Cutaneous injury-related structural changes and their progression following topical nitrogen mustard exposure in hairless and haired mice

Abstract

To identify effective therapies against sulfur mustard (SM)-induced skin injuries, various animals have been used to assess the cutaneous pathology and related histopathological changes of SM injuries. However, these efforts to establish relevant skin injury endpoints for efficacy studies have been limited mainly due to the restricted assess of SM. Therefore, we employed the SM analog nitrogen mustard (NM), a primary vesicating and bifunctional alkylating agent, to establish relevant endpoints for efficient efficacy studies. Our published studies show that NM (3.2 mg) exposure for 12-120 h in both the hairless SKH-1 and haired C57BL/6 mice caused clinical sequelae of toxicity similar to SM exposure in humans. The NM-induced cutaneous pathology-related structural changes were further analyzed in this study and quantified morphometrically (as percent length or area of epidermis or dermis) of skin sections in mice showing these lesions. H&E stained skin sections of both hairless and haired mice showed that NM (12-120 h) exposure caused epidermal histopathological effects such as increased epidermal thickness, epidermal-dermal separation, necrotic/dead epidermis, epidermal denuding, scab formation, parakeratosis (24-120 h), hyperkeratosis (12-120 h), and acanthosis with hyperplasia (72-120 h). Similar NM exposure in both mice caused dermal changes including necrosis, edema, increase in inflammatory cells, and red blood cell extravasation. These NM-induced cutaneous histopathological features are comparable to the reported lesions from SM exposure in humans and animal models. This study advocates the usefulness of these histopathological parameters observed due to NM exposure in screening and optimization of rescue therapies against NM and SM skin injuries.

Figure 1. NM exposure causes epidermal thickness and microvesication in the skin of SKH-1 hairless and C57BL/6 mice.

Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µm) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and C are representative H&E stained skin sections (400× magnification) showing epidermal thickness from vehicle control and 24 h exposed NM exposed skin tissue in SKH-1 and C57BL/6 mice, respectively. Panel E (i–iii) and G (i–iii) are representative H&E stained skin sections (100× magnification), and E (iv–vi) and G (iv–vi) are representative H&E stained skin sections (400× magnification) showing microvesication from vehicle control and 12–120 h NM exposed skin tissue in mice. These NM-related histopathological changes were assessed as detailed under materials and methods, and calculated as percent length of mice skin epidermis showing epidermal thickness (B and D) and microvesication (F and H). Data presented are mean ± SEM of 3–5 animals in each group. *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; e, epidermis; d, dermis; red arrows, microvesication (epidermal and dermal separation); pink arrows, microvesication within the epidermal layer.

Figure 2. NM exposure causes epidermal cell death, epidermal denuding and ulceration, and scab formation in the skin of SKH-1 and C57BL/6 mice.

Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µM) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and C, E and G and I and K are representative H&E stained skin sections (100 or 400× magnification) showing epidermal cell death, epidermal denuding and scab formation, respectively from vehicle control and 72 or 120 h NM exposed skin tissue in SKH-1 and C57BL/6 mice. These NM-related histopathological changes were assessed as detailed under materials and methods, and calculated as percent length of mice skin epidermis showing epidermal cell death (B and D) epidermal denuding and ulceration (F and H), or as incidences of scab formation/outgrowths (J and L). Data presented are mean ± SEM of 3–5 animals in each group. *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; e, epidermis; d, dermis; green arrows, necrosis/dead epidermal layer or epidermal denuding or scab formation.

Figure 3. NM exposure causes parakeratosis and hyperkeratosis in the skin epidermis of SKH-1 and C57BL/6 mice.

Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µM) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and C (i–iii) and E and G (i–iii) are representative H&E stained skin sections (100 or 400× magnification) showing parakeratosis and hyperkeratosis, respectively, from vehicle control as well as NM exposed (72 and 120 h) skin tissue in mice. These NM-related histopathological changes were assessed as detailed under materials and methods, and calculated as percent length of mice skin epidermis showing parakeratosis (B and D) and hyperkeratosis (F and H). Data presented are mean ± SEM of 3–5 animals in each group. *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; e, epidermis; d, dermis; green arrows, parakeratosis, hypercornification or acanthosis.

Figure 4. NM exposure causes acanthosis in the skin epidermis of SKH-1 and C57BL/6 mice.

Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µM) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and B are representative H&E stained skin sections (100 or 400× magnification) showing acanthosis from vehicle control as well as NM exposed (72 and 120 h) skin tissue in mice. This NM-related histopathological change was assessed as detailed under materials and methods, and calculated as percent length of mice skin epidermis showing acanthosis (B and D). Data presented are mean ± SEM of 3–5 animals in each group. *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; e, epidermis; d, dermis; green arrows, acanthosis.

Figure 5. NM exposure causes necrosis, dermal thickness, recruitment of inflammatory cells and extravasated red blood cells in the dermal region of SKH-1 and C57BL/6 mice skin.

Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µM) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and C, E and F, G and H, and I and J are representative H&E stained skin sections from vehicle control as well as NM exposed skin tissue in mice for dermal necrosis (with presence of breaking down inflammatory cells, mainly neutrophils), dermal thickness, inflammatory cells, and extravasated red blood cells. These NM-related histopathological changes were assessed as detailed under materials and methods, and calculated as percent area of mice skin dermis showing necrosis (B and D), inflammatory cells (Table 1), and extravasated red blood cells (Table 2). *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; d, dermis; green arrows, inflammatory cells; red arrows, extravasated red blood cells.