Temgesic

Mice were anesthetized by inhalation of 2–4% isoflurane (Abbott Laboratories). They received Temgesic (Reckitt Benckiser Pharmaceuticals Ltd.) in isotonic sterile saline (9 mg/ml NaCl, Fresenius Kabi) for pain relief and the back of the neck was shaved. A 30-gauge needle (bent 55° with a 2 mm tip) attached to a 50 μl Hamilton syringe was used to perform intrathecal injection into the cisterna magna, for administration into the cerebrospinal fluid. After the injections, mice received subcutaneous injection of 1 ml of isotonic sterile saline for prevention of dehydration.
Mice were intrathecally injected with CpG (ODN 1585, class A, Invivogen), Imiquimod (R837, Invivogen). Control mice received vehicle alone, either phosphate buffered saline (PBS) or 1 × Hanks balanced salt solutions (HBSS) (Gibco).
In trial experiments evaluated by in vivo imaging of luciferase reporter mice, IFNβ expression in response to CpG and Imiquimod was dose-dependent (not shown). Based on the IFNβ expression, the optimal dose for CpG and Imiquimod was determined to be 10 and 50 μg, respectively, and these were used throughout the study.

Ciproxifan hydrochloride (AOB 33754; Aobious, Gloucester, MA, USA) and pitolisant hydrochloride (AOB2752; Aobious, Gloucester, MA, USA) were dissolved in saline. Drug doses correspond to free bases of the compounds. Injections were given intraperitoneally (i.p.) and the injection volume was 0.01 ml/g body weight.
Alpha-fluoromethylhistidine hydrochloride (αFMH) was a kind gift from Dr. J. Kollonitch (Merck Sharp & Dohme, Rahway, N.J., USA) and was also dissolved in saline. The drug dose 50 mg/kg corresponds to salt of the compound.
Drugs used in the surgeries were lidocaine (10 mg/ml, Orion Pharma, Finland), carprofen (50 mg/ml, Rimadyl, Pfizer, USA), and buprenorphine (0.3 mg/ml, Temgesic, Reckitt Benckiser, Slough, UK) and isoflurane (induction, 5%; maintenance, 1.8–2.5%; Attane, Piramal Healthcare, Bethlehem, PA, USA).

Animals were removed from anaesthesia and, once lucid, treated with subcutaneous non-steroidal anti-inflammatory (NSAID, 0.7 mg/kg, 50 mg/mL every 12 h, Carprofen, Norbrook, Australia) and intramuscular Buprenorphine (Temgesic, 1.0 mL, 324 μg/mL Buprenorphine hydrochloride, Reckitt Benckiser, Australia) for pain relief, and intramuscular Depocillin for antibiosis (1 mL/25 kg every 12 h, Procaine benzylpenicillin, Intervet, Australia). NSAID and antibiotic treatment was continued for 3 days post-operatively, and as required thereafter. Clinical assessment was carried out twice daily to determine animal wellbeing, including urine and faecal output, food and water intake, and signs of apathy. Animals remained in indoor housing for 3 days post-operatively, after which they were returned to protected outdoor pens and housed individually.

The abdominal incision was sutured with 6-0 Prolene, and surgical staples were used to close the skin (427631, Aichele Medico AG). All mice received subcutaneous buprenorphine (0.05 mg/kg Temgesic, Reckitt Benckiser AG, Switzerland) 15 minutes before surgery and −6-8 hours following surgery. During the night, mice received paracetamol (2 mg/ml Dafalgan, UPSA) and buprenorphine (0.009 mg/ml Temgesic, Reckitt Benckiser AG, Switzerland) in the drinking water for 48 hours postoperatively. During the day, mice received subcutaneous buprenorphine injection in the morning and evening for 48 hours postoperatively. Blood samples from the tail vein were taken pre-operatively, and at 3 h and 24 h postoperatively using a capillary tube. Serum was separated by centrifugation (2000g for 20 minutes at 4 °C) and flash-frozen in liquid nitrogen before being stored at −80 °C. On the second- or third-day following surgery for renal IRI and 24 h following surgery for hepatic IRI, mice were euthanized under anesthesia via cervical dislocation, followed by exsanguination, and perfused with PBS. Of note, the surviving mice involved in the survival experiment were euthanized after 8 days. The remaining kidney was collected and cut in half transversally. One half was flash-frozen in liquid nitrogen, and the other was fixed in 10% neutral buffered formalin and paraffin-embedded for histology.

Eleven lop‐eared rabbits of mixed sexes with a mean body weight of 4.29 kg were used in this study. Four implants were inserted on each rabbit's skull bone according to a randomized scheme. Preoperatively, the rabbit skulls were shaved and disinfected with chlorhexidine (5 mg/ml, Pharmacia AB, Stockholm, Sweden). The animals were anaesthetized by intramuscular injection with a mixture of 0.15 ml/kg medetomidine (1 mg/ml Dormitor; Orion Pharma, Sollentuna, Sweden) and 0.35 ml/kg ketamine hydrochloride (50 mg/ml Ketalar; Pfizer AB, Sollentuna, Sweden). At each insertion site 0.5 ml Lidocaine hydrochloride 2% (Xylocaine 10 mg/ml; AstraZeneca AB, Södertälje, Sweden) was used as local anesthetics. Sterile conditions were maintained during surgical procedures. A 3‐cm long incision through the skin and the periosteum was made along the central line on top of the skull. The periosteum was dissected from the bone. Four space titanium blocks were fixed to the skull with 1 titanium screws each (Figure 1). The blocks were covered by periosteum that was closed in position using Vicryl 4‐0 (Ethicon, 2000, Nordstedt, Germany). The skin was closed with continuous stitching. Postoperatively, buprenorphine hydrochloride (0.5 ml Temgesic; Reckitt Benckiser, Slough, UK) was administered as an analgesic for 3 days. No antibiotics were used. The rabbits were kept in single cages.