Larry W. Mitcheltree

Therapeutic approaches to dermatotoxicity by sulfur mustard I. Modulation of sulfur mustard‐induced cutaneous injury in the mouse ear vesicant model†**

The mouse ear edema model is recognized for its usefulness in studying skin responses and damage following exposure to chemical irritants, and for evaluating pharmacological agents against chemically induced skin injury. We recently modified the mouse ear edema model for use with sulfur mustard (HD) and used this model to study the protective effect of 33 topically applied compounds comprising five pharmaceutical strategies (anti‐inflammatories, protease inhibitors, scavengers/chelators, poly(ADP‐ribose) polymerase (PARP) inhibitors, calcium modulators/chelators) against HD‐induced dermatotoxicity. Pharmacological modulation of HD injury in mouse ears was established by a reduction in edema or histopathology (epidermal necrosis and epidermal‐dermal separation) at 24 h following topical liquid HD exposure. Ten of the 33 compounds administered as single topical pretreatments up to 2 h prior to HD challenge produced significant reductions in edema. Five of these ten also produced significant reductions in histological endpoints. Three candidates (olvanil, indomethacin, hydrocortisone) showing protection at 24 h were evaluated further for ‘extended protection’ at 48 and 72 h after HD challenge and showed significant modulation of edema at 48 h but not at 72 h. Olvanil also showed significant reductions in histology at 48 and 72 h. Olvanil and indomethacin were shown to reduce significantly the edema at 24 h post‐exposure when administered topically 10 min after HD challenge, with olvanil additionally protecting against epidermal necrosis. These results demonstrate prophylactic and treatment effects of pharmacological agents against HD‐induced skin injury in an in vivo model and support the continued use of the mouse ear vesicant model (MEVM) for evaluating medical countermeasures against HD. Published in 2000 by John Wiley & Sons, Ltd.

Niacinamide Pretreatment Reduces Microvesicle Formation in Hairless Guinea Pigs Cutaneously Exposed to Sulfur Mustard

It has been proposed that sulfur mustard (HD) may indirectly activate poly(ADP-ribose) polymerase (PADPRP) by alkylating cellular DNA (Papirmeister et al., 1985). Activation of PADPRP results in the depletion of cellular NAD+, which initiates a series of biochemical processes that have been proposed to culminate in blister formation. Preventing PADPRP activation and NAD+ depletion should inhibit blister formation. Niacinamide is both an inhibitor of PADPRP and a precursor for NAD+ synthesis. The present study was undertaken to determine whether niacinamide can protect against HD-induced microvesication in cutaneously exposed hairless guinea pigs. Each site was exposed to HD for 8 min by means of a vapor cup. Niacinamide (750 mg/kg, ip) given as a 30-min pretreatment inhibited microvesicle formation by 50% after HD application. However, niacinamide given 2 hr after HD application did not reduce microvesicle formation. There was no benefit when niacinamide was given as both a pretreatment and treatment when compared to niacinamide given only as a pretreatment. The reduction in microvesication 24 hr after HD did not correlate with skin NAD+ content. Niacinamide did not reduce the degree of erythema or edema. Ballooning degeneration of basal epidermal cells was present in some niacinamide pretreated HD exposure sites. These results suggest that niacinamide may only be effective as a pretreatment compound to reduce the incidence of HD-induced microvesicle formation. Maintenance of skin NAD+ content may not be solely responsible for inhibiting microvesicle formation and inhibition of PADPRP may be of greater importance.

Systemic administration of candidate antivesicants to protect against topically applied sulfur mustard in the mouse ear vesicant model (MEVM)

The mouse ear vesicant model (MEVM) provides a quantitative edema response as well as histopathological and biochemical endpoints as measurements of inflammation and tissue damage following exposure to the chemical warfare agent sulfur mustard (HD). In the MEVM, several topically applied anti‐inflammatory agents provided a significant degree of protection against HD‐induced edema and dermal–epidermal separation. This study evaluated the protective effects of three of these pharmacological compounds when administered systemically in the MEVM. Alzet osmotic pumps were used to deliver a subcutaneous dose of the appropriate anti‐inflammatory agent, starting 24 h before exposure to sulfur mustard and continuing until 24 h post‐exposure to HD. Twenty‐four hours after pump implantation, 5 μl of a 195 mM (0.16 mg) solution of sulfur mustard (density = 1.27 g ml−1; MW = 159; purity = 97.5%) in methylene chloride was applied to the inner surface of the right ear of each mouse. Sulfur mustard injury in the mouse ear was measured by both edema response (fluid accumulation) and histopathological damage (necrosis, epidermal–dermal separation). The systemic administration of hydrocortisone, indomethacin and olvanil provided a significant reduction in edema (24%, 26% and 22%, respectively) from the positive control. Compared to HD‐positive controls, hydrocortisone, indomethacin and olvanil caused a significant reduction in subepidermal blisters (71%, 52% and 57%, respectively) whereas only hydrocortisone produced a significant reduction in contralateral epidermal necrosis (41%). We show here that these anti‐inflammatory drugs are effective when administered systemically in the MEVM. Published in 2000 by John Wiley & Sons, Ltd.

Characterization of the Sulfur Mustard Vapor Induced Cutaneous Lesions on Hairless Guinea Pigs

The identification of antivesicant compounds has been hindered by the lack of a suitable in vivo model. Our laboratory has been evaluating the hairless guinea pig as a useful animal model to mimic the human cutaneous response to sulfur mustard (HD) exposure. The characterization of two cutaneous responses to HD, quantification of erythema using a reflectance color meter, and pathology by light microscopy, is described in this manuscript. Fifty-two hairless guinea pigs were exposed to saturated HD vapor using a vapor cup technique. The dose response of HD vapor was established by varying the duration (2–11 min) of skin exposure to an HD vapor concentration of 0.77 g/m3 under occluded caps. The degree of erythema was determined by measuring the increase in the reflectance meters a (relative parameter measuring the degree of redness) response for each exposure site at 4, 5, 6, and 24-h postexposure. A linear increase in erythema was observed between the 2– and 4-min HD exposure doses for each post HD observ...

Sulfur mustard-induced microvesication in hairless guinea pigs: Effect of short-term niacinamide administration

It has been postulated that sulfur mustard (HD) damage may activate poly(ADP-ribose) polymerase (PADPRP), resulting in depletion of cellular NAD+. This biochemical alteration is postulated to result in blister (vesicle) formation. It has been previously demonstrated that niacinamide (NAM), an inhibitor of PADPRP and a precursor for NAD+ synthesis, may be useful as a pretreatment compound to reduce HD-induced microvesication. The present study was undertaken to determine whether niacinamides protective action could be extended beyond 24 hr and if the degree of microvesication is related to changes in skin NAD+ content. HD exposures were made by vapor cup to hairless guinea pigs. Niacinamide (750 mg/kg, ip) given as a 30-min pretreatment did not reduce the degree of microvesication 72 hr after HD compared to saline controls. However, niacinamide given as a 30-min pretreatment and at 6-, 24-, and 48-hr after HD, exhibited a 28% reduction in microvesication 72 hr after HD. Skin NAD+ content at 72 hr after HD was depleted by approximately 53% in the saline and NAM-treated groups. Skin NAD+ content was depleted despite NAM administration. Niacinamide did not reduce the degree of erythema at 48 or 72 hr. These results suggest that niacinamides protective effect against HD-induced microvesication may be extended for at least 72 hr, but NAM levels must be sustained during the post-HD period. The link between maintenance of skin NAD+ and reductions in microvesication is still uncertain.

Noninvasive methods for determining lesion depth from vesicant exposure

Before sulfur mustard (HD) injuries can be effectively treated, assessment of lesion depth must occur. Accurate depth assessment is important because it dictates how aggressive treatment needs to be to minimize or prevent cosmetic and functional deficits. Depth of injury typically is assessed by physical examination. Diagnosing very superficial and very deep lesions is relatively easy for the experienced burn surgeon. Lesions of intermediate depth, however, are often problematic in determining the need for grafting. This study was a preliminary evaluation of two noninvasive bioengineering methodologies, laser Doppler perfusion imaging (LDPI) and indocyanine green fluorescence imaging (ICGFI), to determine their ability to accurately diagnose depth of sulfur mustard lesions in a weanling swine model. Histological evaluation was used to assess the accuracy of the imaging techniques in determining burn depth. Six female weanling swine (8–12 kg) were exposed to 400 &mgr;l of neat sulfur mustard on six ventral sites for 2, 8, 30, or 60 minutes. This exposure regimen produced lesions of varying depths from superficial to deep dermal. Evaluations of lesion depth using the bioengineering techniques were conducted at 24, 48, and 72 hours after exposure. After euthanasia at 72 hours after exposure, skin biopsies were taken from each site and processed for routine hematoxylin and eosin histological evaluation to determine the true depth of the lesion. Results demonstrated that LDPI and ICGFI were useful tools to characterize skin perfusion and provided a good estimate of HD lesion depth. Traditional LDPI and the novel prototype ICGFI instrumentation used in this study produced images of blood flow through skin lesions, which provided a useful assessment of burn depth. LDPI and ICGFI accurately predicted the need for aggressive treatment (30- and 60-minute HD lesions) and nonaggressive treatment (2- and 8-minute HD lesions) for the lesions generated in this study. Histological evaluation confirmed the accuracy of the assessment. The ICGFI instrument offers several advantages over LDPI including real-time blood flow imaging, low cost, small size, portability, and not requiring the patient to be repositioned. A negative, however, is the need for intravenous dye injection. Although this would not be an issue in a hospital, it may be problematic in a mass casualty field setting. Additional experiments are required to determine the exposure time necessary to produce a graded series of partial-thickness HD lesions and to optimize instrumental parameters. The data generated in this follow-on study will allow for a full assessment of the potential LDPI and ICGFI hold for predicting the need for aggressive treatment after HD exposure. The lasting message is that objective imaging techniques can augment the visual judgment of burn depth.

A 7-day mouse model to assess protection from sulfur mustard (SM) skin injury

The mouse ear vesicant model (MEVM) is a screening tool used to identify protective compounds against acute sulfur mustard (SM)‐induced skin injury. It provides endpoints of edema and histopathology 24 h following a topical SM exposure to assess protection against inflammation and tissue damage. To further evaluate successful compounds, the MEVM was modified for use as a 7‐day model. Dose response studies were conducted with SM to select an optimal challenge dose for the new model. Due to severity of SM‐induced tissue damage by Day 7, edema and histopathology were determined unreliable endpoints. Therefore, a modified Draize scoring system (no damage to extensive necrosis) was incorporated as an endpoint to evaluate tissue damage out to Day 7. To aid in optimal SM dose selection, retro synthetic capsaicin (RSCAP), a protective compound in the MEVM, was evaluated as a treatment 15 min before exposure to 0.06, 0.08, and 0.16 mg SM. The RSCAP compound provided similar significant protection at Day 7 against the 0.06‐ (42% reduction) and 0.08‐mg doses (32% reduction), but was not effective against the severely necrotizing 0.16‐mg SM dose. Based on these results, an optimum SM dose of 0.08 mg was selected. Retro synthetic capsaicin and two pharmacologically inactive analogs were tested as topical treatments 15 min prior to SM challenge. The RSCAP compound significantly reduced injury, whereas the inactive analogs had no protective effect. The RSCAP also significantly reduced SM injury when administered topically 10 min after SM challenge. These data support the use of the 7‐day mouse ear vesicant treatment model (MEVTM) in evaluating candidate antivesicant compounds.

A VAPOR EXPOSURE MODEL FOR NEONATAL MICE

Sulfur mustard (HD) is a vesicant compound that was first used as a chemical warfare agent in World War I. (Papirmeister et al. 1991). Numerous animal models have been used to study HD-induced vesication. In this article, we describe modifications of the vapor cup model of Mershon and colleagues (1990) to establish a new vapor cup model for use in neonatal mice. The need to develop this model resulted from the development of gene-targeted knockout mice that can be used to evaluate the function of specific genes and their contribution to HD-induced pathology. However, the knockouts are haired mice; therefore, it is necessary to perform vapor exposures on the pups prior to their growing hair. Neonatal mice were anesthetized with isofluorane inhalation and placed in sternal recumbency on a 37°C isothermal pad to maintain body heat during exposure. The vapor cup consisted of a 1.5-mL microfuge tube cap (8 mm inside diameter) modified using a Dremel tool to contour its rim to better fit the curve of a mouse pups back. The inside of the cap was fitted with an 8-mm disk of Whatman #2 filter paper, and the rim of the cap was coated with a thin bead of Thomas Lubriseal grease. Ten μL of neat HD was placed on the filter paper disk, and the cup was immediately inverted and placed onto the back of an anesthetized mouse pup. Exposure times varied from 10 to 30 min. At 24 h postexposure, the mice were euthanized; the HD-exposed skin was removed and fixed in 10% neutral buffered formalin. Following a minimum of 24 h of formalin fixation, the skin sections were bisected across the exposed area. The sections were embedded in paraffin with the central straight-cut surfaces being the focus of histological evaluation. The amount of damage associated with the HD vapor cup exposure varied with time in a dose response fashion. Typical damage consisted of varying amounts of epidermal necrosis at the basal cell level, with occasional separation of epidermis from dermis (microvesication). In severe cases there was complete coagulation of the epidermis and no microvesication. This model should prove useful in identifying the biochemical mechanism of action of HD and ultimately aid in the evaluation of treatment compounds. It may also provide a relevant exposure model for other compounds for which the assessment of vapor-induced damage is necessitated.