Vetorphale

Animals underwent deep intraperitoneal anesthesia with an anesthetic mixture of three drugs: medetomidine (Domitor, Nippon Zenyaku Kogyo, Tokyo, Japan), midazolam (Dormicum, AstellasPharma, Tokyo, Japan), and butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan). We mixed medetomidine 0.15 mg, midazolam 2 mg, and butorphanol 2.5 mg/kg b.w./rat and added saline (Otsuka Pharmaceutical Factory, Tokushima, Japan) to adjust the mixture to a volume of 0.5 ml/100 g b.w./rat. The rats were perfused transcardially with saline, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The brains were taken out and were fixed with 4% PFA in 0.1 M PB at room temperature (RT) for 4 hr. The brains were immersed in 10% sucrose at 4°C overnight and subsequently were immersed in 20% sucrose at 4°C for 3 days. The brains were cut at 40 μm thickness with a freezing microtome (Microm HM 430; Thermo Scientific, Germany).

Syrian hamsters (male, 4 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan). Baseline body weights, respiratory parameters, and SpO₂ were measured before infection. For the virus infection experiments, hamsters were euthanized by intramuscular injection of a mixture of 0.15 mg/kg medetomidine hydrochloride (Domitor^®, Nippon Zenyaku Kogyo), 2.0 mg/kg midazolam (Dormicum^®, FUJIFILM Wako Chemicals), and 2.5 mg/kg butorphanol (Vetorphale^®, Meiji Seika Pharma) or 0.15 mg/kg medetomidine hydrochloride, 2.0 mg/kg alphaxaone (Alfaxan®, Jurox), and 2.5 mg/kg butorphanol. The B.1.1 virus, Omicron subvariants (5 × 1000 TCID₅₀ in 100 µl), and saline (100 µl) were intranasally inoculated under anesthesia. Oral swabs were collected at the indicated timepoints. Body weight was recorded daily by 7 d.p.i., and at 10 and 14 d.p.i. Enhanced pause (Penh, see below), the ratio of time to peak expiratory flow relative to the total expiratory time (Rpef, see below), and subcutaneous oxygen saturation (SpO₂, see below) were monitored on 1, 3, 5, and 7 d.p.i. Lung tissues were collected at 2 and 5 d.p.i. Viral RNA load in the oral swabs and respiratory tissues was determined by RT-qPCR. Viral titers in the lung periphery were determined using TCID₅₀. These tissues were also used for histopathological and IHC analyses (see below).

Adult male and female Wistar–Imamichi rats for breeding were purchased from the Institute for Animal Reproduction (Ibaraki, Japan). Offspring derived from mating in our facility were housed with dams in the same cages until weaning at three weeks of age. After 8–10 weeks of age, female rats were confirmed to exhibit at least two standard four-day estrous cycles by daily vaginal smears. All animals were bred and housed in a room with a controlled temperature (24 ± 2 °C) and a 14-h light/10-h dark cycle (lights on 06:00 h). Standard diet and tap water were available ad libitum. All animals were euthanized under deep anesthesia by intraperitoneal injection of sodium pentobarbital (64.8 mg/kg body weight) and butorphanol (2.5 mg/kg body weight, Vetorphale, Meiji Seika Pharma, Tokyo, Japan). The time periods for euthanasia ranged from 15:00 to 18:00 h. All animal experimental procedures were approved by the Committee on the Care and Use of Experimental Animals of Nippon Medical School and were conducted in compliance with Guidance on Animal Bioethics (based on the guidelines for humane treatment of experimental animals issued by the US National Institutes of Health) of Nippon Medical School (permit number 27-173; approved 22 June 2015).

In vivo studies were approved by the Kitasato University School of Medicine and Hospital Ethics Committee (Approval number 2017-098). Wistar rats, 10 weeks old, were anesthetized with 0.3 mg/kg medetomidine (Domitor, Nippon Zenyaku Kogyo, Fukushima, Japan), 0.5 mg/kg butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), and 0.5 mg/kg midazolam (Midazolam Sandoz, Sandoz, Tokyo, Japan). CPC/VCM and PMMA/VCM were implanted between the fascia and the muscle of the femur on the left and right side of 40 rats, respectively. The fascia and skin were sutured and the animals were allowed to move freely in their cages immediately after the surgery. Test specimens were removed on days 1, 7, 28, and 56, after sacrificing the animals (n = 10 each).