Sevorane

Ten-week-old female C57BL/6NCrl mice (mass 18–22 g) were purchased from Charles River (Sulzfeld, Germany). Animals were kept on a 12-h day/night rhythm and fed with a phytoestrogen-reduced mouse diet (ssniff, Soest, Germany).
Prior to (surgical intervention) surgery, animals received a subcutaneous application of carprofen (Rimadyl®, Pfizer, Berlin, Germany) (5 mg/kg body mass). Animals were anaesthetized under constant sevoflurane inhalation (Sevorane®, Abbott, Wiesbaden, Germany). After median laparotomy, the hilum of the liver was exposed to access the portal vein. One million tumour cells in a volume of 100 μl PBS buffer were injected slowly into the portal vein using a 30 G needle.
In the study group, seven animals were implanted with tumour cells. The control group encompassed five animals which underwent the same procedures (sham-OP), but were only injected with buffer solution. All animals were sacrificed after 4 weeks. Explanted livers were sliced for macroscopic assessment, photographic documentation of the section planes, and further processing. Tissue samples from the tumour core of the liver metastases derived from CMT-93 as well as matched unharmed liver tissue (macroscopically tumour-free liver) were excised, snap frozen for whole transcriptome sequencing analysis (RNA-seq), or frozen in 2-methylbutane at −70 °C for immunolabelling.

Acute arthritis was induced by the injection of a suspension of 0.3 mg sterile CPP crystals (InvivoGen, Aurogene, Italy) in 20 μL PBS into the right ankle joint of the mice. Injections were performed under inhalant anesthesia (Sevorane^®, Abbott, 4% induction, 1.5% maintenance) using a Fluovac respiratory system (Harvard Apparatus, Holliston, MA, USA) and microliter syringes #705 (Hamilton, Reno, USA) with 27 G beveled needles. Animals were randomized into 4 groups (n = 8 per group) receiving: (1) i.a. CPP crystals, (2) i.a. CPP crystals + PD, (3) i.a. CPP crystals + colchicine (control drug), (4) i.a. PBS (control group).
Ankle swelling was measured at different time points using a precision digital caliper (Kroeplin Gmbh, Schlüchtern, Germany). To avoid any attribution bias, ankle swelling was measured by an investigator who was blinded to the group allocation. Forty-eight hours after the injection of CPP crystals (peak of the acute phase) (preliminary experiments) mice were euthanized, and peripheral blood and ankle joints were collected for inflammatory cytokine assessment and histological analysis, respectively.

Intubation was performed in both groups by administering 2–2.5 mg/kg propofol (Lipuro, Braun), 1–1.5 μg/kg fentanyl (Talinat, VEM), and 0.6 mg/kg rocuronium (Esmeron, Alessandroorsini) intravenously to the patients in both groups. Anesthesia was maintained with sevoflurane (Sevorane, Abbott) in a mixture of O₂/air in a 50/50% concentration and remifentanil (Ultiva, VLD) infusion (0.01–0.1 μg/kg/min). The mechanical-ventilator settings were adjusted to provide a 6–8 mL/kg tidal volume and a 30–35 mmHg end-tidal CO₂ level. If the pulse or mean arterial blood pressure increased by 20% from the preoperative baseline value, 25 μg bolus fentanyl and 0.1 mg/kg rocuronium were administered intravenously. Arthroscopic shoulder surgery was performed by the same surgical team using the same technique, in the beach chair position. To prevent nausea and vomiting, 4 mg ondansetron was administered intravenously. After extubation, the patients with sufficient spontaneous respiration were taken to the recovery room.

The C57BL/6 mice were anesthetized with sevoflurane (Sevorane®, Abbott) and euthanized by terminal anesthesia with CO₂. Washing of the peritoneal cavity was performed through a small incision in the cavity where 5 mL of RPMI-1640 (Sigma) was injected at 4 °C with the aid of a 24G needle and 5-mL syringe. After homogenizing the liquid inside the animal’s peritoneal cavity, the injected RPMI-1640 (Sigma) was collected with the aid of a syringe and needle into a 15-mL tube and placed on ice (4 °C), to prevent cell adhesion to the tube. Shortly afterwards, the recovered volume was centrifuged for 5 min at 1500 rpm, 4 °C. The pellet was resuspended in 5 mL of RPMI-1640 (Sigma) without SFB, and the total cell count was obtained in a Neubauer chamber. 5 × 10⁵ macrophages were plated in each well of the 24-well plate and, after 1 h (necessary for the cells to adhere to the plate surface), the RPMI without FBS was removed, the cells were washed 3 times with PBS at 37 °C, and 500 μL of RPMI-1640 (Sigma) with 10% FBS (Invitrogen) was added to each well. After 24 h, wells were washed with PBS at 37 °C to purify the culture, removing the B1 lymphocytes and leaving only the macrophages.