Atopic Dermatitis Induction in Mice

AD was induced in mice by repeated local exposure of Dermatophagoides farinae extract (DFE; house dust mite extract) and 2,4-dinitrochlorobenzene (DNCB) to the ears, as previously described [17 (link)]. A schematic of the experimental procedure is provided in Figure 1. For the induction of AD, the mice were divided into four groups (control, AD-only, EF-2001-only, and AD + EF-2001), and the surfaces of both earlobes were stripped five times with surgical tape (Nichiban, Tokyo, Japan). After stripping, 20 µL of 1% DNCB was painted onto each ear, followed by 20 µL of DFE (10 mg/mL) four days later. DFE and DNCB treatment was administered once a week for four weeks. Animals received EF-2001 (2 mg/kg orally administered) throughout the four weeks of AD induction.
The ear thickness was measured 24 h after DNCB or DFE application with a dial thickness gauge (Kori Seiki MFG, Co., Tokyo, Japan). At days 14 and 28, blood samples were collected by orbital puncture. Plasma samples were prepared from the cardiac puncture under ketamine/xylazine anesthesia and stored at −70 °C for further analysis. After blood collection, the ears were removed and used for histopathological analysis. The serum immunoglobulin (Ig)E, DFE specific IgE and IgG2a levels were measured at days 14 and 28 after the first induction using an IgE enzyme-linked immunoassay kit (Bethyl Laboratories, Inc., Montgomery, TX, USA) according to the manufacturer’s instructions.