2.2 Induction of Topical AD-Like Skin Dermatitis in Mice by 2,4-dinitrochlorobenzene (DNCB)
The experimental atopic dermatitis (AD)-like skin lesions were established by using 2,4-dinitrochlorobenzene (DNCB) according to the literature [17] with slight modification.
Mice were divided into three groups (6 mice/group): TRPA1−/− and WT groups were treated with DNCB; the negative control group was treated with 3:1 mixture of acetone and olive oil without DNCB. Briefly, after 1 week of acclimation, the dorsal hair of mice was completely shaved with a clipper before sensitization. For the sensitization process, 150 μl of 2% DNCB dissolved in a 3:1 mixture of acetone and olive oil was applied to the dorsal skin, and 10 μl per ear was applied to both ears. After four days, 0.5% DNCB was applied to challenge the dorsal skin (150 μl) and both ears (10 μl per ear) at the interval of two days for 20 days.
The treatment lasted for 21 days, after which the animals were sacrificed and their blood, ears and dorsal skin tissue samples were collected for analysis.
2.3 Evaluation of dermatitis, ear thickness and scratching behavior
The severity of dorsal skin lesions was evaluated based on four symptoms: edema, erythema/hemorrhage, scarring/dryness, and excoriation/erosion. A total dermatitis score was defined as the sum of the scores of individual symptoms (0, no symptoms; 1, mild symptoms; 2, moderate symptoms; 3, severe symptoms) ranging from 0 to 12.
The ear thickness of the mouse was measured by an electronic caliper (Shanghai, China) with 0.1 mm accuracy. The scratching frequencies were measured according to the previously described criteria [13] with some modification. The mice were acclimated for 1 h by being individually placed into a recording cage, after which D NCB was applied to their dorsal skin and ears. After 30 minutes, the number of scratching bouts at the DNCB site was measured and recorded for 10 minutes.