TPA-induced mouse ear edema model

Male ICR mice were housed at a constant temperature (23ºC) and relative humidity (60%) with a fixed 12 h light:12 h dark cycle and had free access to food and water. All experimental procedures involving animals and their care conformed to the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research and Quarantine Service of Korea and were approved by the Hallym Medical Center Institutional Animal Care and Use Committee.

TPA-induced mouse-ear edema models were prepared as described previously (27, 48). To examine the effects of PEP-1-GLRX1 against TPA-induced ear edema, we divided the mice into four groups (n = 5 per group). The experimental groups were as follows: (1) normal control mice; (2) TPA-induced ear-edema mice; (3) TPA ＋ control GLRX1-treated mice; and (4) TPA ＋ PEP-1-GLRX1-treated mice. TPA (1.0 µg) dissolved in 20 ml of acetone was applied to the inner and outer surfaces of the ears of the mice every day for 3 days. PEP-1-GLRX1 (10 µg) protein was topically applied to the ears of mice every day 1 h after TPA treatment. After the final treatment with TPA and PEP-1-GLRX1, we sacrificed the mice to obtain ear biopsies . Ear thicknesses were measured using a digital thickness gauge (Mitutoyo Corporation, Toyko, Japan). Ear weights were measured after 5-mm diameter ear biopsies were obtained from each group using a punch (Kai Industries, Gifu, Japan). For histological analysis, ear biopsies were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at a thickness of 5 µm, and then stained with Histidine and hematoxylin and eosin.