Medetomidine

Mice were deeply anaesthetised with a mixture of 0.3 mg/kg medetomidine (Kyoritsu Seiyaku, Tokyo, Japan), 4 mg/kg midazolam (Astellas Pharma, Tokyo, Japan), and 5 mg/kg butorphanol (Meiji Seika Pharma, Tokyo, Japan), and urine was collected through urinary bladder puncture. Mice were euthanised by exsanguination from femoral artery. Blood and kidneys were collected for serological and histopathological analysis, respectively.
The harvested kidneys were cut into small slices and fixed with 10% neutral buffer formalin (NBF), 4% paraformaldehyde (PFA), or 2.5% glutaraldehyde (GTA) in 0.1 M phosphate buffer (PB) for histopathological analysis, immunofluorescence staining, and ultrastructural analysis, respectively.

Six-week-old, male Wistar rats (Nippon Bio-Supp. Center, Tokyo, Japan) weighting 150–170 g were used in this study. After one week of acclimatization, the left ureter was ligated, and the rats were sacrificed 10 d after the operation. The anesthesia used for the operation was a combination of midazolam (4 mg/kg; Sandoz, Tokyo, Japan), butorphanol (1 mg/kg, Meiji Seika Parma Co., Ltd., Tokyo, Japan), and medetomidine (Kyoritsu Seiyaku, Tokyo, Japan) and was injected intraperitoneally. This study was approved by the Center for Disease Biology and Integrative Medicine. All the protocol was followed by “Regulations for the Conduct of Animal Experiments” and “Manual for the Conduct of Animal Experiments” in the University of Tokyo. All persons involved in this animal experiment attended the training course of animal experiment in the University of Tokyo (Rika Miura: 17,253, Imari Mimura: 17,250). Animal experiments was conducted in accordance with the protocols approved by the Animal Experiment Committee and the Ethics Committee of the University of Tokyo (approve number P18-060). All methods are reported in accordance with ARRIVE guidelines.

The methods in the present study were approved by the Institutional Animal Care and Use Committee of Fukuyama University (H26-A-5, 2019-A-5), and the animals were treated in accordance with the guidelines of the United States National Institutes of Health regarding the care and use of animals for experimental procedures.
In total, 292 male ddY mice (Nihon SLC, Shizuoka, Japan) were used. Mice were housed at 22 ± 1°C under a 12-h light and dark cycle with free access to food and water. The mice were anesthetized by intraperitoneal injection of a mixture of medetomidine (0.3 mg/kg, Kyoritsu Seiyaku, Tokyo, Japan), midazolam (4 mg/kg, Alfresa, Osaka, Japan), and butorphanol tartrate (5 mg/kg, Meiji Seika Pharma, Tokyo, Japan), and then placed in a stereotaxic frame (Narishige, Tokyo, Japan). Collagenase type VII (0.03 U/3 μL saline) or the same amount of saline was injected via a Hamilton syringe into the unilateral striatum (0.2 mm anterior, 2.2 mm lateral from the bregma suture, and 3.5 mm depth from the skull) for 3 min [11 (link)]. The syringe was slowly removed 1 min after the injection, and then the scalp incision was sutured. An IDO inhibitor, 1-MT (1 mg), was dissolved in saline using 0.1 N NaOH solution and injected subcutaneously 1 h after collagenase injection and then daily. Control mice were injected with saline instead of 1-MT.