Time course pathogenesis of sulphur mustard-induced skin lesions in mouse model

Abstract

Sulphur mustard (SM) is a bifunctional alkylating agent that causes cutaneous blistering in humans and animals. In this study, we have presented closer views on pathogenesis of SM-induced skin injury in a mouse model. SM diluted in acetone was applied once dermally at a dose of 5 or 10 mg/kg to Swiss albino mice. Skin was dissected out at 0, 1, 3, 6, 12, 24, 48, 72 and 168 hours, post-SM exposure for studying histopathological changes and immunohistochemistry of inflammatory-reparative biomarkers, namely, transforming growth factor alpha (TGF-α), fibroblast growth factor (FGF), endothelial nitric oxide synthase (eNOS) and interlukin 6 (IL-6). Histopathological changes were similar to other mammalian species and basal cell damage resembled the histopathological signs observed with vesication in human skin. Inflammatory cell recruitment at the site of injury was supported by differential expressions of IL-6 at various stages. Time-dependent expressions of eNOS played pivotal roles in all the events of wound healing of SM-induced skin lesions. TGF-α and FGF were strongly associated with keratinocyte migration, re-epithelialisation, angiogenesis, fibroblast proliferation and cell differentiation. Furthermore, quantification of the tissue leukocytosis and DNA damage along with semiquantitative estimation of re-epithelialisation, fibroplasia and neovascularisation on histomorphologic scale could be efficiently used for screening the efficacy of orphan drugs against SM-induced skin injury.

Keywords: DNA damage; FGF; IL-6; Sulphur mustard; TGF-α; eNOS.

Figure 1

Leukocyte response in sulphur mustard (SM) (5 or 10 mg/kg diluted in acetone) exposed mouse skin at various time intervals. Same alphabets on bars indicate non‐significant (P < 0·05) difference between time intervals at same dose shown with the same colour bar. Asterisks indicate significantly (P < 0·05) higher values in 10 mg/kg as compared to 5 mg/kg at given time intervals and for given parameter (n = 6)

Figure 2

Immunohistological grading of interlukin 6 (IL‐6), fibroblast growth factor (FGF), endothelial nitric oxide synthase (eNOS) and transforming growth factor alpha (TGFα) in sulphur mustard (SM; 5 or 10 mg/kg diluted in acetone) exposed mouse skin at various time intervals. Values in y‐axis represent grading on arbitrary scale [0 (absent);1(1 < area ≤ 10%); 2 (10 <mboxarea≤ 30%); 3 (30 < area ≤ 50%); 4 (50 <mboxarea)] (n = 6).

Figure 3

Photomicrograph of sulphur mustard (SM) (10 mg/kg) treated mouse skin sections taken at various time intervals, haematoxylin and eosin (H&E). Bar = 25 µm. (A) 0 hours (control) showing normal arrangement of epidermis, dermis and adnexa. (B) 3 hours showing hyperplasia of epidermal cells, pyknotic nuclei of basal cells (arrow) and oedema of dermis along with mild inflammatory reaction. (C) 6 hours showing coagulative necrosis of epidermis follicular degeneration (arrow), dermoepidermal separation (arrow head) and excessive infiltration of inflammatory cells. (D) 24 hours showing hyperkeratinisation (arrow), necrosis of epidermis, dermoepidermal separation (arrow head) along with transmigrated monocytoid cells and neutrophils. (E) 72 hours showing coagulative necrosis of epidermis extending deep into the dermis, follicular degeneration (arrow) that were roofed by necrosed inflammatory cells entrapped in fibrinous exudates (arrow head). (F) 168 hours showing coagulative necrosis of epidermis and dermis, atrophied adnexa (arrow head) and necrosed inflammatory cells in dermis roofed by eschar tissue (arrow).

Figure 4

Localization of basophils and mast cells in toluidine blue‐stained skin sections. Bar = 25 µm. (A) 0 hours (control) showing intact mast cells in dermis (arrow). (B) 3 hours showing degranulating mast cells (arrow) in dermis after sulphur mustard (SM) (5 mg/kg) treatment. (C) 03 hours showing degranulating mast cell (arrow) in dermis after SM (10 mg/kg) treatment. (D) 168 hours showing metachromatic regranulating basophils (arrow).

Figure 5

TUNEL positive cells in mice skin sections taken at various time intervals post‐sulphur mustard (SM) (A) 5 mg/kg and (B) 10 mg/kg exposure.

Figure 6

Immunohistological localization of transforming growth factor alpha (TGF‐α) in sulphur mustard (SM) (5 or 10 mg/kg, diluted in acetone) treated mouse skin sections taken at various time intervals. Bar = 25 µm. (A) 0 hours showing marked expression of TGF‐α (arrow) in epidermal cells and hair follicles of mouse skin after SM (5 mg/kg) exposure. (B) 24 hours after SM (5 mg/kg) treatment showing increased immunoreactivity of TGF‐α in dermis by fibroblast cells (arrow) and macrophages (arrowhead) and reduced immunoreactivity in epidermal cells and hair follicles compared to (A). (C) 24 hours (SM 10 mg/kg) showing similar immunoreactivity of TGF‐α in dermis by fibroblast and infiltrating macrophages as shown by mice skin after SM (5 mg/kg) exposure in (B). (D) 168 hours (SM 10 mg/kg) showing marked increase in immunosignals of TGF‐α diffused throughout the dermis compared to (B) and (C).

Figure 7

Immunohistological localization of endothelial nitric oxide synthase (eNOS) and interlukin 6 (IL‐6) in sulphur mustard (SM) (5 or 10 mg/kg, diluted in acetone) treated mouse skin sections taken at various time intervals. Bar = 25 µm. (A) Negative control (without eNOS primary antibody). (B) eNOS expression at 72 hours after SM (5 mg/kg) treatment showing immunoreactivity in infiltrated inflammatory cells and blood vessel endothelium (circles). (C) Negative control (without IL‐6 primary antibody). (D) 3 hours after SM (5 mg/kg) treatment showing IL‐6 positive signals by epidermal cells, hair follicle (arrow) and few transmigrated inflammatory cells. (E) 168 hours (SM 5 mg/kg) showing weak IL‐6 immunoreactivity compared to (D). (F) 168 hours (SM 10 mg/kg) showing mild IL‐6 immunosignals in upper part of dermis adnexa only (arrow). Note decrease in intensity as compared to (D).

Figure 8

Immunohistological localization of fibroblast growth factor (FGF) in sulphur mustard (SM) (5 or 10 mg/kg, diluted in acetone) treated mouse skin sections taken at various time intervals. Bar = 25 µm. (A) Negative control (without primary FGF antibody). (B) 0 hours after SM (10 mg/kg) treatment showing similar FGF immunoreactivity as shown by (A). (C) 48 hours after SM (5 mg/kg) exposure showing mild streaks of FGF immunoreactivity (arrow) diffused throughout the dermis. Note increase in immunorectivity of FGF as compared to (B). (D) 48 hours after SM (10 mg/kg) exposure showing mild streaks of FGF immunoreactivity (arrow) similar to (C). (E) 168 hours (SM 5 mg/kg) showing enhanced FGF immunoreactivity (arrow) compared to (C). (F) 168 hours (SM 10 mg/kg) showing moderate FGF (arrow) expression that was more intense than (D) and similar to (E).

Figure 9

Photomicrograph of sulphur mustard (SM) (5 mg/kg) treated mouse skin sections taken at various time intervals, haematoxylin and eosin (H&E). Bar = 25 µm. (A) 1 hour showing ballooning of epidermal cells, acanthosis in epidermis (arrow), oedema of dermis and a few transmigrated neutrophils. (B) 3 hours showing hyperplasia of epidermal cells (arrow), and oedema of dermis along with mild inflammatory reaction. (C) 6 hours showing ballooning of epidermal cells, dermoepidermal seperation (arrow head) and infiltration of inflammatory cells in dermis. (D) 12 hours showing pyknosis of nuclei in basal cells (arrow), dermoepidermal separation (arrow head), oedema and transmigrated inflammatory cells in dermis. (E) 72 hours showing necrosis of epidermis and dermis and necrosed inflammatory cells entrapped in fibrinous exudates (arrow head) forming the eschar tissue. (F) 168 hours showing eschar tissue (arrow) covering the ulcer, coagulative necrosis of epidermis and dermis with atrophied adnexa and remnants of necrosed transmigrated inflammatory cells in dermis.

Figure 10

Photomicrograph of sulphur mustard (SM) (5 or 10 mg/kg) treated mouse skin sections taken at various time intervals. Masson trichrome. Bar = 25 µm. (A) 12 hours (SM 5 mg/kg) showing dermoepidermal separation, oedema and blue‐stained sparse arrangement of collagen along with monocytoid cell infiltration. (B) 12 hours (SM 10 mg/kg) showing dermoepidermal separation, necrosis of epidermal cells and oedema. Severity of lesions was more compared to (A). (C) 72 hours (SM 5 mg/kg) showing decreased collagen content compared to the 12‐hour stage. (D) 72 hours (SM 10 mg/kg) showing decreased collagen content compared to the 12‐hour stage and 72 hours of SM (5 mg/kg). Severe epidermal necrosis (arrow) (E) 168 hours (SM 5 mg/kg) showing necrosis of epidermis (arrow), proliferation of fibroblast (deep blue colour) and neoangiogenesis (arrow head). (F) 168 hours (SM 10 mg/kg) showing eschar tissue covering the ulcer, with minimal dermal tissue (blue) atrophied adnexa and transmigrated inflammatory cells.

Figure 11

Photomicrograph of TUNEL positive cells. Bar = 25 µm. (A) Negative control (without rTdT enzyme). (B) TUNEL positive cells in epidermis of mice skin section (arrow) 72 hours after SM (5 mg/kg) exposure.