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. ^{Reference Marlow, Mershon and Mitcheltree51} 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. ^{Reference Smith, Casillas and Graham18} The hairless guinea pig skin is considered morphologically more like human skin, ^{Reference Snider, Matthews and Braue31,Reference Smith, Graham and Moeller52} which has prompted greater use of these animals to understand the mechanism of action of SM and for the development of countermeasures. ^{Reference Barillo, Croutch and Reid53}

Table 1. Effects of sulfur mustard on guinea pig skin

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. ^{Reference Mishra, Rir-sima-ah and March54} 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. ^{Reference Laskin, Black and Jan2,Reference Pohanka, Stetina and Svobodova55} This is followed by the appearance of anti-inflammatory/wound repair macrophages. ^{Reference Biyashev, Onay and Dala56} 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. ^{Reference Dachir, Cohen and Sahar57}

Of interest are studies by Graham et al. ^{Reference Graham, Bryant and Brave47} 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. ^{Reference Mouret, Wartell and Batal38,Reference Joseph, Composto and Perez48} 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. ^{Reference Yourick, Dawson and Mitcheltree58–Reference Yourick, Dawson and Benton60}

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. ^{Reference Mershon, Mitcheltree and Petrali15,Reference Chang, Soriano and Hahn61,Reference Petrali, Oglesby and Hamilton62} More detailed information has been reported on the effects of SM on hair follicles and sebaceous glands in the mouse model. ^{Reference Joseph, Composto and Heck23,Reference Joseph, Gerecke and Heck63} 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. ^{Reference Joseph, Heck and Cervelli64} 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.

Table 2. Effects of sulfur mustard on rat skin

Table 3. Effects of sulfur mustard on mouse skin

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

Table 5. Effects of sulfur mustard on rabbit skin

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. ^{Reference Monteiro-Riviere, Inman and Babin36,Reference Casillas, Mitcheltree and Stemler65–Reference Patrick, Burkhalter and Maibach67} 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. ^{Reference Rajnoch, Ferguson and Metcalfe68} After a latency period, edema, measured by changes in ear weight, epidermal necrosis, and epidermal-dermal separation are assessed. ^{Reference Casillas, Mitcheltree and Stemler65,Reference Casillas, Kiser and Truxall69} Transmission electron microscopy and immunohistochemistry have been used to identify biomarkers of injury, as well as mechanisms of subepidermal blister formation. ^{Reference Monteiro-Riviere, Inman and Babin36,Reference Sabourin, Petrali and Casillas70}

In rabbits, dorsal and ventral skin and ear skin have been used to investigate SM injury and the formation of microblisters. ^{Reference Zlotogorski, Goldenhersh and Shafran71–Reference Sun, Sun and Zheng75} In each exposure scenario, SM damage has been assessed visually by monitoring erythema, wound healing, and histopathology. ^{Reference Vogt, Dannenberg and Schofield19,Reference Liu, Wannemacher and Snider73,Reference Sun, Sun and Zheng75,Reference Schoene, Bruckert and Schreiber76} In the rabbit models, depending on the dose, SM damages the superficial microvasculature as measured by Evans blue dye extravasation and leakage of erythrocytes. ^{Reference Vogt, Dannenberg and Schofield19,Reference Dachir, Cohen and Fishbeine77} 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. ^{Reference Nabai and Ghahary78} 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. ^{Reference Schoene, Bruckert and Schreiber76}

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. ^{Reference Summerfield, Meurens and Ricklin79–Reference Rittie81} Pig skin hair is coarser than human hair but has a similar distribution. ^{Reference Stricker-Krongrad, Shoemake and Bouchard41,Reference Summerfield, Meurens and Ricklin79,Reference Khiao In, Richardson and Loewa82} Although humans have eccrine glands distributed throughout their skin, swine eccrine glands are primarily found in the snout, lips, and carpal organ. ^{Reference Seaton, Hocking and Gibran80} 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. ^{Reference Takeo, Lee and Ito83} However, as with other animal models, SM is unable to form true blisters, a characteristic sign of toxicity in humans following vesicant exposure. ^{Reference Kehe and Balszuweit6,Reference Graham and Schoneboom7,Reference Kehe, Thiermann and Balszuweit84}

Table 6. Effects of sulfur mustard on pig skin

Table 7. Effects of sulfur mustard on pig skin

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. ^{Reference Pramudita, Shimizu and Tanabe85} 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. ^{Reference Graham, Schomacker and Glatter49,Reference Hall, Lydon and Dalton50} Histopathological changes include skin structure, epithelial and basement membrane integrity, and expression of markers of proliferation and differentiation of keratinocytes during wound healing. ^{Reference Laskin, Wahler and Croutch40,Reference Sabourin, Danne and Buxton86–Reference Chilcott, Dalton and Ashley88} 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. ^{Reference Plahovinsak, Buccullato and Reid89}

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. ^{Reference Barillo, Croutch and Reid53} Silver ions in the product also serve as an antimicrobial, reducing infections at the wound site. ^{Reference Barillo, Pozza and Margaret-Brandt91}

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. ^{Reference Graham, Stevenson and Mitcheltree33,Reference Barillo, Croutch and Barillo92} 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

Table 8. Skin histopathology scoring for evaluating sulfur mustard countermeasures using Göttingen minipigs^a

^a Scoring system^{Reference Graham, Stevenson and Mitcheltree33,Reference Barillo, Croutch and Barillo92}

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.