development of a mouse model for sulfur mustard-induced ocular injury and long-term clinical...

140

Introduction

Sulfur mustard (SM, bis-(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent that covalently modifies DNA, proteins, and other macromolecules (1–3). SM can induce severe ocular injury, and the features of this injury have been well documented in humans (4,5) and rabbit models (6–8). The injury appears to be biphasic, consisting of an acute and delayed injury

phase. Acute injury is characterized by inflammation, epithelial loss, corneal edema, limbal engorgement reepithelialization, and finally early neovascularization. After initial recovery phase, a delayed injury phase ensues which is characterized by inflammation, corneal edema, corneal erosions and infiltrates, advanced corneal neovascularization, and finally corneal opacities and scars which severely compromised eye function.

RESEARCH ARTICLE

Development of a mouse model for sulfur mustard-induced ocular injury and long-term clinical analysis of injury progression

Albert Leonard Ruff1, Anthony John Jarecke2, David Joseph Hilber2, Christin Coleen Rothwell1, Sarah Lynn Beach1, and James Franklin Dillman III1

1USAMRICD, Cellular and Molecular Biology, Gunpowder, USA and 2US Army Public Health Command, Gunpowder, USA

AbstractContext: Sulfur mustard (SM) is a highly reactive vesicating agent that can induce severe ocular injury. The clinical features of this injury have been well documented, but the molecular basis for this pathology is not well understood. Identification and validation of specific targets is necessary in the effort to develop effective therapeutics for this injury. Currently used rabbit models are not well suited for many molecular studies because the necessary reagents are not widely available. However, these reagents are widely available for the mouse model. Objective: Our objective is to develop a mouse model of SM-induced ocular injury suitable for the study of the molecular mechanisms of injury and the evaluation of therapeutics. Materials and Methods: Ocular exposure to sulfur mustard vapor was accomplished by using a vapor cup method. Dose response studies were conducted in female BALB/c mice. An exposure dose which produced moderate injury was selected for further study as moderate injury was determined to be amenable to studying the beneficial effects of potential therapeutics. Histopathology and inflammatory markers were evaluated for up to 28 days after exposure, while clinical injury progression was evaluated for 1 year post-exposure. Results: A biphasic ocular injury was observed in mice exposed to SM. Acute phase SM ocular injury in mice was characterized by significant corneal epithelium loss, corneal edema, limbal engorgement, and ocular inflammation. This was followed by a brief recovery phase. A delayed injury phase then ensued in the following weeks to months and was characterized by keratitis, stromal edema, infiltrates, neovascularization, and eventual corneal scarring. Discussion and Conclusions: SM-induced ocular injury in mice is consistent with observations of SM-induced ocular injury in humans and rabbit models. However, in the mouse model, the SM ocular injury, a more rapid onset of the delayed injury phase was observed. We have developed an animal model of SM injury that is suitable for studies to elucidate molecular mechanisms of injury and identify potential therapeutic targets.Keywords: Sulfur mustard, mice, ocular injury, inflammation

Address for Correspondence: Albert Leonard Ruff, USAMRICD, Cellular and Molecular Biology, 3100 Ricketts Point Road, Gunpowder, 21010 USA. E-mail: [email protected]

(Received 31 July 2012; revised 27 August 2012; accepted 15 September 2012)

Cutaneous and Ocular Toxicology, 2013; 32(2): 140–149© 2013 Informa Healthcare USA, Inc.ISSN 1556-9527 print/ISSN 1556-9535 onlineDOI: 10.3109/15569527.2012.731666

Cutaneous and Ocular Toxicology

32

2

140

149

31July2012

27August2012

15September2012

1556-9527

1556-9535

© 2013 Informa Healthcare USA, Inc.

10.3109/15569527.2012.731666

2013

Mouse model for sulfur mustard-induced ocular injury

A. L. Ruff et al.

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.

Mouse model for sulfur mustard-induced ocular injury 141


Recent work by Kadar et al. suggests that the delayed injury phase in SM ocular exposure may be due to limbal stem cell deficiency secondary to the SM-induce loss of corneal sensory innervation (9).

It has been hypothesized that DNA damage is the primary initiator of the cellular response to SM exposure (10–12); however, the molecular mechanisms that con-nect SM-induced DNA damage to the observed clinical injury remain largely unknown. There have been several microarray studies of SM-induced injury, in vivo and in vitro, models of skin and lung mapping SM response pathways (13,14). Some studies have identified roles for Fas, caspase, and calcium signaling in SM-induced cell death (15–17). Other studies, including some by our own laboratory, have investigated molecules such as p53, NF-kB, JNK, and p38 (18–20). While these studies are beginning to shed light on the molecular mechanisms of SM-induced inflammation for those organ systems, similar studies in ocular models are lacking. Thus, the molecular mechanisms of SM-induced ocular injury remain unclear. A better understanding of the major sig-naling pathways in ocular SM injury is imperative to the therapeutic development for this injury.

More knowledge is needed regarding the molecular mechanisms of this injury for the identification of poten-tial therapeutic targets. As discussed, rabbit models are not well suited for these types of studies because the necessary reagents are not widely available for this spe-cies. Fortunately, many reagents for studying cellular and molecular events (e.g. microarrays, monoclonal antibodies, knockout animals) are widely available for the mouse. In addition, mice are widely used to study ocular disease and injuries including corneal neovascularization and scarring, which are prominent features of SM-induced ocular injury. Many of the cellular and molecular events implicated in the pathogenesis of human eye disease/injury have cor-related well to mouse models (21–25). For these reasons, the development of a mouse model of SM-induced ocular injury is beneficial and would be more advantageous for the development of human treatment/therapeutics.

Methods

MiceFemale BALB/c mice (19–21 g, Charles Rivers Laboratories, Wilmington, MA) were used in this study. Mice were studied and maintained in an animal care facility that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All research was conducted in compliance with the “Guide for Care and Use of Laboratory Animals,” revised 1996, and guidelines in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Vapor cup constructionVapor cups were constructed from 0.2 ml PCR tubes with the cap of the PCR tube serving as the vapor cup.

Whatman filter paper was placed in the cap to serve as a reservoir for liquid SM. A Viton O-ring (1.9 mm width, 2.8 mm ID) was attached to the rim of the cap using instant adhesive. The O-ring served as a smooth surface to avoid possible scratching of the cornea during expo-sure. An o-ring size was selected that would expose the maximum area of the cornea while confidently avoid-ing exposure of the limbus in order to ensure consistent exposure across animals (Figure 1).

Pain controlOsmotic pumps (Alzet model 1002, Durect Corporation, Cupertino, CA, USA), loaded with a 1 mg/ml solution of buprenorphine were implanted subcutaneously on the backs of mice 1 day before exposure. The daily dose of buprenorphine by this method was 0.3 mg/kg/day for 14 days.

Exposures and controlsMice were anesthetized with xylazine 7.5 mg/kg and ketamine 75 mg/kg for exposure. Neat SM was applied to the Whatman filter paper in the vapor cup under magnification. The vapor cup was then inverted onto a glass slide for 30 s to allow SM vapor to build up in the cup. The mouse eye was gently protruded and the vapor cup placed over the eye for the indicated time. During the exposure, the vapor cup was briefly removed at 30-s intervals to blink the eye. After exposure, eyes were blinked regularly until the animal recovered from anesthesia. Negative control animals were implanted with osmotic pumps, anesthetized as for exposure, and

Figure 1. SM vapor cup construction and fit. Vapor cup components and construction; 0.2 ml PCR tubes, Whatman filter paper, and Viton o-rings were assembled as described in the methods (A). (B) shows a cross section graphical representation of vapor cup fit to the mouse eye. The dashed grey line represents the approximate area of the cornea that is exposed to SM vapor.

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.

142 A. L. Ruff et al.


exposed to an empty vapor cup for 2 min (n = 5). Naïve animals were also included in the study to control for any effects from the vapor cup alone (n = 5).

Clinical observationsEyes were evaluated by slit lamp microscopy and scored post-exposure on day 2, 7, 14, 21, and 28; at weeks 6, 8, 10, 12, 14, and 16, and then monthly thereafter. A Topcon SL-D7 slit lamp microscope (Topcon, Paramus, NJ, USA) equipped with a 40X objective, dedicated Nikon D200 digital camera system, and integrated through-the-slit flash was used for the clinical examinations. A green filter was used for acquisition of some images to better visual-ize corneal blood vessels, and an exciter filter was used with fluorescein staining to evaluate epithelial injury. A cornea neovascularization scoring system was developed to grade injury according to three main criteria: clock hour extent, density, and centricity of neovascularization (see Table 1). The cornea was divided into three equidis-tant zones using a template overlaid on the images for scoring. Zone 1 was the outer 1/3 of the cornea next to the limbus, Zone 2 was the intermediate area, and Zone 3 was the center of the cornea. Each animal was scored independently by two different investigators and aver-aged. The data are expressed as the average score of the group over time +/− SD (n = 5). Ischemia of blood vessels in the iris was scored by the clock hour extent in which ischemia was observed. Data were analyzed for statisti-cal significance using a one-way analysis of variance (ANOVA) followed by Bonferonni’s multiple comparison tests. Only raw images were used for all scoring. Adobe Photoshop Elements were used to convert images to black and white for printing purposes. This program was also used to adjust the contrast of the images in Figure 3 (identical settings used for all images) to better visualize the corneal blood vessels for printing purposes.

HistopathologyRight eyes of mice were exposed to SM vapor for 1.25 min. Eyes were enucleated from 1 to 28 days after SM-exposure. Histopathology processing and

scoring were performed by the Research Support Division, USAMRICD. Immediately after enucleation, eyes were fixed by immersion in Davidson’s fixative for 4 h then processed by routine histological methods. Paraffin tissue sections were cut at 6 microns and stained using hematoxylin and eosin. Parameters of corneal injury were scored using a system standard at USAMRICD as follows: 0 = No lesion; 1 = Minimal (1–10%); 2 = Mild (11–25%); 3 = Moderate (26–45%); and 4= Severe (> 45%). The per-cent indicates the percentage of the area of the cornea that is affected. The parameters scored include corneal epi-thelial necrosis (cells in the continuum of degeneration to necrosis/apoptosis), corneal epithelium attenuation (reduced number of epithelial layers), stroma necrosis/loss (fibrocyte necrosis/loss), stroma edema (thicken-ing of the stroma), stroma inflammation (presence of inflammatory cell infiltrates), and stroma deformity (loss of stroma architecture from edema, scar, ulceration, etc.). The data are expressed as the average score over time +/− SD (n = 3) and analyzed by one-way ANOVA followed by Dunnett’s comparison tests.

Inflammatory mediator analysisRight eyes of mice were exposed to SM vapor for 1.25 min. Eyes were enucleated from 2 to 24 days after SM-exposure. Whole eyes were homogenized in Epilife Medium (Invitrogen, Carlsbad, CA, USA) contain-ing 0.1% Triton X-100 and a protease inhibitor cock-tail (Roche Applied Science, Indianapolis, IN, USA). Lysates were cleared by centrifugation in a refrigerated microfuge at maximum speed. Samples were assayed using a bead-based multiplex cytokine assay (Milliplex, Millipore, Billerica, MA, USA) and analyzed using a Bio-Plex System array reader with Bio-Plex Manager 4.0 software (Bio-Rad Laboratories, Hercules, CA, USA). The data were expressed as the average +/− standard deviation (SD) over time for (n = 9 for interleukin [IL]-1 alpha [IL-1α], IL-1β, monocyte chemotactic protein-1 [MCP-1], and keratinocyte chemoattractant [KC]; n = 3 for IL-6, secreted vascular cell adhesion molecule-1 [sVCAM-1], and matrix metalloproteinase 9 [MMP9]). Data are expressed as pg/ml for all markers except MMP9 which is expressed as fluorescence intensity (MMP9 was measured in a separate assay). Data were analyzed for statistical significance using a one-way ANOVA followed by Bonferonni’s multiple comparison tests.

Results

The right eyes of mice were exposed to SM vapor using a vapor cup method. Preliminary dose response studies tested 0.5, 1, and 2 µl of SM in the vapor cup and exposure dura-tions up to 6 min. These results showed that the duration of exposure, not the amount of SM in the vapor cup, was the primary determinant of dose (data not shown). This finding was not unexpected because all three volumes appeared to fully wet the surface of the reservoir (though with different degrees of saturation), and the area of from which the SM

Table 1. Neovascularization scoring system.Criteria ScoreClock hour extent One point for each clock hour with at

least one neo-vessel in Zone 1*Density One additional point added for each

clock hour with more than three vessels in Zone 1*

Centricity Three points if at least one vessel enters Zone 2*Five points if at least one vessel enters Zone 3*One additional point is added for each additional vessel entering Zones 2 or 3

* The cornea was divided into three equidistant zones for scoring. Zone 1 was the circumference of the cornea next to the limbus, Zone 3 was the center of the cornea, and Zone 2 was the intermediate area.

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.



vaporized was constant regardless of the volume added. Preliminary studies also showed that, exposure durations in excess of 2 min produced very severe injury exhibiting a rapid progression of lens swelling and iris changes in addi-tion to features of injury described below (data not shown). Subsequent dose response studies used 1 µl of SM in the vapor cup and exposure durations of 1.75 min or less. The 1.25 min exposure duration was the highest dose that pro-duced an apparent moderate injury while avoiding a more severe injury and was therefore selected for the histopathol-ogy and inflammatory marker studies.

Epithelial injuryExposure durations of 1 min or greater produced an overt loss of cornea epithelium (Figure 2A). Animals exposed to SM vapor for 30 s showed no loss of cornea epithelium and only minor stippling. Corneal epithelial cell loss in SM injury is maximal at day 2 and reepithelialization occurs by day 7. While fuller corneal clarity is achieved by day 14 (Figure 2B), it is coincident with corneal edema and epithelial disruptions. Some recurrent fluorescein staining was observed in exposure doses of 2 min and greater, but not with lower doses (data not shown). These were recurrent epithelial defects consistent with the reepithelialization of the cornea.

Corneal neovascularizationSM-induced corneal neovascularization (CN) was scored for the 1.0-, 1.25-, 1.5-, and 1.75-min exposure dura-tion experimental groups for time points out to 1 year

post-exposure (Figure 3). No differences were observed between negative controls and naïve controls, and only the scores for the negative controls are shown (indicated as 0 min on the graphs).

The blood vessels in the cornea are readily distin-guishable from blood vessels on the iris under slit lamp microscopy. They are also readily distinguishable in photomicrographs for each animal by observing changes over time. Vessels in the iris generally remain the same or become ischemic. However, corneal vessels can be challenging to discriminate in photomicrographs with-out these frames of reference. In the photomicrographs provided, the cornea blood vessels appear dark and sinu-ous over the iris vessels which are generally lighter and straighter except for day 2 where they are engorged and sinuous. Blood vessels appear prominent in Zone 1 by 2 days post-exposure and encompass virtually the entire clock hour extent around the eye (Figure 3A). Although it is possible that some of these vessels are the result of neo-vascularization, it is more likely that they are pre-existing vessels that were not apparent prior to exposure but have now become engorged. Thus, limbal engorgement and early vascularization in Zone 1 were characteristic of the acute injury. By 7–14 days post exposure, many of the vessels in Zone 1 regress. CN progresses centrally thereafter in a sectoral fashion. At later time points, CN was observed to consistently progress into Zones 2 and 3, which was characteristic of the delayed injury response. For these reasons, vessel scoring data are expressed as a circumference score (Figure 3B) and a centricity score (Figure 3C). The circumference score in all exposed animals is significantly greater than in negative con-trols (p < 0.001) and injury varies significantly over time (p < 0.001). Data from the 1.5- and 1.75-min exposure doses track similarly over the first 4 to 6 weeks as do the data from the 1.0- to 1.25-min exposure doses. Mice in the 1.5- and 1.75-minute exposure doses were eutha-nized by week 8 for humane purposes due to the severity of ocular injury progression (see Figure 5). All exposure groups were significantly different from the control group (p < 0.001). There was no significant difference between the 1.5- and 1.75-min dose groups. The response of the 1.0-min dose group was significantly different from the responses of the 1.25-, 1.5- and 1.75- min dose groups (p < 0.05, p < 0.001, and p < 0.001, respectively), and the 1.25-minute dose group was also significantly different from the responses of 1.5- and 1.75-minute dose groups (p < 0.05 and p < 0.01, respectively). The centricity scores increased for all dose groups over the first 8 weeks of the study. At that point, the centricity scores for the 1.0- and 1.25-min dose groups appear to level out until week 14. At 2 to 4 months, resurgence of CN occurs and then is followed by some degree of remission. All exposure groups were significantly different from the control group (p < 0.001 for 1.0- and 1.25-min groups, and p < 0.05 for 1.5- and 1.75-min groups). There were no other signifi-cant differences between exposure groups for the cen-tricity scores.

Figure 2. SM-induced cornea epithelial injury. Data shown are representative of the average response (n = 5). Images were captured 2 days post-exposure when epithelial injury was maximal. (A) shows SM-induced dose-dependent epithelial injury. (B) shows SM-induced epithelial injury over time from a single representative animal exposed to SM vapor as previously described for 1.75 min.

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.



Other corneal, iris, and lens responsesAll mice exhibited iris engorgement and some degree of lens swelling was apparent at day 2 and day 21 in Figure 3A (see heavy arrows). Lens swelling was more pronounced and persisted (week 1–2) in longer exposures. Scarring of the cornea also progressed over time. It is most visible at day 42 in this figure (see bracket,}). The number of weeks after SM exposure that scar formation was observed was variable (Table 2), but occurred as early as 3 weeks post-exposure and as late as 20 weeks post-exposure. Occasional hemorrhages can also be observed at various time points (see day 28 >). Iris engorgement generally receded by week 1–2. Iris atrophy was noted in many mice by weeks 1–3 and was more likely to occur earlier with higher doses (data not shown). By week 4, iris atrophy generally leveled off for all exposures. Some mice exhibited pupil displace-ment by weeks 2–3 which was also more noticeable with

longer exposures. Similar to CN, the data for iris ischemia from the 1.5-to 1.75-min exposure doses track similarly as do the data from the 1.0- to 1.25- min exposure doses (Figure 4). Ischemia in the 1.0- and 1.25-min groups affected primarily the more minor vessels in the iris whereas ischemia in the two highest doses involved both the major and minor vessels. Interestingly, the gross ability of the iris to contract did not appear to be affected by the ischemia. Contractile rate or other aspects of iris function may have been affected, but these were not assessed in our study. All exposure groups were significantly different from the negative control group (p < 0.001 for 1.25-, 1.5-, and 1.75-min groups, and p < 0.05 for the 1.0-min group). The results for the 1.0- and 1.25-min groups were significantly different from the 1.5- and 1.75- min groups (p < 0.001). There was no significant difference between the 1.0- and 1.25-min groups and the 1.5- and 1.75-min groups.

Figure 3. SM-induced cornea neovascularization. Right eyes of mice were exposed to SM vapor for 1, 1.25, 1.5, and 1.75 min as previously described. Eyes were evaluated by slit lamp microscopy from 2 to 28 days post-exposure. Negative control animals were exposed to an empty vapor cup for 1.25 min. Adobe Photoshop Elements were used to adjust the contrast of the images (identical settings used for all images) to better visualize the corneal blood vessels for printing purposes. (A) shows SM-induced cornea neovascularization and keratitis over time. Enlarged images of Day 2 and Day 42 are shown for better visualization. Results shown are repeated measures from a single representative animal exposed to SM vapor for 1.75 minutes as this animal best showed all aspects of injury from the same camera angle over time. Key: CN (→), lens swelling (heavy arrows), scar (}), iris hemorrhages (>). Vessel scoring data are expressed as a circumference score (vessels appearing early in Zone 1, B) and a centricity score (vessels in Zones 2 and 3, C). Each animal was scored independently by two different investigators and averaged, and the data are expressed as the average score of the group over time +/− SD (n = 5).

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.



EuthanizationPreliminary studies showed that the corneas of exposed mice could perforate secondary to ulcer formation in the area of the infiltrate. Therefore, mice were eutha-nized over the course of the experiment when any signs appeared that the cornea may perforate to preclude this event or there was onset of other signs indicative of severe progressive injury. The number of animals in each group and the time to euthanasia are shown in Figure 5. A dose response is clear, and there is a direct correlation between the exposure time and time to eutha-nasia. No mice needed to be euthanized in the naïve (data not shown) or empty vapor cup control groups. Time to euthanasia was significantly different between

control groups and the 1.25-, 1.5-, and 1.75- min expo-sure doses (p < 0.001). There was also a significant dif-ference between the 1-min exposure dose and the other exposure doses (p < 0.001) but no significant differences were seen between the controls and the 1-min exposure dose. The 1.25-min exposure dose was also significantly different from the 1.5- and 1.75-min exposure doses (p < 0.01 and p < 0.001, respectively). No significant difference could be found between the 1.5- and 1.75-min exposure doses.

HistopathologyHistopathological analyses showed that cornea epithe-lium necrosis and stroma fibrocyte necrosis/loss were maximal at the earliest time points examined (1 and 2 days post-exposure), and were significant relative to unexposed controls (Figure 6). Cornea epithelium attenuation, stromal edema, and stromal inflammation appear to significantly increase over the first few days following exposure. Scores for all these features return to baseline on the order of 1 to 3 weeks following exposure. All these injuries then resurge from 3 to 4 weeks post-exposure and are accompanied by a significant increase in stroma deformity.

Inflammatory markersInflammatory markers in whole eye lysates were ana-lyzed using bead-based antibody capture assays. The analytes assayed included IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon gamma [IFNγ], KC, tumor necrosis factor alpha [TNFα], monocyte chemotactic protein-1 [MCP-1], macrophage inflammatory protein 1 beta [MIP-1β], regulated and normal T cell expressed and secreted [RANTES], vascular endothelial growth fac-tor [VEGF], secreted endothelial selectin [sE-selectin], secreted vascular cell adhesion protein 1 [sVCAM-1], secreted intercellular adhesion molecule 1 [sICAM-1],

Figure 5. Time to euthanasia. Right eyes of mice were exposed to SM vapor for 1 to 1.75 min as previously described. Mice were euthanized for humane reasons when signs of severe injury progression were eminent (e.g. prior to cornea perforation).

Table 2. Time post-exposure of scar formationDose Mouse Time post-exposure1.0 min A Week 6

B Week 6C Week 8D Week 6E Week 3

1.25 min A Week 6B Week 20C Week 3D Week 10E Week 6

1.5 min A Week 6B* *Week 8, no scarC* *Week 8, no scarD Week 6E Week 6

1.75 min A Week 6B* *Week 3, no scarC* *Week 4, no scarD Week 6E* *Week 3, no scar

*Euthanized at indicated time point.

Figure 4. Iris ischemia. Right eyes of mice were exposed to SM vapor for 1–1.75 min as previously described. Ischemia of blood vessels in the iris was scored by the clock hour extent in which ischemia was observed. The apparent improvement in the 1.25-min dose group at later time points is primarily due to euthanization of animals due to injury progression. The data are expressed as the average score of the group over time +/− SD (n = 5).

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.



MMP9, and plasminogen activator inhibitor-1 [PAI-1]. Of these, seven markers were observed to be significantly up-regulated in eyes exposed to SM vapor for 1.25 min: IL-1α, IL-1β, IL-6, KC, MCP-1, MMP9, and sVCAM-1 (Figure 7). These were up-regulated at the earliest time points and decreased to baseline levels within 1–2 weeks after exposure. Although some showed a small trend of increased expression at later time points, only KC was significantly up-regulated after the recovery phase.

Discussion

Our findings of SM-induced ocular injury in mice are consistent with observations of SM-induced ocular injury in humans and rabbit models, although delayed SM-induced ocular injury appears on the order of weeks to months, and months to years, in rabbits and humans, respectively (4,7). The more rapid progression of injury in the mouse model was expected because injuries in mice

generally progress more rapidly than in larger mammals and humans. A related feature of the mouse model is the phenomenon of early versus late onset of delayed pathol-ogy. In humans, some victims have been reported to develop delayed pathology within months after exposure whereas in other victims symptoms appear much later, as long as 20 or more years after exposure (4,5). In the rabbit model used by Kadar et al., only half of exposed corneas develop delayed pathology (7,9). We observed a similar phenomenon in that only half of the animals with moderate injury (the 1.0- and 1.25-min exposure groups) showed delayed pathology by 6-8 weeks post-exposure (see Table 3), but by 12 weeks post-exposure all animals in our model had developed delayed injury. It is possible that the onset of delayed pathology could be observed in all animals in this rabbit model, but the onset would likely take considerably more time given that the lifespan of rabbits is much longer than that of mice (1–2 year for mice and 8–12 years for rabbits). Our findings and those

Figure 6. SM-induced cornea histopathology score. Right eyes of mice were exposed to SM vapor for 1.25 min as previously described. Eyes were harvested from 1 to 28 days post-exposure. Eyes were fixed and sectioned for standard H&E staining for histopathology analysis. Parameters were scored 0–4 using the scoring system described in the text. The data are expressed as the average score over time +/− SD (n = 3). *p < 0.05, **p < 0.01.

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.



with the rabbit model used by Kadar et al. are slightly different than those in the rabbit model used by McNutt et al. and Milhorn et al. in which roughly 90% of rabbits develop delayed pathology (6,8). However, the two rabbit models use different exposure methodology and achieve

different severities of injury. Overall, our mouse model most closely mimics the rabbit model used by Kadar et al. and moderate injury in humans.

We observed that a 30-s exposure to SM vapor pro-duced a mild transient ocular injury showing no signifi-cant loss of epithelium post exposure, minimal to no iris changes and/or lens swelling, and only minor vascular changes (e.g. engorgement of limbal blood vessels). Moderate injury was characterized by an initial loss of corneal epithelium that quickly regenerates, minimal to mild transient lens swelling that may return later, iris changes (minimal pupil dislodgement, atrophy), and moderate neovascular changes to include initial limbal engorgement that regresses but is then followed by the

Figure 7. SM-induced inflammatory markers. Right eyes of mice were exposed to SM vapor for 1.25 min as previously described. Eyes were harvested from 2 to 24 days post-exposure. Whole eyes were homogenized in Epilife medium with 0.1% Triton X-100 and protease inhibitor cocktail. Lysates were analyzed by multiplex bead-based antibody assay (Milliplex, Millipore, Billerica, MA, USA). The data are expressed as pg/ml over time for the average for IL-1a, IL-1b, MCP-1, and KC; n = 3 for IL-6, and sVCAM-1 +/− SD (n = 9). Data for MMP9 are expressed as fluorescence intensity over time for the average +/− SD (n = 9). *p < 0.05, **p < 0.01, ***p < 0.001.

Table 3. Incidence of delayed pathology at 6–8 weeks post-exposure

DoseMice with delayed

pathologyMice without delayed

pathology1.0 min 2 31.25 min 3 21.5 min 5 01.75 min 5 0

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.



return of neovascularization marked by denser vessels moving into the central corneal area. Late term changes include the formation of a corneal infiltrate near the apex of the neovascular vessels that regresses over time, often leaving significant corneal scarring, or occasionally will worsen to the point of requiring euthanization 4 or more months after exposure. Severe injury displayed a loss and healing of the corneal epithelium similar to moderate injury, but all other symptoms were more severe. There was a shorter recovery phase between the healing of the epithelium and the onset of delayed symptoms. There was mild to moderate lens swelling that regressed but then progressed severely in 3–12 weeks warranting euth-anization. This injury was also characterized by more pronounced iris changes (pupil dislodgement, more and earlier onset of atrophy) and neovascular changes. These animals required euthanization at early time points often before the formation of a corneal scar. It was observed that the 1.25-min exposure time was the highest dose that produced moderate injury with examples of early and late onset of delayed injury, and this dose was selected for histopathology and inflammatory marker analyses to further characterize this model. This was the high-est dose that showed only minimal lens swelling or iris changes. These features of lens swelling and iris changes are characteristic of severe injuries that were prominent, at least variably, in the 1.5 min and greater exposure dose groups. Early discrete infiltrative scarring of the nasal or nasal-central cornea was noted at 6 weeks for the major-ity of mice. This infiltrative scarring inevitably led to formation of a large, discrete infiltrative keratitis fed by neo-vascular vessels. The infiltrate had associated edema and increased stromal deformity over time but after a period of several months the edema subsided and neo-vascularization regressed resulting in a large scarred and deformed area. The degree of iris atrophy and transient lens swelling seen did not seem to correlate with the degree or timing of the corneal infiltrates and scarring.

We elected to analyze whole eye lysates for inflamma-tory markers. Since the analysis of corneal button lysates would have required the pooling of several corneas, the analysis whole eye lysates was chosen in an attempt to not confound the results by the mixing of corneas from the two different types of responding animals, early ver-sus late onset of delayed pathology, which could not be discriminated at the time points for the molecular analy-ses. This method provided data on overall eye inflam-mation and allowed the measurement of analytes from a single eye. A disadvantage of this method is that it does not provide information on cornea specific markers that could be important for understanding cornea pathology. In spite of the differences between the mouse and rab-bit models and the difference in methods, the profile of inflammatory cytokines and histopathological findings of our mouse model shares a high degree of similarity with recent findings of the rabbit model used by McNutt et al (26). Both models show expression of IL-1β, TNFα, IL-6, IL-8, and MMP9 and exhibited cornea epithelial

necrosis, stromal inflammation, cornea epithelial atten-uation, and stroma deformity in SM injured eyes.

The development of this mouse model provides not only a foundation for future in-depth studies, but also a model system ready for immediate use in the testing of therapeutics and treatments for this injury. This model will enable the analysis of SM-induced molecular and cellular events that lead to clinical ocular injury and will accelerate therapeutic development. Future work should further characterize histopathological findings of injury progression over longer time points and inves-tigate molecular or cellular markers implicated in cornea pathology.

Conclusions

We determined that a moderate ocular injury versus severe injury would be most useful for the study of thera-peutics because a moderate injury is more likely to be effectively treatable whereas a severe injury may be only treatable by cornea transplant. Our clinical and histopa-thology data show that acute SM-induced ocular injury in mice is characterized by significant corneal epithelium loss. Corneal edema, limbal engorgement and ocular inflammation, consistent with keratitis and chemical burns, were also observed. During the reepithelialization of the cornea, early stages of cornea neovascularization were seen and progressed over time. This was followed by a brief recovery phase. A delayed injury phase then ensued in the following weeks to months and was char-acterized by keratitis, stromal edema, infiltrates, neovas-cularization and eventual corneal scarring. This process is suggestive of neurotrophic keratitis which is often associated with chemical burns.

Declaration of interest

The authors report no conflicts of interest.

References 1. Deaton MA, Bowman PD, Jones GP, Powanda MC. Stress protein

synthesis in human keratinocytes treated with sodium arsenite, phenyldichloroarsine, and nitrogen mustard. Fundam Appl Toxicol 1990;14:471–476.

2. Dillman JF, McGary KL, Schlager JJ. Sulfur mustard induces the formation of keratin aggregates in human epidermal keratinocytes. Toxicol Appl Pharmacol 2003;193:228–236.

3. Langenberg JP, van der Schans GP, Spruit HE, Kuijpers WC, Mars-Groenendijk RH, van Dijk-Knijnenburg HC et al. Toxicokinetics of sulfur mustard and its DNA-adducts in the hairless guinea pig. Drug Chem Toxicol 1998;21 Suppl 1:131–147.

4. Papirmeister B, Feister A, Robinson S, Ford R. Medical defense against mustard gas: toxic mechanisms and pharmacological implications. Boca Raton: CRC Press, 1991.

5. Sidell F, Urbanetti J, Smith W, Hurst C. Chapter 7 Vesicants. In: Sidell F, Takafuji E, Franz D, eds. Medical Aspects of Chemical and Biological Warfare. Washington, D.C.: Office of the Surgeon General, 1997, 197–228.

6. Milhorn D, Hamilton T, Nelson M, McNutt P. Progression of ocular sulfur mustard injury: development of a model system. Ann N Y Acad Sci 2010;1194:72–80.

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.



7. Kadar T, Turetz J, Fishbine E, Sahar R, Chapman S, Amir A. Characterization of acute and delayed ocular lesions induced by sulfur mustard in rabbits. Curr Eye Res 2001;22:42–53.

8. McNutt P, Hamilton T, Nelson M, Adkins A, Swartz A, Lawrence R et al. Pathogenesis of acute and delayed corneal lesions after ocular exposure to sulfur mustard vapor. Cornea 2012;31:280–290.

9. Kadar T, Dachir S, Cohen L, Sahar R, Fishbine E, Cohen M et al. Ocular injuries following sulfur mustard exposure–pathological mechanism and potential therapy. Toxicology 2009;263:59–69.

10. Fox M, Scott D. The genetic toxicology of nitrogen and sulphur mustard. Mutat Res 1980;75:131–168.

11. Lawley PD, Brookes P. Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids. Biochem J 1968;109:433–447.

12. Papirmeister B, Westling AW, Schroer J. Mustard: the relevance of DNA damage to the development of the skin lesion. Edgewood Arsenal: U.S. Army Medical Research Laboratory, 1969.

13. Dillman JF, Phillips CS, Dorsch LM, Croxton MD, Hege AI, Sylvester AJ et al. Genomic analysis of rodent pulmonary tissue following bis-(2-chloroethyl) sulfide exposure. Chem Res Toxicol 2005;18:28–34.

14. Dillman JF, Hege AI, Phillips CS, Orzolek LD, Sylvester AJ, Bossone C et al. Microarray analysis of mouse ear tissue exposed to bis-(2-chloroethyl) sulfide: gene expression profiles correlate with treatment efficacy and an established clinical endpoint. J Pharmacol Exp Ther 2006;317:76–87.

15. Simbulan-Rosenthal CM, Ray R, Benton B, Soeda E, Daher A, Anderson D et al. Calmodulin mediates sulfur mustard toxicity in human keratinocytes. Toxicology 2006;227:21–35.

16. Rosenthal DS, Velena A, Chou FP, Schlegel R, Ray R, Benton B et al. Expression of dominant-negative Fas-associated death domain blocks human keratinocyte apoptosis and vesication induced by sulfur mustard. J Biol Chem 2003;278:8531–8540.

17. Ray R, Simbulan-Rosenthal CM, Keyser BM, Benton B, Anderson D, Holmes W et al. Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop. Toxicology 2010;271:94–99.

18. Minsavage GD, Dillman JF. Bifunctional alkylating agent-induced p53 and nonclassical nuclear factor kappaB responses and cell death are altered by caffeic acid phenethyl ester: a potential role for antioxidant/electrophilic response-element signaling. J Pharmacol Exp Ther 2007;321:202–212.

19. Rebholz B, Kehe K, Ruzicka T, Rupec RA. Role of NF-kappaB/RelA and MAPK pathways in keratinocytes in response to sulfur mustard. J Invest Dermatol 2008;128:1626–1632.

20. Ruff AL, Dillman JF. Sulfur mustard induced cytokine production and cell death: investigating the potential roles of the p38, p53, and NF-kappaB signaling pathways with RNA interference. J Biochem Mol Toxicol 2010;24:155–164.

21. Banerjee K, Biswas PS, Kim B, Lee S, Rouse BT. CXCR2-/- mice show enhanced susceptibility to herpetic stromal keratitis: a role for IL-6-induced neovascularization. J Immunol 2004;172:1237–1245.

22. Biswas PS, Banerjee K, Kinchington PR, Rouse BT. Involvement of IL-6 in the paracrine production of VEGF in ocular HSV-1 infection. Exp Eye Res 2006;82:46–54.

23. Fini ME, Stramer BM. How the cornea heals: cornea-specific repair mechanisms affecting surgical outcomes. Cornea 2005;24:S2–S11.

24. Stramer BM, Zieske JD, Jung JC, Austin JS, Fini ME. Molecular mechanisms controlling the fibrotic repair phenotype in cornea: implications for surgical outcomes. Invest Ophthalmol Vis Sci 2003;44:4237–4246.

25. Torres PF, Kijlstra A. The role of cytokines in corneal immunopathology. Ocul Immunol Inflamm 2001;9:9–24.

26. McNutt P, Lyman M, Swartz A, Tuznik K, Kniffin D, Whitten K et al. Architectural and biochemical expressions of mustard gas keratopathy: preclinical indicators and pathogenic mechanisms. PLoS ONE 2012;7:e42837.

Cut

aneo

us a

nd O

cula

r T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Que

ensl

and

on 1

0/08

/13

For

pers

onal

use

onl

y.

development of a mouse model for sulfur mustard-induced ocular injury and long-term clinical...

Documents