Midazolam

Of the total of 664 mice, 323 underwent a bilateral ovariectomy or a sham surgery in the present study. The mice were anesthetized with a mixture of 0.18 mg/kg medetomidine hydrochloride (Wako, Osaka, Japan), 2.4 mg/kg midazolam (Wako), and 3 mg/kg butorphanol tartrate (Meiji Seika Pharma, Tokyo). The three-mix anesthetic was injected subcutaneously (6 μl/g). At ≥ 1 week after the surgery, we performed the vaginal cytology experiment, and we confirmed that the cyclicity had stopped in the ovariectomized mice and remained at a stage resembling diestrus (Fig. 1d).

Method for extracapsular clear lens extraction (ECLE) from rat and mouse eyes was established, confirming the histological observation of rat PCO in our previous study [19 (link)]. Eighteen 7-week-old female albino SD rats and twelve 7-week-old C57BL/6J mice were used as PCO animal models. ECLE was performed in both eyes of all rats and mice anesthetized with intraperitoneal administration of a combination anesthetic prepared with 0.3 mg/kg body weight medetomidine, 4.0 mg/kg body weight midazolam, and 5.0 mg/kg body weight butorphanol (Wako, Osaka, Japan). Animals were sacrificed at either 0 (after the surgery was completed) (day 0), 7 (1 week (W)), or 14 days (2W) after surgery by administering a lethal dose of CO₂. Mice exhibited no signs of distress during euthanasia. Euthanasia by CO₂ inhalation was performed according to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. Lens capsules with LECs removed from all eyes were used as PCO samples. All PCO samples from right eyes were processed for microarray studies (n = 6 at each time point) and all PCO samples from left eyes were used for reverse transcription-quantitative PCR (RT-qPCR) (n = 4 at each time point) and protein blotting (n = 2 at each time point).

The anesthesia mixture used was prepared by combining the following: 2 mL midazolam (5 mg/mL; FUJIFILM Wako Co., Ltd.), Vetorphale 2.5 mL (5 mg/mL; Meiji Seika Pharma Co., Ltd.), medetomidine hydrochloride 0.75 mL (1 mg/ml; Kyoritsu Seiyaku), normal saline 19.75 mL (Otsuka Pharmaceutical Co., Ltd.). The mice were anesthetized by subcutaneously injecting the anesthesia mixer at a dose of 0.1 mL/10 g. The animal study was reviewed and approved by Tokyo Medical University Animal Care and Use Committee.

All experiments were conducted in accordance with the ethical standards of our university (Animal Care Committee of Kanazawa University, AP-183983) and with international standards for animal welfare and institutional guidelines. EC-14 was cultured in Luria–Bertani broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) for 12 h and then seeded into new broth and incubated at 37 °C for 12–14 h with shaking. Subsequently, EC-14 was collected by centrifugation at 8000× g for 5 min at 4 °C and suspended for inoculation into mice. Male Jcl:ICR mice (n = 4) (4 weeks old; CLEA Japan, Tokyo, Japan) were purchased 7 days prior to the experiments and subjected to immunosuppression treatment with 150 mg/kg and 100 mg/kg of endoxan (Shionogi) at 4 days and 1 day prior to infection, respectively.
EC-14 (approximately 5 × 10⁶ CFU/100 µL) was injected into the muscle of the hind leg of mice under anesthesia. Mice were anesthetized using a mixture of butorphanol tartrate, midazolam, and medetomidine hydrochloride (Fujifilm Wako Pure Chemical Corp., Osaka, Japan). Mice were euthanized at 2, 6, 8 and 24 h after infection. Subsequently, the infected muscle was collected and homogenized in PBS. The number of CFU in the homogenized samples was determined by dilution in PBS and plating on agar medium.