mitigate damages caused by SM exposure.

SM toxicity begins with corneal epithelial defects or sloughing of corneal surface tissue upon contact with the eye (Balali-Mood and Hefazi, 2006; Mann and Pullinger, 1942; Pechura and Rall, 1993). SM penetrates deeper into the cornea two to 6 h post-exposure, causing corneal edema, iritis, iris vessel dilation, and iris necrosis (Balali-Mood and Hefazi, 2006; Mann and Pullinger, 1942; Pechura and Rall, 1993). In this study, we replicated ocular mustard vapor toxicity in our rabbit animal model by exposing the whole eyes to 200 mg-min/m3 of SM-vapor for 8 min. The median incapacitating (ICt50) dose of SM is 100–200 mg min/m³/based on human clinical studies (Balali-Mood and Hefazi, 2006; Mann and Pullinger, 1942; Pechura and Rall, 1993). The spectrum of MGK is dependent upon SM-vapor exposure, duration, and concentration in which mild acute toxicity demonstrates mild conjunctivitis with scattered punctate corneal epithelial erosions. Vision loss and permanent corneal scarring can occur with increased SM concentrations and exposure duration (Javadi et al., 2005; McNutt et al., 2012a, 2012b; Safarinejad et al., 2001).

Rabbit eyes exposure to SM showed a loss of epithelial cells and delayed epithelial cell restoration for up to 14 days. This could be seen by positive fluorescein staining and defects in the basal epithelial layer, including loss of cell density and formation of a hyperreflective barrier observed via in vivo confocal microscopy. Clinical changes in the corneal epithelium after TED-only, SM-only, and SM + TED had consistent outcomes on day-3 and day-7 (Tripathi et al., 2020). In vivo confocal microscopy or rabbit corneal epithelium had consistent results with fluorescence staining in SM-only and SM + TED groups. This study further investigated epithelial defects with in vivo confocal microscopy that showed a snow-like pattern of damage in the basal epithelium similar to corneal pathologies seen in bacterial keratitis (Chen et al., 2017). This may be a possible cause of delayed reepithelization seen on day-14 or causing recurrent epithelial erosions further supported by the studies depicting snow-like spots in the basal epithelium in human patients diagnosed with limbal stem cell deficiency (Bonnet et al., 2021). The delay in corneal epithelium regeneration and snow-like spots were not as prevalent in corneas treated with TED after SM exposure.

Similarly, morphological changes in the corneal stroma were consistent and had consistent outcomes on day-3 and day-7. The previous study was limited to observing in vivo corneal stroma changes via slit-lamp microscopy and H&E imaging. In this study, we used optical coherence tomography imaging to confirm changes seen in vivo. SM-only rabbits increased corneal thickness on day-3, day-7, and day-14. The use of TED mitigated SM toxicity of the cornea. The present study advanced our knowledge of stromal damage after SM toxicity via in vivo confocal microscopy of the corneal stroma, which allowed us to observe the cellular change in the stroma in live rabbits over time. We could see SM caused a loss of corneal transparency due to a build-up of cell debris and stress fibers in the corneal stroma at day-3, day-7, and day-14. Moreover, the SM group showed stress fibers, cellular debris, and active cells up till day-14, which may be a key finding in fibrosis formation post-SM exposure. While the SM + TED group showed improved stroma post SM exposure on day-7, and day-14, a partially restored stroma was present with keratocyte-like cells.

PSR staining and TEM techniques were used to examine gross anatomical changes and quantify the ultrastructural stromal changes in the cornea. PSR staining showed an increase in collagen III (COL III) expression after SM exposure, which signifies active corneal wound repair (Lorenzo-Martín et al., 2019; Ma et al., 2018). A massive increase in COL III supports the theory that “healed tissue” differs from “unexposed tissue”. COL III has less tensile strength, increased light scattering, and requires more adhesive compounds like vimentin and elastin than collagen I (COL I) (Karsdal et al., 2017; Voloshenyuk et al., 2011). Thus, persistently increased COL III leads to a more fragile cornea and may play a role in the delayed onset of MGK. TEM showed significant changes in collagen diameter and inter-fibular distance (IFD), the distance between individual collagen fibrils in the corneal stroma. The changes in collagen size and IFD may result in blurry and disabled vision (Gronkiewicz et al., 2017, 2016; Meek et al., 2005). Several studies suggested that changes in stromal architecture are critical events during corneal wound healing, thus describing uniform collagen architecture essential for corneal transparency (Meek, 2009; Meek and Knupp, 2015; Michelacci, 2003; Sinha et al., 2021). Future molecular analyses of extracellular matrix studies are required to identify the mechanisms during corneal wound healing after SM exposure. TED application following SM vapor exposure has been shown to mitigate the amount of COL III produced on day-3, day-7, and day-14. Moreover, the change in IFD was significantly mitigated on day-14. The consistent IFD supports that corneal transparency was restored earlier in the SM + TED group compared to the SM only group.

The present study also showed defects in the corneal endothelium after SM exposure which were not assessed in our previous work. In conjunction with previous studies, in vivo confocal imaging of SM exposure caused endothelial cells to undergo pleomorphism and polymorphism (Kadar et al., 2013; McNutt et al., 2020). The structural changes cause defects in their pump function, which may be a possible cause of increased corneal edema after SM toxicity (Bonanno, 2012). Moreover, studies showed that corneal edema positively correlates with loss of endothelial density (McNutt et al., 2021). The cornea is an ectatic tissue; therefore, the inability to hydrate or dehydrate leads to compromised vision (Kamil and Mohan, 2021). Further, compromised endothelial pump function may contribute to the significant increase in IFD between collagen fibrils, which is evident as TED application mitigated stromal collagen IFD changes by day-14, whereas a significant increase in IFD can be seen in SM-only groups. Thus, topically applied drugs (TED) can be used to mitigate damage caused to the corneal endothelium following mustard gas exposure.

In our previous study, we observed the beneficial effects of TED on SM-exposed rabbit corneas in vivo for seven days using classical clinical examinations and histological techniques to examine the corneal stroma and epithelium (Tripathi et al., 2020). This study further extended our preliminary findings for up to 14 days using more advanced multimodal techniques like in vivo confocal microscopy (using HRT3) and optical coherence tomography (using Spectralis). These techniques allowed us to perform detailed observations of SM damage in each layer of the cornea and the beneficial effects of TED treatment in live rabbits. Previous human studies have shown that delayed MGK is marked by a significant decrease in measured and corrected IOP (Jadidi et al., 2019). Contrary to this, we did not find any significant changes in IOP this study. Additionally, TED treated, SM exposed, or SM + TED eyes did not have a significant change in tear production. In the present study, a mitigated and moderated wound response was observed in rabbit corneas with SM + TED on day-14 compared to SM-only group. Rabbit corneas received TED treatment twice daily for the first seven days. Thus, day-14 data shows the enduring effects of TED on SM exposure to the cornea. On day-14, SM-exposed corneas had persistent epithelial damage, reduced corneal thickness, the repopulation of keratocyte-like cells in the stroma, and a mild recovery in the characteristic collagen fibril architecture in the stroma. These changes are consistent with previous in vivo rabbit studies (Banin et al., 2003; Goldich et al., 2013; Goswami et al., 2019; McNutt et al., 2021, 2020). However, on day-14, TED treatment to SM-exposed eye (SM + TED) restored the epithelium, corneal thickness, stromal content, and collagen architecture. Additionally, in vivo confocal imaging suggested that TED drops mitigated damage to the corneal endothelium further strengthening the effectiveness of TED up to 14 days after SM vapor exposure.

In conclusion, this study showed that SM exposure causes structural and cellular defects in the corneal epithelium, stroma, and endothelium in situ, possibly, describing the in vivo symptoms for MGK pathology. Thus, topical eye drops (TED) mitigated SM toxicity to the cornea and allowed for a more moderate wound healing response restoring the unique ultrastructural organization of the corneal stroma. Furthermore, TED was well tolerated by the eye during the seven-day application process and did not cause any post-application side-effects previously reported with steroids (Dinning, 1976; Phulke et al., 2017). Molecular studies are underway to examine the mechanism of action of TED in mitigating corneal stroma damage caused by SM exposure.

Data availability

Data will be made available on request.