C57bl 6 strain

Animals were male mice of the C57BL/6 strain (Charles River, L’Arbresle, France). They were housed by groups of 10 in collective cages (425 mm × 276 mm × 153 mm; 820 cm²) until they were 10 months old, in a temperature-controlled colony room (22 ± 1 C), under a 12:12 light–dark cycle (lights on at 7:00 a.m). They were provided with food and water or alcohol ad libitum. All procedures were carried out during the light phase of the cycle. Two weeks before the experiments, they were housed individually (331 mm × 159 × mm 132 mm; 335 cm²).
All experimental procedures were performed between 8:00 and 12:00 a.m. to prevent any circadian rhythm side effects on GC levels and were conducted in accordance with the European Union Directive 2010/63/European Union for animal experiments and local ethical committee (#5012089).

A total of 132 fecal samples were collected from female mice (n = 66) of the C57Bl/6 strain (Charles River, Calco, LC, Italy) and included in the experimental set reported in Figure 1. All procedures for animal housing and maintenance were performed in compliance with previous studies [46 (link),47 (link),48 (link)]. The age of the animals at the start of the study was between 8 and 12 weeks. The mice were monitored daily, and fecal samples were collected every 15 days for the S100B assay and 16S amplicon sequencing analysis. In order to test the effect of Pentamidine (PTM), a S100B inhibitor, as indicated in Figure 2, PTM was administered intraperitoneally (4 mg/kg) to 25 mice (50 fecal samples) vs. 28 untreated controls (as a part of the previous experimental set involving a total of 66 animals), following a previously established protocol [47 (link)]. Additionally, in a pilot test, 300 µL of a solution (phosphate-buffered saline—PBS) containing 200 µg of S100B (Sigma, St. Louis, MO, USA) or of vehicle (phosphate-buffered saline—PBS) was orally administered, respectively, to 3 and 2 C57Bl/6 mice. After 15 days, fecal samples (n = 10) were collected and analyzed for microbiota biodiversity.

Male mice (C57BL6 strain, Charles River, Sulzfeld, Germany), aged 3 (young), 6 (middle-aged) and 13–15 (old) months were used in these experiments. All procedures were carried out in compliance with national regulations on animal welfare and experimentation and European Union Directive 2010/63/EU. Animals were housed in pairs under standard laboratory conditions with ad libitum access to water, unless specifically mentioned. Behavioral tests (see below), each carried out in different batches of animals, were conducted during the animals' daily period of activity (diurnal phase of darkness; lights off: 7 a.m.) after 1 week of habituation to the experimental room and experimenter. In keeping with standard procedures, mice were placed on a calorie-restriction schedule to reduce body weights by 10–15% (body weights monitored daily), unless specifically stated otherwise. Animals that displayed overt signs of pathology (cf. Ladiges et al., 2013 (link)) during autopsy at the end of each experiment were excluded from final analyses; the exact number of animals used in each experiment is given in the Results Section and corresponding figure legends.

Male mice from the C57Bl/6 strain (4–6 weeks old) were used in this study, supplied by Charles River Italia (Calco, Italy). The mice were housed in an environment controlled for light (12 am to 12 pm) and temperature (22 ± 2 °C); food (Mucedola Global Diet 2018; Harlan, Corezzana, Italy) and water were provided for consumption ad libitum. All animal experiments were conducted in conformity with the “Guiding Principles for Research Involving Animals and human Beings” and were approved by the Local Ethics Committee of the University of Siena and the Italian Health Ministry (no. 186/2015-PR).

Cortical neuronal cultures were prepared from dissected embryos at E15, using wild-type naive time pregnant mice of the C57BL/6 strain (Charles River) and left to mature for 2 weeks before experiments conducted. Time pregnant mice were housed overnight in standard conditions and anaesthetized using isoflurane. Procedures were carried out according to the Institutional Animal Care Committee at the Vollum Institute (Oregon Health and Sciences University). A mixture of male and female embryos were used. Whole cortices were removed in HBSS-HEPES buffer, washed 10x and incubated in Trypsin/DNase for 5 mins at 37°C. Tissue was washed 3x before mechanical dissociation using standard pipetting procedure. Cells were centrifuged at 800rpm for 5 mins and resuspended into a single cell solution in MEM with L-glutamine, 10% Horse serum and 10% Fetal Bovine Serum to a density of 0.5 × 10⁶. Cells were plated onto Poly-L-lysine coated coverslips or plates and incubated at 37°C for 24 h. Media was then replaced with Neurobasal media containing B-27 and Glutamax, replenished every 2-3 days for 2 weeks.

All experiments were performed in accordance with guidelines and approvals from the University of Toronto Animal Care Committee and the Canadian Council on Animal Care (University of Toronto Protocol #20012022). Animals of both sexes from wild-type mice, C57/Bl6 strain (Charles River Laboratories) were used throughout. Animals were housed in the Faculty of Arts and Science Biosciences Facility (BSF) in a 12 hr light: 12 hr d cycle, with 2–5 animals/cage.

Caerulein (Sigma-Aldrich) hyperstimulation was induced in both male and female mice of the C57BL/6 strain from Charles River Laboratories, 6- to 8-weeks of age, weighing 20 to 25 g. Caerulein (50 µg/kg) was given by 8 hourly intra-peritoneal injections for two consecutive days. Day 0 was defined as 24 hours after the last injection. Pancreas tissue was collected from the head of the pancreas (less than a 5 mm portion of the pancreas immediately adjacent to the duodenum⁶⁴ ) at baseline (non-injured state), day 3 (injury), and at days 7 and 14 post-injury. Upon resection, the tissue was immediately partitioned for RNA extraction and tissue fixation. All mice were maintained on a 12-hour light-dark cycle and had free access to standard laboratory chow and water. All methods and animal experiments were performed in accordance with guidelines approved by the University of Pittsburgh School of Medicine and an animal protocol approved by the University of Pittsburgh Institutional Animal Care and Use Committee. All experiments were performed in accordance with relevant guidelines and regulations.