Sodium pentobarbital

Large White Landrace Cross gilts (n = 6; 98 ± 7 days GA; term = 115 days) were anaesthetised with an intramuscular injection (I.M.) of 20 mg/kg ketamine and inhalation of isoflurane. Gilts were intubated and general anaesthesia was maintained using isoflurane with 2 L/min O₂ and 4 L/min medical air. Gilts were positioned on the operating table on their backs, an incision was made along the abdomen, the uterus was incised, and a fetal head was exposed. Fetal pigs (n = 24) were cannulated via the UV, and venous blood was sampled for partial pressure of oxygen (PO₂), partial pressure of carbon dioxide (PCO₂), oxygen saturation (SO₂), pH, hemoglobin (Hb), bicarbonate (HCO₃⁻), base excess (BE), sodium (Na⁺), potassium (K⁺), and calcium (Ca²⁺) as previously described (Charest-Pekeski et al., 2021 (link); Darby et al., 2021 (link)). In a subset of fetuses (n = 21, 105 ± 7 days GA), the carotid artery (CA) was instrumented and fetal BP, and HR were measured and continuously recorded in LabChart 8 Pro (ADInstruments Inc., Colorado Springs, United States) (Charest-Pekeski et al., 2021 (link)). Following in utero experiments gilts and their fetal pigs were humanely euthanized with an intravenous overdose of sodium pentobarbital (Virbac, New South Wales, Australia).

All tissue collection was carried out under RNase-free conditions. At the time of sacrifice, rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.; Virbac Animal Health), and then tissue from peripheral organs (liver, lung, spleen, and skeletal muscle), the cerebellum, and brainstem (BS) was collected from each animal, pooled within group into a 1.5 ml RNase-free tube, and snap frozen on dry ice. The rest of the brain from each animal was frozen on dry ice and stored at −80°C. Frozen brains were then individually mounted in a cryostat, warmed to −4°C and 500 µm coronal sections were cut. Tissue samples from the preoptic area (POA), amygdala (Amyg), bed nucleus of the stria terminalis (BNST), dorsal hippocampus (dHipp), and cingulate cortex (CC) were punch dissected from these coronal sections using either 12 ga (2.4 mm, ID) or 15 ga (1.6 mm, ID) punches, and the tissue from the same region within each group was pooled into 1.5 ml RNase-free tubes. The tubes first were kept on dry ice to prevent thawing of the tissue and subsequently were stored at −80°C for later analysis.

All experiments were approved by the Animal Ethics Committee of Macquarie University and conducted in accordance with the Australian code for the care and use of animals for scientific purposes (8th Edition). The study was conducted and reported according to the ARRIVE guidelines. Lewis and LPK rats of both sexes (n = 33 male Lewis, n = 22 female Lewis, n = 32 male LPK, n = 29 female LPK, total = 116) were obtained from the Animal Resource Centre, Perth, Australia and housed at the Central Animal Facility of Macquarie University on a 12-h light–dark cycle (lights on 6 am), at 22 ± 2 °C with access to food and water  ad libitum. At the end of the study, animals were euthanised with an i.p. injection of 20% (v/v) solution of sodium pentobarbital (100 mg/kg, Virbac, NSW, Australia) prior to the collection of blood and tissues.

Large White piglets were obtained from The University of Queensland Gatton Piggery. Approval for this study was granted by The University of Queensland Animal Ethics Committee (MED/UQCCR/132/16/RBWH) and was carried out in accordance with National Health and Medical Research Council (NHMRC) guidelines (Australia) and ARRIVE guidelines.
Term piglets were born spontaneously and collected on first day of life (postnatal day 1 (P1); < 18 h). IUGR piglets were defined by birth bodyweight (< 10th percentile) [35 (link)–37 (link)]. Litter-matched pairs were obtained from multiple sows (n = 11). The IUGR piglet model is well-established and characterised by our group and others [11 (link), 33 (link)]. This model is caused by placental insufficiency [33 (link)]; the most common cause of IUGR in the human population. On either P1 (NG n = 6; IUGR n = 6) or P4 (NG n = 6; IUGR n = 6) (equal males and females in each group), piglets were euthanased via an intracardiac injection of sodium pentobarbital (650 mg/kg; Lethabarb, Virbac, Australia). The brain was immediately removed, weighed, hemisected and coronally sliced. The right hemisphere sections were immersion fixed in 4% paraformaldehyde as previously described [38 (link)]. The parietal cortex from the left hemisphere was snap frozen in liquid nitrogen and stored at − 80 °C for molecular studies.

Mice were killed by an overdose of sodium pentobarbital (240 mg/kg, Virbac, Australia) and lung lobes from saline or HDM challenged mice were dissected and finely minced in a petri dish. Lung cells were dissociated in 4 ml of Liberase^™ (Sigma-Aldrich, United States) at 37°C. Following removal of residual tissue debris and erythrocytes, single cell suspensions were stained with an antibody cocktail including Anti-CD45-FITC, Anti-F4/80-APC, Anti-CD11b-eFluor450, and anti-CD11c-PE-Cy7 (Thermo Fisher Scientific, United States). Alveolar macrophages were sorted using the FACSAria^™ Fusion (BD Biosciences, United States) under a strict gating strategy. Briefly, dead cells, cellular debris, and cell doublets were excluded using PI viability dye, side scatter (SSC), and forward scatter (FCS) gates. From this population of live singlets, haemopoietic cells were selected based on high CD45⁺ expression. All F4/80- cells were then excluded from the analysis, and alveolar macrophages were differentiated based upon CD11c^high and CD11b^low expression. Freshly isolated alveolar macrophages were used for mRNA extraction.