Fentanyl

Between 08.00 and 10.00 h, unfasted pregnant dams (n = 10 per diet, per age) were weighed and anaesthetized (10 μl g⁻¹ fentanyl‐fluanisone:midazolam in sterile water, 1:1:2; i.p.; Janssen Animal Health, Beerse, Belgium). A cardiac blood sample was taken before death by cervical dislocation. Liver and retroperitoneal white adipose tissue (WAT) were dissected, weighed and snap frozen in liquid nitrogen for western blotting. Maternal skeletal muscle (biceps femoris) was also dissected and snap frozen. Blood glucose concentrations were measured using a hand‐held glucometer (One Touch Ultra; LifeScan, Johnson & Johnson Medical Ltd, Livingston, UK). At D19, the maternal liver was also fixed in 4% paraformaldehyde, dehydrated and embedded into paraffin wax before sectioning at 8 μm and staining with haematoxylin and eosin. Tissues and blood taken from dams in the previously published cohort (Sferruzzi‐Perri et al. 2013), contributed to the samples used for biometrical and biochemical analyses on the mother and placental gene expression.

Three Cftr mutant and three WT mice (female, aged 10 - 12 weeks, body weight 17.7 - 20.8 g) were used. There were no significant differences in age or body weight between mutant and WT mice. For particle inhalation, animals were deeply anaesthetized by intraperitoneal (i.p.) injection of a mixture of medetomidine (Domitor®, Pfizer GmbH, Karlsruhe, Germany; 500 μg/Kg), midazolam (Dormicum®, Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany; 5 mg/Kg) and Fentanyl (Fentanyl®, Janssen-Cilag GmbH, Neuss, Germany; 50 μg/Kg). For lung fixation immediately after inhalation, anesthesia was deepened with i.p. injection of a mixture of xylazin (Rompun®, Bayer Vital GmbH, Leverkusen; 5 mg/Kg) and ketamine (Ketamin 10%, WDT eG, Garbsen, Germany; 100 mg/Kg) prior to exsanguination by cutting the abdominal aorta. For 24-h examinations, anesthesia was antagonized by subcutaneous injection of atipamezole (Antisedan®, Pfizer GmbH Karlsruhe, Germany; 2.5 mg/kg), flumazenil (Anexate®, Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany; 500 μg/Kg), and naloxone (Narcanti®, Janssen Animal Health, Neuss, Germany; 1.2 mg/Kg). For lung fixation at 24 h, a mixture of xylazin and ketamine was injected prior to euthanasia by exsanguination as above.

All experiments were performed in accordance with the German legislation governing animal studies. Official permission was granted from the governmental animal care office (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany). Female German landrace pigs from a disease-free barrier breeding facility were housed in ventilated rooms and allowed to acclimatize to their surroundings for a minimum of 5 days before surgery. The animals, weighing around 60 kg, were fasted 12 hrs prior to the experiments. For premedication, the animals received an intramuscular injection of 4 mg kg⁻¹ azaperone (Stresnil, Janssen, Germany). Anesthesia was induced by intravenous injection of 3 mg kg⁻¹ propofol followed by oral intubation. The animals were ventilated with 40% oxygen at 20–26 bpm and a tidal volume of 10 mL kg⁻¹ to keep the end tidal partial carbon dioxide tension (pCO2) between 36 and 42 mmHg. Anesthesia was maintained with isoflurane at a concentration of 1%–1.5% (Forane, Abbott, Germany) and fentanyl (fentanyl, Janssen, Germany) at a concentration of 3-4 mg kg⁻¹. To compensate for basic fluid requirements volume, animals received Ringer's lactate (RL) solution at a rate of 4 mL kg⁻¹; after laparotomy, the constant infusion rate was set to 8 ml kg⁻¹ and not changed until infliction of trauma.