Atipamezole

Immunodeficient female mice (BALB/c-nu/nu; 8–20 weeks old) were purchased from CLEA Japan Ltd. (Tokyo, Japan). Mice were kept under a 12-h light/12-h dark schedule (lights on from 7:00 a.m. to 7:00 p.m.) and allowed food and water ad libitum. All animal experiments were performed in agreement with the guidelines of Kagoshima University Committee on Recombinant DNA Security and approved by the Animal Care and Experimentation Committee of Kagoshima University (permit no. 25,035 and 25036; dated 8 August 2013 permission no. 16008; valid from 9 August 2016 to 31 March 2019). All surgeries were performed following intraperitoneal (IP) injection of three anesthetics (medetomidine (0.75 mg/kg; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), midazolam (4 mg/kg; Sandoz K.K., Tokyo, Japan), and butorphanol (5 mg/kg; Meiji Seika Pharma Co., Ltd., Tokyo, Japan)). Consciousness was restored by subcutaneous injection of atipamezole (3.75 mg/kg; Nippon Zenyaku Kogyo Co., Ltd.), an antagonist of medetomidine; then, mice were warmed using an electric plate warmer. All efforts were made to minimize suffering.

Medetomidine (Nippon Zenyaku Kogyo, Fukushima, Japan) 0.375 mg/kg, midazolam (Sandoz K.K., Yamagata, Japan) 2.0 mg/kg and butorphanol (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) 2.5 mg/kg were administered intraperitoneally to induce anesthesia according to a previously published protocol [58 (link)]. Rapid recovery from anesthesia was achieved by administration of atipamezole (0.75 mg/kg), an antagonist of Medetomidine (Nippon Zenyaku Kogyo Co., Ltd., Tokyo, Japan). Prior to euthanasia, the animals were deeply anesthetized using sodium pentobarbital (Kyoritsu Seiyaku Corporation, Tokyo, Japan) 75 mg/kg, administered intraperitoneally.

Endometrial transplantation was performed as described previously.¹⁵, ²⁷ Briefly, mice were anaesthetized by intraperitoneal (i.p.) injection of a mixture of 0.75 mg/kg medetomidine hydrochloride (Nippon Zenyaku Kogyo Co., Ltd.), 4.0 mg/kg midazolam (Astellas Pharma Inc) and 5.0 mg/kg butorphanol (Meiji Seika Pharma Co., Ltd.). Then, through paravertebral incisions, the mice were bilaterally ovariectomized to exclude the effects of endogenous oestrogen and menstrual cycle. After the procedure, the effect of medetomidine was reversed by i.p. injection of 0.75 mg/kg atipamezole (Nippon Zenyaku Kogyo Co., Ltd.), and the animals were allowed to recover. All donor and recipient mice received subcutaneous injections of estradiol dipropionate (100 mg/kg) in sesame oil (Obahormone Depot, Aska) every week from the time of ovariectomy. Seven days after ovariectomy, the uterine horns from the donor were removed. A round endometrial fragment (3 mm in diameter) was transplanted to each side of the peritoneal wall of recipient mice and secured using 7‐0 polypropylene sutures (Ethicon, Johnson & Johnson). The day of implantation was defined as day 0. WT or RAMP1^−/− recipient mice bearing an implant from donor WT or RAMP1^−/− mice are referred to hereafter using the terms WT → WT and RAMP1^−/− → RAMP1^−/−.

Priming the system of the apheresis machine with normal saline followed by autologous blood is necessary to ensure hemodynamic stability during PBMC collection. Therefore, two weeks before the experiment, about 200 mL autologous blood (20 mL/kg) was collected from each dog in a sterile blood-collection bag with acid–citrate–dextrose formula A (ACD-A, Terumo BCT) and kept at 4 °C until the day of apheresis. Before this sampling, the dogs were sedated with atipamezole 10~20 µg/kg (Nippon Zenyaku Kogyo Co., Fukushima, Japan) and awakened with medetomidine 10~20 µg/kg (Nippon Zenyaku Kogyo Co., Fukushima, Japan). The food was withheld from all dogs 12 h before the apheresis process, while water was provided ad libitum. On the day of apheresis, to customize the length of the PBMC apheresis process, individual data (HCT%, body weight, height ≥ 80 cm, and gender) were collected to establish each dog’s total blood volume (TBV) and its optimal pump rates considering the flow rate of the anticoagulant (AC) [33 (link)].

Institute of Cancer Research (ICR) male mice (five-week-old; Clea Japan, Inc., Tokyo, Japan) were used. They were kept under a 12 h light/12 h dark schedule (lights on from 0700 h to 1900 h) and allowed food and water ad libitum. In the experiment, mice were used under sufficient anesthesia after the intraperitoneal (IP) injection of the combination of three anesthetics (medetomidine (0.75 mg/kg; Nippon Zenyaku Kogyo Co. Ltd., Fukushima, Japan), midazolam (4 mg/kg; Sandoz K.K., Tokyo, Japan) and butorphanol (5 mg/kg; Meiji Seika Pharma Co., Ltd., Tokyo, Japan)). Recovery of the mice from anesthesia was performed by the IP injection of atipamezole (3.75 mg/kg; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), an antagonist of medetomidine, followed by warming with an electric plate warmer.
All animal experiments were performed at the National Defense Medical College (Saitama, Japan), in accordance with the guidelines of National Defense Medical College Committee on Recombinant DNA Security, and approved by The Care and Use of Laboratory Animals (permission no. 12002, valid from 3 July 2012 to 31 March 2015; and no. 15002, valid from 13 July 2015 to 31 March 2018). All efforts were made to minimize the number of animals used and their suffering.

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.