Isoflurane

Surgery was performed under anesthesia inhalation (1–2% Isoflurane, CP-Pharma, and 100 mL min⁻¹ carbogen) and 1 mg kg⁻¹ body weight p.o. Meloxicam (0.5 mg mL⁻¹ suspension, CP-Pharma) was given 1 h before surgery for pain relief. A midline incision was used to open the abdomen and expose the bile duct and liver. After the surgical procedure (details for different models are given below), the abdominal layers were closed with 4-0 antibacterial sutures (Ethicon) and Bupivacaine (2–4 mg kg⁻¹ body weight, PUREN Pharma) was administered intra-incisional for topical anesthesia. In addition, the animals received Ringer acetate (20 mL kg⁻¹, Berlin Chemie AG) subcutaneously for fluid resuscitation and were offered an additional heat source (warm lamp) during the recovery phase. Regular and soaked food was available on the ground for the animals at all times after surgery. The animals were scored and weighed a minimum of twice daily. The scores were designed to reflect post-surgical conditions and specific symptoms of surgical intervention. Supplementary Table S3 provides additional information on the model regarding the change in body weight, ALAT, and ASAT can be found in.

At PND14 juvenile F1 mice were administered PBS (Control group; Lonza, Basel, Switzerland) or influenza A virus (IAV group). For intranasal (i.n.) inoculation, the mice were lightly anaesthetized by inhaling Isoflurane (cp-pharma), and 25 µL virus suspension (1500 FFU IAV in PBS) or PBS were administered through droplets into the nose of the animals which were subsequently scored and weighed daily for 14 days. After weaning, the mice were kept in same-sex and same-treatment cages to avoid cross-contamination. On PND56 animals underwent a hetero- or homotypical challenge with either PBS or IAV and subsequently scored until sacrifice (Supplementary Data 4). The animals were sacrificed on PND60/61 by cervical dislocation. Immeadiatly after sacrifice, whole blood was collected by cardiac puncture as well as bronchoalveolar lavage (BAL) fluid by inserting a flexible tube through a cut in the trachea and washing the lungs with 700μL PBS. Following this complete brains, thymus, lungs, adrenal glands, and the distal tip of the left lobe of the liver were harvested and stored at –80°C before analysis.

Equines were sedated with 0.04 mg/kg BW romifidine (Sedivet, Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim am Rhein, Germany) and 0.03 mg/kg BW butorphanol (Alvegesic, CP-Pharma Handelsgesellschaft GmbH, Burgdorf, Germany). General anaesthesia was induced with 0.08 mg/kg BW diazepam (Ziapam, Laboratoire TVM, Lempdes, France) and 3 mg/kg BW ketamine (Ursotamin, Serumwerk Bernburg AG, Bernburg, Germany). Animals were orotracheally intubated, and anaesthesia was maintained with isoflurane (CP-Pharma). The rp and mc AT (~ 5 g at each depot) samples were collected in dorsal recumbency 15 ± 1 h after LPS infusion. Liver tissue was also sampled for another part of this study at the same time. Both sc AT samples (~ 5 g at each depot) were taken in lateral recumbency 15.5 ± 1 h after LPS infusion. In subsequent evaluations, to avoid scar tissue, sc AT was collected on the other body side (t1) or at least with 5 cm distance from the first sampling point (t2). Each biopsy specimen was immediately flash-frozen in liquid nitrogen (− 196 °C) and stored at − 80 °C until analysis. After surgery, all animals were treated orally with 0.55 mg/kg BW flunixin (Flunidol, CP-Pharma) twice a day for 3 days to reduce post-surgical pain and were assessed for pain on base of a modified pain score [31 (link)].

Adult male Sprague–Dawley rats and adult male C57BL/6J mice (both from the Department for Laboratory Animal Science and Genetics, Himberg, Austria), TRPA1^−/− and TRPV1^−/− mice were used. Heterozygous TRPV1^+/− mice were a generous gift from John B. Davis [45 (link)], and TRPA1^+/− animals from David P. Corey [46 (link)]. Animals were back-crossed on C57BL/6J for more than ten generations. Breeding, euthanasia, and all procedures of animal handling were performed according to regulations of animal care and welfare. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). For in vitro experiments mice were euthanized by cervical dislocation, preceded by anaesthesia by exposure to isoflurane (CP Pharma, Burgdorf, Germany). Animals were housed with a 12:12 h light-dark cycle and access to food and water ad libitum.

All animal studies (Government of Upper Bavaria; no. 55.2-1-54-2531-25-12), experiments, and procedures were reviewed and approved by the Oberbayern Animal Welfare Committee in Munich, Germany. We confirm that all experiments were performed in accordance with relevant guidelines and regulations.
Female BALB/c mice, with a weight of 18.8–26.4 grams, were delivered from Charles River Laboratory (Sulzfeld, Germany) and allowed to adapt for 1 week before the start of the experiments at the age of 40 weeks. The mice were kept under standard housing conditions.
For orbital preparations, mice were anesthetized and euthanized with isoflurane (CP-Pharma) at a concentration of 1.5–2%. Complete dissection of the orbital and periorbital areas was performed, as described previously [42 (link)]. In brief, the heads of the animals were dissected and the skin, the connective tissue around, the brain, and the teeth were removed but left all orbital tissues, eyelids, and adjacent tissues intact. Trimmings were made with coronary cuttings at the positions of Bregma +1.95 and +5.85 mm. Tissue blocks were fixed in 4% paraformaldehyde at 4°C temperature overnight. Decalcification in 15% EDTA (w/v) (ethylenediaminetetraacetic acid) was carried out for 21 days with three times of changing solution, each for one week.

We used five independent experimental groups containing randomly selected animals: (1) MH-sC: mice received magnetic hyperthermia (MH) following intratumoral application of MNPs (RCL-01, 1 mg Fe per 100 mm³ tumor volume) and weekly systemic chemotherapy (50 mg gemcitabine, i.p., 30 mg nab-paclitaxel, i.v., per kg body weight) for 7 weeks; (2) MH: mice treated with magnetic hyperthermia only; (3) sC: mice treated for 7 weeks with systemic chemotherapy only; (4) M: mice injected with MNPs only; (5) N: non-treated controls (no MNPs, MH, or sC). There were 5 mice per group (Table 1).
After orthotopic implantation into the pancreas, tumor volume measurements were conducted via ultrasound imaging. All tumor volume measurements were normalized to the respective tumor volume at experimental day 0 (Figure 1). Whole-body near-infrared optical and CT imaging (IVIS^® Spectrum CT, PerkinElmer, Inc., Hopkinton, MA, USA) was performed on day 0. The tumor treatment parameters are shown in Table 2. Finally, animals were sacrificed on the post-observation day 30. During all experimental procedures, animals were anesthetized with isoflurane (2–2.5 (v/v)%, CP-Pharma, Burgdorf, Germany).