Depilatory double-disc mouse model for evaluation of vesicant dermal injury pharmacotherapy countermeasures

Abstract

Background: Sulfur mustard (SM) is a chemical warfare vesicant that severely injures exposed eyes, lungs, and skin. Mechlorethamine hydrochloride (NM) is widely used as an SM surrogate. This study aimed to develop a depilatory double-disc (DDD) NM skin burn model for investigating vesicant pharmacotherapy countermeasures.

Methods: Hair removal method (clipping only versus clipping followed by a depilatory), the effect of acetone in the vesicant administration vehicle, NM dose (0.5-20 μmol), vehicle volume (5-20 μl), and time course (0.5-21 days) were investigated using male and female CD-1 mice. Edema, an indicator of burn response, was assessed by biopsy skin weight. The ideal NM dose to induce partial-thickness burns was assessed by edema and histopathologic evaluation. The optimized DDD model was validated using an established reagent, NDH-4338, a cyclooxygenase, inducible nitric oxide synthase, and acetylcholinesterase inhibitor prodrug.

Results: Clipping/depilatory resulted in a 5-fold higher skin edematous response and was highly reproducible (18-fold lower %CV) compared to clipping alone. Acetone did not affect edema formation. Peak edema occurred 24-48 h after NM administration using optimized dosing methods and volume. Ideal partial-thickness burns were achieved with 5 μmol of NM and responded to treatment with NDH-4338. No differences in burn edematous responses were observed between males and females.

Conclusion: A highly reproducible and sensitive partial-thickness skin burn model was developed for assessing vesicant pharmacotherapy countermeasures. This model provides clinically relevant wound severity and eliminates the need for organic solvents that induce changes to the skin barrier function.

Keywords: edema; mice; skin; vesicant.

FIGURE 1

Effect of hair removal method on mechlorethamine hydrochloride (NM) induced edema. Each female CD‐1 mouse was given two wounds by applying 20 μl (20:80 water: acetone) containing 20 μmol of NM on two filter discs covered by a single thermoplastic strip. Punch biopsy weights of the NM treated area were used to assess skin edema. Naive control mouse skin weights for both conditions, clipped with and without Nair were subtracted from the respective skin weights of the NM treated groups. Representative photographs are presented of mice 72 h after NM exposure. Clipped mice (A) exhibited mild burns, while mice that were clipped and treated with Nair (B) presented more severe skin burns. Clipping‐only versus clipping‐Nair hair removal methods were assessed for NM induced skin edema in mice (C). Edema in the clipping‐Nair group was significantly higher than the clipping‐only group. Burns were also more reproducible (%CV = 9.36% versus 168%, respectively). Columns represent group mean punch biopsy values ± SD, n = 6/group, two experimental and two control groups, 24 mice total. These data indicate that clipping and depilatory elicit a more severe and reproducible edematous response than that of clipping alone. *p < 0.05 as determined by Wilcoxon matched‐pairs signed rank test.

FIGURE 2

Mechlorethamine hydrochloride (NM) vehicle choice exhibited minor changes in skin burn appearance. Clipped and depilated female CD‐1 mice were each given two burns by applying NM solubilized in a water vehicle, with and without acetone pretreatment, or a water‐acetone mixture vehicle. The progression of representative wounds from each group (n = 5/group, three experimental groups, two control groups, 18 mice total) was presented over 3 days. One group (NM in Water) received 15 μmol of NM in 15 μl of ultrapure water. The second group (Acetone Pre‐treatment + NM in Water) received 25 μl of acetone that was allowed to dry for 3 min before receiving 15 μmol of NM in 15 μl of ultrapure water. The third group (NM in 20:80 Water: Acetone) received 15 μmol of NM in 15 μl of 20:80 ultrapure water:acetone. The vast majority of burns in the water vehicle groups, with and without the acetone pretreatment, were identical in terms of area and symmetry. Burns in the NM in 20:80 Water:Acetone group were more spread out from the designated burn area when compared to the others (A), possibly caused by the organic solvent distributing the vesicant outside of the filter disc. Punch biopsy weights of the NM treated area were used to determine the impact of each vehicle on edema 72 h after vesicant exposure (B). Columns represent group mean punch biopsy values ± SD, n = 5/group, three experimental groups, two control groups, 18 mice total. These 18 mice were the same mice from the Figure 2A study. Naive control mouse average punch biopsy weights were subtracted from those of the NM treated groups. One‐way ANOVA with Kruskal‐Wallis test and Dunn's multiple comparisons test determined no significant difference between the three groups, suggesting that acetone was not needed to produce edema.

FIGURE 3

Time course of mechlorethamine hydrochloride (NM) induced skin injury. Clipped and depilated female CD‐1 mice were euthanized at the indicated time points. Each mouse was given two wounds by 15 μl of 0.1 M acetate buffer (pH 4.0) containing 15 (A) or 5 (B) μmol of NM. These NM administration parameters represent the finalized depilatory double‐disc (DDD) model with a modified dose. Columns represent group mean punch biopsy values ± SD, n = 5/group, eleven experimental groups, two control groups, 59 mice total. Naive control group (CTL) average punch biopsy weights were subtracted from the values. *p < 0.05 as determined by one‐way ANOVA with Kruskal‐Wallis test and Dunn's multiple comparisons test.

FIGURE 4

Mechlorethamine hydrochloride (NM) dose–response evaluation showed 2.5 μmol was the lowest dose to induce significant edema. Punch biopsy weights, offset by those of the naive control group (CTL), of the NM treated area were used to assess skin edema in clipped and depilated female CD‐1 mice 24 h after NM administration. Each dose was administered in 15 μl of 0.1 M acetate buffer (pH 4.0). These NM administration parameters represent the finalized depilatory double‐disc (DDD) model with varied doses. The lowest dose that produced significant edema formation was 2.5 μmol and was therefore selected as the lower limit of the subsequent dose–response study to confirm burn severity with histology. Columns represent group mean punch biopsy values ± SD, n = 4–16/group, six experimental groups, one control group, 54 mice total. *p < 0.05 as determined by one‐way ANOVA with Kruskal‐Wallis test and Dunn's multiple comparisons test.

FIGURE 5

Mechlorethamine hydrochloride (NM) induced vesication. Each NM dose was administered to clipped and depilated female CD‐1 mice in 15 μl of 0.1 M acetate buffer (pH 4.0). These NM administration parameters represent the finalized depilatory double‐disc (DDD) model with varied doses. The endpoint of the data presented was 24 h after NM administration. Hematoxylin and eosin stained skin samples were examined under a light microscope. Representative images were presented from each group (n = 4, four experimental groups, one control group, 20 mice total). These 20 mice were included in the Figure 4 studies. The 2.5 μmol, 5 μmol, 10 μmol, and 15 μmol groups showed vesication, whereby the dermal‐epidermal separation is shown by arrowheads.

FIGURE 6

Prodrug anti‐inflammatory efficacy in mice exposed to mechlorethamine hydrochloride (NM). Clipped and depilated female CD‐1 mice were given two wounds by 15 μl of 0.1 M acetate buffer (pH 4.0) containing 5 μmol of NM. These NM administration parameters represent the finalized depilatory double‐disc (DDD) model. The 1% NDH‐4338 w/v group (NM + 4338) and vehicle only control group (NM + vehicle) received 20 μl of treatment, starting 1 h after vesicant administration, four times over course of 24 h. The untreated vesicant control group received NM only (NM). Punch biopsy weights of the NM treated area, offset by those of the untreated naive control group, were used to assess skin edema. Following the 24‐h treatment course, the 1% NS 4338 group yielded a significantly lower punch biopsy weight than the NM control group. There was no significant difference between the vehicle and NM control groups, indicating that the positive anti‐inflammatory effect observed in the experimental group was attributable to the prodrug and not the formulation vehicle. Columns represent group mean punch biopsy values ± SD, n = 4/group, one experimental group, three control groups, 16 mice total. *p < 0.05 as determined by one‐way ANOVA with Kruskal‐Wallis test and Dunn's multiple comparisons test.