Ketamine

In all the surgical procedures, mice were anaesthetized with 5% isoflurane (Sigma Aldrich, CAS: 26675-46-7) by volume for induction, injected i.p. with ketamine/xylazine (60 mg/kg ketamine, Sigma Aldrich, CAS: 1867-66-9 and 5 mg/kg xylazine, Sigma Aldrich, CAS: 23076-35-9), head fixed to a stereotaxic apparatus (David Kopf instruments) and then maintained anesthetized by continuous isoflurane (~1.25%) inhalation throughout the surgical procedure. Eye ointment was used to protect the mice eyes (Duratears, Vetmarket) and body temperature was recorded and maintained by a heating pad (FHC, DC temperature controller) at 34 °C throughout the surgery. At the beginning of each surgical procedure the mice were injected subcutaneously with Carprofen (5 mg/kg, Sigma Aldrich, CAS:53716-49-7) to reduce inflammation and pain. At the end of each surgical procedure the mice were injected subcutaneously with Buprenorphine (0.05 mg/kg, Sigma Aldrich, CAS:52485-79-7) to reduce pain. The mice were then allowed to recover in their home cage for at least 1–2 weeks before the subsequent surgical procedure or experiment began.

Sirolimus was purchased from MedChem Express, LLC (Princeton, NJ). The drug was dissolved in dimethyl sulfoxide (DMSO) at 50 μg/μL (55 mM) and stored in at −20 °C. This concentrated stock was diluted to a final concentration of 0.5 μg/μL in dH₂O immediately before use. Ketamine (50 mg/mL), xylazine (suspended in methanol at 10 mg/mL), and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Ketamine-xylazine solution consisted of 80 μL (4 mg) Ketamine plus 20 μL (0.2 mg) xylazine and stored at 4 °C for a maximum of 2 weeks. The solution was diluted 1:10 with dH₂O immediately prior to use. Mice were injected intraperitoneally with 10 μL/g (40 μg/g Ketamine +2 μg/g xylazine) before inoculation and other surgical procedures. Adequate sedation was confirmed before intranasal inoculations by the absence of footpad reflexes. Mice were held in a supine position (at a 45° angle) with the back supported by the palm and the neck skin fold by the thumb and index finger. The inoculum was slowly released from a 10-μL micropipette as two small drops covering the two nostrils. The mice were allowed to inhale the volume without forming bubbles. They were then maintained in the same position until they regained consciousness and their rapid breathing returned to normal.

IL-37 transgenic mouse with C57BL/6 background were generated. C57/BL6 mice were used as controls. All female mice were maintained in animal facility of Tongji University (Shanghai, China) and used at the age of 6 to 10 weeks. Mice were anesthetized by injection of a mixture of Ketamine (Sigma Chemical Co.) and were then infected i.n. with 4×10⁷ viable CFUs of BCG or with PBS as control. The animal protocol was approved by the Committee on the Ethics of Animal Experiments of the Shanghai Pulmonary Hospital affiliated to Tongji University (Permit Number: 2013–003). All surgery was performed under anesthesia by injection of a mixture of Ketamine (Sigma Chemical Co.) and all efforts were made to minimize suffering.

Female wild‐type C57BL/6 mice (20–25 g) were obtained from HFK Bio‐Technology Company (Beijing, China) and housed in the SPF‐grade animal facility under the controlled conditions as follows: 70% ± 4% relative humidity, 24 ± 1°C and a 12‐h light/dark cycle. The mice were given free access to tap water and food. All the animal‐related procedures were conducted following the guidelines established by the Institutional Animal Care and Use Committee of Suzhou Ninth Hospital Affiliated to Soochow University (wj2016065), and all animal‐related experiments were performed following the ARRIVE guidelines. In the ketamine‐induced model, the mice were administered with 30 mg/kg/day ketamine (Sigma‐Aldrich) via intraperitoneal injection for 12 weeks as previously described.¹² The mice in its control group were injected with normal saline. In the CYP‐induced model, the mice were administered with 300 mg/kg/day CYP (Thermo Company) via intraperitoneal injection for 7 days as previously described,¹³ and the mice in its control group were injected with normal saline. In the LPS‐induced model, the mice were administered with 25 mg/kg LPS (Thermo Company) via intraperitoneal injection for 24 h as previously described. The mice in its control group were injected with normal saline. The mice were euthanized by carbon dioxide inhalation, and the bladders were removed after treatment.¹⁰

All animals were fed ad libitum and kept in a cabinet at 50–70% humidity, at a temperature of 19–26 °C in a cycle of 12 h light/12 h dark. This study adheres to the guidelines established by the Brazilian College of Animal Experimentation (COBEA) and was approved by the Ethical Committee of the School Medicine of the Federal University of São Paulo (UNIFESP, protocol no. 1949-11).
Eight-to-twelve-week-old male Wistar rats (250–300 g) were divided into two groups (6 animals each). The control group received phosphate-buffered saline (PBS), while the treated group received 540 mg/kg/day of diacetyl (Cat B8530-7; Sigma Aldrich, St. Louis, MO, USA) dissolved in PBS Both groups were dosed using gavage. The concentration of 540 mg/Kg/day of 2,3-butanedione and the treatment period of four weeks was based on the experiment conducted by Colley and Cols (Colley et al., 1969 (link)).
After four weeks of treatment, the animals were anesthetized with ketamine and xylazine (Sigma Aldrich, St. Louis, MO, USA) and sacrificed. The lung tissue was collected and immediately frozen in liquid nitrogen and stored at –80 °C.

Fertilized oocytes were washed in 2 % L-Cysteine (pH 8.0) to remove the jelly coat. Xenopus laevis embryos were collected and staged according to Nieuwkoop and Faber [12 (link)].
Experiments were conducted in 0.3× Modified Barth’s Saline (MBS, 88 mM NaCl, 1 mM KCl, 0.7 mM CaCl2, 1 mM MgSO4, 2.5 mM NaHCO3, and 5 mM HEPES, pH 7.8). Working solutions of ketamine (Sigma, St Louis, MO, USA; cat.no.K2753) were dissolved in 0.3 × MBS, and the treatment was administered at various concentrations from stage 8 (before the gastrula stage) to stage 21 (complete neural tube closure). After treatment, the embryos were transferred to 0.3× MBS and cultured in six-well plates at 21 °C; each well contained 10 embryos in 5 ml of 0.3× MBS. The solution and drug were changed every 24 h. Each treatment group was tested 3 separate times.