Medetomidine

The mice were anesthetized with an intraperitoneal injection of mixed anesthetic agent (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol; Fujifilm Wako Pure Chemical Co., Osaka, Japan), and transcardially perfused with a fixative containing 4% paraformaldehyde and 1.5% glutaraldehyde in phosphate-buffered saline (PBS). After perfusion, the liver and white adipose tissue were removed and allowed to stand in the same fixative for one day. The tissues were rinsed several times with PBS and embedded in paraffin. Tissues were cut into 5-μm-thick sections, mounted on slides, and stained with hematoxylin eosin (HE).

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).

For the orthotopic syngeneic mouse models, the recipient C57BL/6 wild-type mouse was anesthetized with 4 mg/kg of midazolam (Wako), 0.3 mg/kg of medetomidine (Wako), and 5 mg/kg of butorphanol (Wako), and placed on a tilted platform. A 50-µL volume of bleomycin (Tokyo Chemical Industry, Tokyo, Japan) at 0.5 mg/mL was administered to the mouse via the trachea with the use of a cannula. At 2 weeks after bleomycin treatment, a single-cell suspension of KC or AC cells 1 × 10⁵ cells in 50 µL of PBS was introduced into the lungs via the trachea as for bleomycin administration (Supplementary Figure S3, Supplementary Video S1). At the end of experiments, tumors were removed from mice and fixed in 4% paraformaldehyde (Wako) overnight.
For the subcutaneous injection model, a single-cell suspension of AC cells 1 × 10⁶ cells in 100 µL of 50% growth factor–reduced was injected under the skin on the dorsal flank of a nude mouse. Tumor volume (mm³) was calculated every 2 days according to the formula: short axis² × long axis × 0.5236. When tumor size reached ~75 mm³, mice were randomly assigned to receive vehicle (Methyl cellulose, Wako) or crizotinib (Pfizer, New York, NY, USA) at 150 mg/kg by daily oral administration.