
==== Front
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Disaster Med Public Health Prep
Disaster Med Public Health Prep
Disaster medicine and public health preparedness
1935-7893
1938-744X

37849329
10.1017/dmp.2023.177
nihpa2005143
Article
Skin Models Used to Define Mechanisms of Action of Sulfur Mustard
Laskin Jeffrey D. PhD 1
Ozkuyumcu Kevin PharmD 2
Zhou Peihong MD 2
Croutch Claire R. PhD 3
Heck Diane E. PhD 2
http://orcid.org/0000-0003-1832-7311
Laskin Debra L. PhD 2
Joseph Laurie B. PhD 2
1 Department of Environmental and Occupational Health and Justice, Rutgers University School of Public Health, Piscataway, NJ, USA;
2 Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Piscataway, NJ, USA
3 MRIGlobal, Kansas City, MO, USA
Author contributions. Jeffrey D. Laskin: conceptualization, funding acquisition, literature search, original manuscript draft writing, review & editing; Kevin Ozkuyumcu: literature search & manuscript draft writing; Peihong Zhou: manuscript review & editing; Claire R. Croutch: manuscript review & editing; Diane E. Heck: manuscript review & editing; Debra L. Laskin: funding acquisition, manuscript review & editing; Laurie B. Joseph: literature search, original manuscript draft writing, review & editing.

Corresponding author: Jeffrey D. Laskin PhD; jlaskin@eohsi.rutgers.edu.
28 6 2024
18 10 2023
18 10 2023
24 9 2024
17 e551e551
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited.
Sulfur mustard (SM) is a threat to both civilian and military populations. Human skin is highly sensitive to SM, causing delayed erythema, edema, and inflammatory cell infiltration, followed by the appearance of large fluid-filled blisters. Skin wound repair is prolonged following blistering, which can result in impaired barrier function. Key to understanding the action of SM in the skin is the development of animal models that have a pathophysiology comparable to humans such that quantitative assessments of therapeutic drugs efficacy can be assessed. Two animal models, hairless guinea pigs and swine, are preferred to evaluate dermal products because their skin is morphologically similar to human skin. In these animal models, SM induces degradation of epidermal and dermal tissues but does not induce overt blistering, only microblistering. Mechanisms of wound healing are distinct in these animal models. Whereas a guinea pig heals by contraction, swine skin, like humans, heals by re-epithelialization. Mice, rats, and rabbits are also used for SM mechanistic studies. However, healing is also mediated by contraction; moreover, only microblistering is observed. Improvements in animal models are essential for the development of therapeutics to mitigate toxicity resulting from cutaneous exposure to SM.

animal models
dermatotoxicity
sulfur mustard
wound healing
==== Body
pmcSulfur mustard (SM, bis 2-chloroethyl sulfide) is a potent skin vesicant synthesized for chemical warfare. As a bifunctional alkylating agent, SM initiates its action by modifying and disrupting cellular macromolecules, including DNA and proteins.1–5 Acute responses of skin to SM are typically characterized by delayed onset erythema and intense itching, followed by the formation of small fluid-filled vesicles; with time, these vesicles coalesce to form pendulous blisters.1,6,7 A necrotic layer and ulceration can form on the affected skin surface following rupture of the blisters. Responses of human skin to SM are multifactorial and depend on the dose and time following exposure, as well as environmental conditions such as temperature and humidity.7,8 Location of exposure sites on the body, variations in skin properties, and underlying disease states, along with age and sex, are all determinants of skin responses to SM.8

To understand the mechanism of action of SM and develop medical countermeasure, various animal models have been utilized, including mice, rats, guinea pigs, rabbits, and pigs.9–12 Unfortunately, there are no simple or common animal models for SM injury that produce true blisters like humans. In this context, in describing early reporting on the use of human subjects for mustard research in 1919, Sollman explained that “experiments on animals was [sic] abandoned after a few trials, since their skin does not react in the same manner as human skin, and the effects that do occur are not easily graded.”8 Blistering is not commonly observed in animals.13,14 To produce true blistering, either unconventional species must be used, or multistep procedures must be undertaken in common animal models.14 For example, it has been reported that blisters can be produced on the skin of frogs, birds, and the inner ears of rabbits,15 on the skin of isolated perfused pig flaps,15,16 and on guinea pig skin that has been thermally burned and allowed to re-epithelialize.16,17 Studies performed with SM on birds and frogs are limiting as their skin is not similar to human skin. For this reason, SM research has relied on the surrogate marker of microblistering or subepidermal blister formation at the dermal-epidermal junction, which occurs in rodents, rabbits, and pigs.18–20 SM is known to damage not only epidermal structures, including the basement membrane, but also stromal and vascular components of the skin tissue.21–23

Translating SM data from animals to humans has been challenging not only because there is little or no blistering, but also due to additional factors such as distinct structural differences in the tissue, unique aspects of the immune system, and mechanisms of wound healing. For example, in mice, the skin and epidermis are thinner when compared to humans, there are fewer epidermal cell layers, a lack of epidermal ridges and eccrine sweat glands, and limited adherence to underlying tissues.24 In humans and pigs, wounds close by formation of granulation tissue followed by re-epithelialization24; in contrast, wound closure in rodents and rabbits is primarily by contraction, in part due to the presence of the panniculus carnosus.25 At later stages, tissue remodeling during wound healing occurs via fibroblast migration and myofibroblast activity.26 It should be noted that contraction is usually defined for incisional wounds27; the role of contraction in thermal and SM injury is not clear since the extent of tissue damage may not allow wound closure by the panniculus carnosus.

In rodent models, both haired and hairless strains have been used; hairless animals are advantageous largely due to the ease of visualizing a cutaneous response.28 Hair removal and associated inflammation are avoided with these animals.29 However, it should be noted that the skin of haired and hairless animal strains can be morphologically different. For example, the epidermis of mice of the most commonly used hairless strain, SKH1, is thicker than haired strains.9 Haired and hairless strains are also genetically and immunologically distinct, complicating efforts to compare results from different laboratories.30 Little information is available on differences in wound healing in response to chemical and thermal injury in haired and hairless mouse strains.

In most animal models, different phases of SM injury can be defined, including latency, erythema/inflammation, microblistering, ulceration/eschar formation, and wound healing. The extent of injury depends on several factors, including the model, location and area of skin exposed to SM, as well as SM dose and methods of administration and environmental conditions when applying SM. Targeting one or more phases of injury is essential in the development of effective countermeasures to mitigate SM toxicity. Both clinical signs and morphological/biochemical parameters have been used to characterize the action of SM in animal models. Clinical signs are evident by visual inspection; at early times, this includes erythema, edema and transepidermal water loss (TEWL), and, at later times, extent of injury and whether injury is superficial, intermediate in depth, or deep dermal injury.29 The integrity of the dermal-epidermal junction, measured by dermal torque, has been demonstrated in SM-treated guinea pigs.31 Laser Doppler imaging has also been used to assess cutaneous blood flow and ballistometry to evaluate mechanical properties of the skin, including rigidity and elasticity in pig models.32,33

Techniques in histology, electron microscopy, and immunohistochemistry have been used to analyze structural alterations in skin exposed to SM. These studies have largely focused on the epidermis, basement membrane, and accessary structures, including hair follicles and sebaceous glands. Early effects of SM in basal cells of the epidermis, as reported in guinea pig skin include nuclear condensation and mitochondrial swelling, disorganization of desmosomes and hemidesmosomes, and widening of intracellular spaces in the basal cell layer.19 At later times, nuclear pyknosis, cell fragmentation, and necrosis extending into suprabasal cells are evident.34 Markers of DNA damage and apoptosis and necrosis also appear in epidermal cells.35 Mediators of inflammation, including prostaglandins and cytokines, are also expressed after SM-induced injury.11,22 Microvesicles appear in the lamina lucida of the basement membrane as a consequence of degeneration of the basal layer.36,37 Proteolysis of basement membrane components, including laminins, collagens, and other anchoring proteins by matrix metalloproteinases, contributes to the disruption of the basal cell layer, microvesication, and ulceration.38,39 At later times, in minipig skin, aberrant epidermal proliferation and differentiation are associated with re-epithelialization including hyperplasia, hyperkeratosis, and parakeratosis. This is thought to contribute to prolonged wound healing.40,41

The dermis and hypodermis are also targets for SM. This is important as the integrity of these tissues is critical for wound healing.22,23,42,43 Leukocyte infiltration, a marker of inflammation, has been observed in the dermis post-SM exposure in all animals studied.19,44–46 In mouse and guinea pig skin, mast cell degranulation is also evident, along with alterations in collagen deposition.38,47,48 In pig skin, SM also disrupts the dermal vasculature and subsequent blood flow, and responses can affect tissue oxygenation, possibly leading to reperfusion injury.49,50 These pathologic responses can impair wound healing, lead to infection, and initiate scarring.

Guinea Pig Skin Model of SM Toxicity

Both haired and hairless guinea pigs have been used to assess SM toxicity with generally similar results (Table 1). Hairless guinea pigs have been reported to be more sensitive to SM in terms of the extent of dermal injury.51 These animals are also more sensitive to SM-induced epidermal necrosis compared to other animal models, including the weanling pig, mouse ear, and hairless mice.18 The hairless guinea pig skin is considered morphologically more like human skin,31,52 which has prompted greater use of these animals to understand the mechanism of action of SM and for the development of countermeasures.53

As indicated above, a characteristic early response of guinea pig skin to SM is a marked inflammatory response, notably, infiltration of neutrophils and macrophages into the tissue.54 Mustards cause the release of inflammatory mediators, including reactive oxygen and reactive nitrogen species, and cytokines such as TNFα and IL-1α, which activate macrophages contributing to tissue injury.2,55 This is followed by the appearance of anti-inflammatory/wound repair macrophages.56 That macrophages can contribute to wound repair is evidenced by findings that intradermal injection of activated human macrophages into SM-treated guinea pig skin can significantly improve clinical signs of tissue damage.57

Of interest are studies by Graham et al.47 showing that SM reduces mast cell numbers in hairless guinea pig skin, suggesting that degranulation may be an early marker of toxicity. These investigators hypothesized that histamine and other mediators released by mast cells may play a role in SM-induced injury. These data are in accord with studies by ours and other laboratories demonstrating mast cell degranulation and reduced number of mast cells in SM-exposed hairless mouse skin.38,48 The use of antihistamine promethazine, in combination with the PARP inhibitor niacinamide, and the non-steroidal anti-inflammatory agent indomethacin in guinea pig skin, decreases mast cell degranulation.58–60

Rat, Mouse, and Rabbit Models of SM Toxicity

In these models, exposure to SM is either by direct application of liquid to the skin or as a vapor (Tables 2–5). Vapor exposures are typically preferred since vapor is the more likely route of exposure during a mass causality scenario. Depending on the dose and environmental conditions, generally similar characteristic responses are observed following treatment of the dorsal skin of rats, mice, and rabbits with SM. Initially, there is a latency period, which is followed by a cutaneous inflammatory response characterized by erythema, edema, and leukocyte infiltration. Subsequently, there is microblister formation, tissue granulation, epidermal necrosis, and, finally, wound repair and tissue remodeling.15,61,62 More detailed information has been reported on the effects of SM on hair follicles and sebaceous glands in the mouse model.23,63 In hair follicles, SM induces epithelial cell karyolysis within the hair root sheath, infundibulum, and isthmus and reduces the numbers of sebocytes in sebaceous glands.64 Significant DNA damage and apoptosis are evident around pilosebaceous units with increased numbers of inflammatory cells surrounding utriculi. These findings may explain, at least in part, depletion of hair follicles in human skin following exposure to SM.

An important method that can partially overcome wound contraction and the need for fur removal is the use of the mouse ear vesicant model (see Table 4). This method is largely based on early studies showing that biological and biochemical processes associated with inflammation can easily be measured following exposure to cutaneous irritants or allergens.36,65–67 In this model, SM is applied to the inner surface of the mouse ear, which is largely free of hair. Ear cartilage appears to prevent wound contraction.68 After a latency period, edema, measured by changes in ear weight, epidermal necrosis, and epidermal-dermal separation are assessed.65,69 Transmission electron microscopy and immunohistochemistry have been used to identify biomarkers of injury, as well as mechanisms of subepidermal blister formation.36,70

In rabbits, dorsal and ventral skin and ear skin have been used to investigate SM injury and the formation of microblisters.71–75 In each exposure scenario, SM damage has been assessed visually by monitoring erythema, wound healing, and histopathology.19,73,75,76 In the rabbit models, depending on the dose, SM damages the superficial microvasculature as measured by Evans blue dye extravasation and leakage of erythrocytes.19,77 SM also damages fibroblasts, possibly disrupting the extracellular matrix. In contrast to the dorsal and ventral skin, rabbit ears have no panniculus carnosus; thus, wound contraction does not contribute to the healing process.78 This model is thought to better reflect wound healing in humans. However, in a continuous flow vapor exposure model, rabbit ears have been reported to be significantly less sensitive than human skin to SM injury.76

Pig Models of SM Toxicity

Pig skin is the most anatomically and physiologically similar to human skin, compared to rodents and rabbits, making it a preferable model for translational research (Tables 6 and 7). From a regulatory standpoint, considerable background data are available on pig skin related to the development of dermatological products, making this model ideal for SM countermeasure research. Pig skin is tightly attached to the subcutaneous connective tissue, contains a relatively thick epidermis, distinct rete ridges and, like human skin, dense elastic fibers in the dermis.79–81 Pig skin hair is coarser than human hair but has a similar distribution.41,79,82 Although humans have eccrine glands distributed throughout their skin, swine eccrine glands are primarily found in the snout, lips, and carpal organ.80 In the skin of both pigs and humans, re-epithelialization during wound healing is associated with basal cell proliferation and differentiation into enucleated granular cells that migrate outward toward the surface of the skin.83 However, as with other animal models, SM is unable to form true blisters, a characteristic sign of toxicity in humans following vesicant exposure.6,7,84

Both dorsal and ventral skin models have been used to assess SM toxicity in pig skin (see Tables 6 and 7). In general, the ventral skin of pigs is thinner and more responsive to SM than dorsal skin.85 The choice of dorsal versus ventral pig skin models is dependent on the type of exposure (eg, liquid vs vapor cap) and the type of injury being investigated (eg, superficial vs intermediate or deep dermal). Both models can be used to assess pharmaceutical preparations. However, dorsal skin is preferable with the use of wound dressings that must be maintained for prolonged periods of time (see further below). Both clinical and histopathological endpoints are used to assess tissue damage. Clinical changes include blood flow, elasticity, skin color, thickness, and spectral properties.49,50 Histopathological changes include skin structure, epithelial and basement membrane integrity, and expression of markers of proliferation and differentiation of keratinocytes during wound healing.40,86–88 Following these endpoints over time will provide information on the wound healing process and the effectiveness of potential countermeasures. Decontaminants, protectants, anti-inflammatory agents, and wound dressing have been evaluated for their ability to mitigate tissue damage induced by SM, often with varying degrees of success.89

Based on pig skin models that have been developed to assess medical countermeasures against SM-induced skin injury, one product, Silverlon® Wound Contact, Burn Contact Dressings, has been approved by the FDA.90 Manufactured as a non-adherent knitted nylon fiber wound dressing coated with metallic silver, Silverlon® is approved for use with decontaminated, unroofed first and second degree burns induced by SM. Silverlon® also acts as an oxygen-permeable sterile barrier, which promotes wound healing.53 Silver ions in the product also serve as an antimicrobial, reducing infections at the wound site.91

Support for Silverlon® in the FDA approval process was based on a pathophysiological scale in the Göttingen minipig vapor cap model (Table 8). Individual endpoints indicate the extent and type of repair and include the appearance of epithelial cells, basement membrane damage, re-epithelialization of the wound, whether abnormal hair follicles are present, extent of dermal inflammation and the presence of rete ridges, vascular proliferation, and hemorrhage.33,92 In the case of Silverlon®, approvals were based on re-epithelialization of the skin and improved appearance of the basement membrane, as well as a reduction in dermal inflammation. Silverlon® has also been FDA approved for radiation dermatitis and cutaneous radiation injury through dry desquamation.93

Summary

Animal models are essential not only for understanding the mechanism of action of SM, but also to develop effective therapeutics. Importantly, therapeutics may be effective at different stages of SM injury (eg, during the latency prior to a cutaneous response, during the inflammatory response, or during wound healing/tissue remodeling) and can be used alone or in combination. For example, Silverlon® is effective for wound healing following the appearance of first- and second-degree burns after exposure to SM. It remains to be determined whether treatments with anti-inflammatory agents prior to the development of SM burns will improve Silverlon®-induced wound healing. Thus far, research in the field is limited as SM is a blistering agent, and none of the animal models form overt blisters in response to this vesicant. Further studies are required to better understand differences between human and animal responses to SM so that more effective countermeasures can be developed that not only enhance wound healing, but also mitigate the blistering response.

Funding statement.

This work was supported by the National Institutes of Health Grants U54AR055073, R01ES004738, R01ES033698, and P30ES005022

Table 1. Effects of sulfur mustard on guinea pig skin

Animal	Model	Citations	Strain/exposure route	Measurements/treatments	PMID #	
Guinea pig	Dorsal skin	Vogt et al., 198419	Guinea pig/liquid	Histopathology, TEM	6233199	
Mershon et al., 199015; Braue et al., 199720, 199817; Snider et al, 199931	Hairless guinea pig/vapor cap	Draize test, histopathology, Nikosky’s signs	2258024
27333584
27332107
10594902	
Cowan et al., 199394
Cowan & Broomfield, 199395	Hairless guinea pig/vapor cap	Increased proteolytic activity, inflammation	8299005
8299000	
Yourick et al., 199159, 199296, 199360, 199558	Hairless guinea pig/vapor cap	Histopathology, erythema, NAD+/NAD+/niacinamide, promethazine, indomethacin	1838996
1440603
8266337
7782559	
Petrali et al., 199362, 199737; Kan et al., 200334	Hairless guinea pig/vapor cap	Histopathology, TEM, basement membrane, basal cell apoptosis	8462065
9144634
12696578	
Smith et al., 199552, 199718	Hairless guinea pig/vapor cap	Histopathology	7593821
9039976	
Kjellstrom et al., 199797	Haired guinea pig, continuous flow vapor	Comparison of standard dressing vs surgical excision vs surgical excision plus autografts	9140575	
Logan et al., 199998	Hairless guinea pig/vapor cap	PK/PD	10234473	
Langenberg et al., 199899	Hairless guinea pig/liquid	Toxicokinetics	10028407	
Sawyer et al., 1999100, 2000101, 2008102; Mi et al., 2003103	Hairless guinea pig/vapor cap	Draize test, pathology, apoptosis, p53/hypothermia, L-NAME, dimercaptosuccinic acid	10413186
10662607
14613718
18516227	
Wormser et al., 1997104, 200228; Brodsky et al., 2006105	Dunken Hartley and hairless guinea pigs/ liquid, vapor cap	Comparative study, toxicokinetics, histopathology/iodine	9049053
12242609
16252085	
Dachir et al., 201077, 201222, 201457	Hairless guinea pig/vapor cap	TEWL, PGE2, MMP-2/9, histopathology/macrophages	20384890
23082902
24641113	
Mishra et al., 2010106	Hairless guinea pig/vapor cap	Immune sensitization, proliferation, cytokine expression	19887117	
Benson et al., 2011a107, 2011b108; Weber et al., 201171	Hairless guinea pig/vapor cap	PK/PD, histopathology, erythema, edema, MMP-2/9, model development	21410818
21598172
21473735	
Barillo et al., 201753	Hairless guinea pig/vapor cap	Skin permeation studies/wound dressings	28846576	

Table 2. Effects of sulfur mustard on rat skin

Animal	Model	Citations	Strain/exposure route	Measurements/treatments	PMID #	
Rat	Dorsal skin	Vojvodić et al., 198574	Albino rats/liquid	Survival time, weight loss, pathology/sodium thiosulfate, vitamin E, heparin sulfate, dexamethasone, promethazine, atropine	4092884	
Black et al., 1992109	Wistar rats/vapor	SM metabolism, urine analysis/ thiodiglycol sulfoxide	1501468	
Hambrook et al., 1992110	Wistar rats/vapor cap	SM metabolism	1615709	
Kumar et al., 2002111	Wistar rats/liquid	LD50/ amifostine, DRDE-07	12269699	
Vijayaraghavan et al., 2005112	Wistar rats/liquid	LD50, histopathology, DNA fragmentation	15629193	
Kulkarni et al., 2006113	Wistar rats/liquid	DNA fragmentation, histopathology/ DRDE-07 analogs	16421877	
Karvaly et al., 2008114	Wistar rats/liquid	SM degradation/barrier creams and ointments	17429799	
Misik et al., 2013115	Wistar rats/liquid	Decontamination protection, LD50/Argos™, Dermogel™, FloraFree™	23078279	
Pohanka et al., 201355	Wistar rats/liquid	Antioxidant depletion in liver, kidney, muscle	22947058	
Yue et al., 2014116, 2015117	Sprague-Dawley rats/liquid	Metabolism, DNA adducts, histopathology, weight loss, bone marrow micronucleus assay	24467472
25650027	
Wang et al., 2015118	Sprague-Dawley rats/liquid	Metabolism, DNA adducts	25955432	
Steinritz et al., 2021119	Wistar rats/liquid	SM creatine kinase B and DNA adducts	33635393	

Table 3. Effects of sulfur mustard on mouse skin

Animal	Models	Citations	Strain/exposure route	Measurements/treatments	PMID #	
Mouse	Dorsal skin	Vijayaraghavan et al., 1991120	Swiss mice/liquid	Survival, body weight, lipid peroxidation/ flavonoids, vitamin E, sodium thiosulfate	1926154	
Smith et al., 199718	SKH1 hairless mice/liquid	Histopathology	9039976	
Rao et al., 199935	Swiss mice/liquid	Systemic DNA damage	10614687	
Blank et al., 2000121	SKH1 hairless mice /vapor cap	Myeloperoxidase, inflammatory mediators	11428626	
Ricketts et al., 2000122	SKH1 hairless mice /vapor cap	Inflammatory mediators	11428647	
Kumar et al., 2001123	Swiss mice/liquid	Oxidative damage/ Trolox, quercetin, GSH	11248218	
Anderson et al., 2002124	CD1 neonatal mice/vapor cap	Histopathology	20597816	
Kumar et al., 2002111
Kulkarni et al., 2006113
Vijayaraghaven et al., 2005112	Swiss mice/liquid	LD50, histopathology, DNA fragmentation/ amifostine, DRDE-07 analogs	12269699
16421877
15629193	
Sharma et al., 2010125	Swiss mice/liquid	Mortality, hematology, GSH/GSSG, DNA fragmentation/amifostine, NAC, melatonin, thiosulphate, DRDE-07	20466873	
Vallet et al., 2012126	SKH-1 hairless mice/vapor cap	Inflammatory mediators	21939433	
Lomash et al., 2013127	Swiss albino mice/ liquid	Histopathology, inflammatory-reparative biomarkers	22672652	
Clery-Barraud et al., 2013128	SKH-1 hairless mice/vapor cap	TEWL, evaporimeter, cutometer, skin color change	22741598	
Mouret et al., 201538
Sauvaigo et al., 2016129,
Batal et al., 20134, 2015130	SKH-1 hairless mice/liquid	TEWL, skin color change, histopathology, inflammatory mediators, DNA repair enzymes, DNA, GSH adducts	25275893
26551547
24141030
25562541	
Das et al., 2016131	C57BL6 mice/liquid	Lethality, wound area, body weight, hematology, bone marrow cellularity/ vitamin D	26940683	
Joseph et al., 201163, 201464, 201623, 201848	SKH-1 hairless mice/vapor cup	Wound healing, inflammatory markers/ anticholinergic prodrug	21672537
24662110
27371823
29127031	

Table 4. Effects of sulfur mustard in the mouse ear vesicant model

Animal	Model	Citations	Strain/exposure route	Measurements/treatments	PMID #	
Mouse	Mouse ear vesicant model	Casillas et al.,200069, Smith et al., 199718	CD1 mice/liquid	Edema, histopathology, inflammatory meditators/Olvanil, steroids, NSAIDs	11428628
9039976	
Monteiro-Riviere et al., 199936	CD1 mice/liquid	Dermal edema, basement membrane proteins	10513676	
Sabourin et al., 200070	CD1 mice/liquid	Inflammatory mediators	11083082	
Ricketts et al., 2000122	CD1 mice/liquid	Inflammatory mediators	11428647	
Powers et al., 200066	CD1 mice/liquid	Serine and cysteine proteases, elastase, metalloproteases	11428632	
Dachir et al., 200411	CD1 mice/liquid	Histopathology, inflammatory mediators/ steroids, NSAIDs	15052605	
Gerecke et al., 2009132	CD1 mice/liquid	Microarrays/MMP2, MMP9 inhibitors	18955075	
Chang et al., 2018133, 2020a39, 2020b61	CD1 mice/liquid	Inflammatory mediators, epidermal hyperplasia, microblisters, laminin γ2 proteolytic fragments/type IV collagenase inhibitor	29935281
32421930
32479919	

Table 5. Effects of sulfur mustard on rabbit skin

Animal	Model	Citations	Strain/exposure route	Measurements/treatments	PMID #	
Rabbit	Dorsal/ventral skin	Vogt et al., 198419	Rabbit/liquid	Histopathology, TEM/ hydrocortisone	6233199	
Vojvodić et al., 198574	Chinchilla rabbit/ liquid	Skin lesions pathology/ sodium thiosulfate, dexamethasone, promethazine	4092884	
Dannenberg et al.,198544
Harada et al., 1985134, 198745
Higuchi et al., 1988135
Tsuruta et al., 1997136
Tanaka et al., 199746	New Zealand white rabbits/liquid	In vivo-in vitro studies, histopathology, release of inflammatory mediators, proteases, chemoattractant	4050973
4050975
2433944
3049342
8796382
9187966	
Chauhan et al., 199643	New Zealand white rabbits /liquid	Histopathology, scanning electron microscopy, extracellular matrix	8956094	
Liu et al., 199973	Rabbits/liquid	Lesion size, erythema/ topical skin protectants	10594900	
Kumar et al., 2010137	New Zealand white rabbits/liquid	Weight change, erythema/ amino alylaminoethane thiols	20164158	
Zhang et al., 2014138
Lin et al., 2014139
Nie et al., 2014140	Domestic rabbits/ liquid	SM metabolism, DNA adducts, GSH adducts	24858262
24361979
24924210	
Sun et al., 201575	New Zealand rabbits/liquid	Histopathology/decontamination with potassium ketoxime	24641121	
Hind limb	Hansen et al., 1951141	Albino rabbits/ liquid	Appearance of lesions/ hypothermia	14923336	
Rabbit ear	Schoene et al., 198976	Albino rabbits/continuous flow vapor exposure cell	Erythema, dose, permeation	2596397	
Zlotogorski et al.,199771	Albino rabbits/ liquid	Draize, edema, erythema, histopathology	9184197	

Table 6. Effects of sulfur mustard on pig skin

Animal	Model	Citations	Strain/exposure route	Measurements/treatments	PMID #	
Pig/minipig	Dorsal skin models	Lindsay et al., 1995142	Yucatan miniature swine/vapor cap	Collagen, glycoprotein, histopathology	7598994	
Smith et al., 199621	Weanling pig/liquid	Histopathology	8902098	
Brown et al., 1997143	Yucatan minipig/vapor cap	Histopathology, transmission electron microscopy	9166101	
Smith et al., 1997a18, 1997b87	Yorkshire cross weanling pig/vapor cap	Basement membrane proteins, proliferation, apoptosis	9039976
9302645	
Logan et al., 2000144	Yorkshire cross weanling pig/vapor cap	Measurement of SM skin off gassing	11428637	
Reid et al., 200042	Yorkshire weanling pig/vapor cap	Clinical evaluation, histopathology	11428629	
Chilcott et al., 2000145, 200788	White pig/vapor cap	TEWL, chromameter, skin reflectance spectroscopy, histopathology/pretreat with barrier cream	10741590
17687688	
Sabourin et al., 200286	Yorkshire weanling pig/vapor cap	Proinflammatory markers	12481301	
Hall et al., 201750	White pig/liquid	Scanning laser Doppler, skin reflectance spectroscopy, thermography, histopathology/WoundStat™	28304107	
Dachir et al., 201710	White pig/vapor cap or liquid	Erythema, histology, cholinesterase inhibition/Dermostyx (IB1)	27417258	
Laskin et al., 202040	Gottingen minipig/vapor cap	Histopathology, keratinocyte proliferation, growth and differentiation markers	32445752	
Barillo et al., 201753, 202092	Gottingen minipig/vapor cap	Skin permeation studies/wound dressings, TEWL, histopathology/ methods of debridement	28846576
31504620	
Dachir et al., 2021146	White pig/vapor cap	Erythema, cholinesterase inhibition/ decontamination- Fuller’s Earth, oxime lotion	33508307	

Table 7. Effects of sulfur mustard on pig skin

Animal	Model	Citations	Strain/exposure route	Measurements/treatments	PMID #	
Pig	Ventral skin models	Graham et al. 200249	Yorkshire cross weanling pig/ vapor cap	Elasticity, scanning laser Doppler, TEWL, chromometer	12005121	
Graham et al., 2006147	Yorkshire weanling cross pig/liquid	Histopathology/laser debridement, hydrocolloid wound dressings	17111042	
Reid et al., 200042, 200732	Yorkshire weanling cross pig/liquid	Chromometer, TEWL, scanning laser Doppler, histopathology	11428629
17374066	
Rogers et al., 2008148	Yorkshire crossbred pig/liquid	Transcript analysis, porcine genome arrays	18988085	
Price et al., 2009149	Yorkshire crossbred pig/liquid	Transcriptional analysis, porcine genome arrays	19694609	
Graham et al., 200933	Yorkshire weanling crossbred pig/vapor cap	Clinical measurements (TEWL, chromometer, torsional ballistometry, ultrasonography), histopathology, basement membrane proteins /Amino-Plex®, Aquacel®	18762227	
Plahovinsak et al., 201689	Yorkshire or Yorkshire crossbred pig/ liquid	TEWL, Draize, chromometer, histopathology/clobetasol propionate, diclofenac sodium, capsaicin	26362124	

Table 8. Skin histopathology scoring for evaluating sulfur mustard countermeasures using Göttingen minipigsa

Marker	Scoring	
Re-epithelialization	0 - epithelium does not completely cover wound (wound is still open)
1 - wound is completely closed with epithelial cell monolayer
2 - wound is completely covered
3 - wound is completely covered by at least 2 layers of epithelial cells and includes at least the presence of some stratum corneum.
4 - wound is completely covered by greater that 2 layers of epithelial cells and includes normal corneum	
Abnormal epidermal cells	dyskeratosis, apoptosis, pyknosis, karyorrhexis
0 - present (over and above control background levels)
1 - absent (as compared to control background incidence)	
Basement membrane	0 - basement membrane is not completely intact
1 - basement membrane is intact with abnormal architecture
2 - basement membrane is intact with normal architecture	
Hair follicles	0 - absent/abnormal as compared to control background
1 - absent/normal as compared to control background	
Dermal inflammation	3 - no increase in leukocytes over that seen in normal untreated skin
2 - increased leukocytes noted in papillary dermis
1 - increased leukocytes in papillary dermis and upper half of reticular dermis
0 - diffuse increased leukocytes extending to all layers of the dermis	
Rete ridges	0 - absent, 1 – present	
Vascular proliferation	1 - absent, 2 – present	
Hemorrhage	1 - absent, 0 – present	
a Scoring system33,92

Conflict(s) of interest. The authors declare no conflicts of interest.
==== Refs
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