Murine Model of Thermal and Electrical Stimulation

Five-week-old male C57BL/6J mice and db/db mice (BKS.Cg-m+/+Leprdb/J: Leprdb/Leprdb mice; Charles River Laboratories, Inc., Kanagawa, Japan), were housed in a vivarium in accordance with the guidelines of the animal facility center of Kumamoto University. The mice were maintained on food (standard or high-fat diet) and water ad libitum. High-fat diet was composed of 14% lard, 14% beef tallow, 25% casein, 20% sucrose, 15% cornstarch, 5% cellulose. Six-week-old or 25-week-old mice were treated with HS and/or MES. A well-ventilated 12 cm×10 cm (width×height) chamber was designed for the animal treatment. Electrical stimulation was delivered to un-anesthetized test animal through a pair of 10-cm diameter electro-conductive and thermo-generative rubber electrodes, which were padded with moist soft cotton cloth. The electrodes can be adjusted to allow contact with the animal (Fig. 1B). The electrodes are connected to a Biometronome™ (Tsuchiya Gum Co., Ltd.) that delivered 12 V (55 pps) of direct current with individual pulse duration of 0.1 ms. The temperature at the surface of the electrodes was adjusted to 42°C. For the control group, mice were sham treated by putting them in the chamber with similar set-up as above for 10 min per session but without conducting HS+MES. All procedures involving these animals were approved by the Animal Care and Use Committee of Kumamoto University (#A19-115).