Medetomidine

Mice left hind leg underwent depilation using hair clipper and localized radiation using an X-ray generator (150 kVp; 20 mA; filter: 0.2 mm Cu and 0.5 mm Al; MBR-1520R-3; Hitachi Power Solutions, Ibaraki, Japan) with a custom-made collimator to expose only the left hind leg (Fig. 1). Mice were anesthetized during radiation exposure using three types of mixed anesthetic agents (0.75 mg/kg of medetomidine [Nippon Zenyaku Kogyo Co., Fukushima, Japan], 4.0 mg/kg of midazolam [Maruishi Pharmaceutical. Co., Osaka, Japan], and 5.0 mg/kg of butorphanol [Meiji Seika Pharma Co., Tokyo, Japan]). In addition, 0.75 mg/kg of atipamezole (Nippon Zenyaku Kogyo Co.) was used to reverse the effects of medetomidine after radiation exposure.

Male C57BL/6J mice aged 8–10 weeks were intratracheally instilled with 80 μl of saline containing 3.2 mg/kg body weight of bleomycin sulfate (Nippon Kayaku Co., Ltd., UK) to induce BLM-ILD or 80 μl of saline alone as the control. Mice were anesthetized with an intraperitoneal injection of 0.75 mg/kg of medetomidine (Nippon Zenyaku Kogyo, Fukushima, Japan), 4.0 mg/kg of midazolam (Sandoz, Tokyo, Japan), and 5.0 mg/kg of vetorphale (Meiji Seika Pharma, Tokyo, Japan) before the procedure, and medetomidine was antagonized by a peritoneal injection of 0.75 mg/kg of atipamezole (Nippon Zenyaku Kogyo, Fukushima, Japan) after the procedure. The lungs were dissected under the anesthesia described above before or on day 1, 3, 7, 10, or 14 after the administration of BLM.

The mice were anesthetized using an intraperitoneal (i.p.) injection of a mixture of 0.75 mg/Kg medetomidine (Nippon Zenyaku Kogyo, Koriyama, Japan), 4 mg/kg midazolam (SANDOZ, Yamagata, Japan) and 5 mg/kg butorphanol tartrate (Meiji Seika Pharma, Tokyo, Japan). To stimulate saliva secretion, 0.5 mg/kg pilocarpine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was intraperitoneally administered. Saliva was collected using a micropipette for 15 min. The volume of saliva was normalized to the bodyweights of the mice.

Six-week-old C57BL/6 male mice (CLEA Japan, Tokyo, Japan) were anesthetized with intraperitoneal injection of combined anesthetics {midazolam 4 mg/kg BW (Sandoz Japan, Tokyo, Japan), medetomidine 0.75 mg/kg BW (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), butorphanol tartrate 5 mg/kg BW (Meiji Seika Pharma Co., Ltd., Tokyo, Japan)]; their pupils were dilated using a single drop of a mixture of tropicamide and phenylephrine (0.5% each; Mydrin-P®; Santen Pharmaceutical Co., Ltd., Osaka Japan) and received 2-μL intravitreal injections of either KCL dissolved in PBS (50 mM) or just PBS as vehicle via an UltraMicroPump (type UMP2) equipped with a MicroSyringe Pump Controller (World Precision Instruments, Sarasota, FL) [31 ]. Twelve animals were used in each group.

After shaving the occipital region of the mice with an electric shaver under anesthetic conditions, the remaining hair was removed using a hair removal cream (Veet, Reckitt Benckiser, Berkshire, UK), and tape stripping was performed 10 times. A mixture of midazolam (Astellas Pharma, Tokyo, Japan), butorphanol (Meiji Pharmaceutical, Tokyo, Japan), and medetomidine (Nippon Zenyaku Kogyo, Fukushima, Japan) was used for giving anesthesia. The treatment was performed weekly. Transdermal sensitization was induced by applying respective samples to the skin after treatment. Three groups such as control, green kiwifruit, and gold kiwifruit were established (n = 5 or 6).
The control group received 5% sodium dodecyl sulfate (SDS) alone, the green kiwifruit group received 4 mg/mL of green kiwifruit extract in 5% SDS, and the gold kiwifruit group received 4 mg/mL of gold kiwifruit extract in 5% SDS. Four times a week for a total of 5 weeks, 50 μL of the sample was applied to the epidermis using a micropipette. Then, the serum was collected weekly. At 5 weeks, all the mice were anesthetized by injecting sodium pentobarbital salt intraperitoneally. The mice were then sacrificed by cervical dislocation. The mice were weighed weekly to determine whether their growth was inhibited.