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.