Fisher 344 rats

All the animal procedures were approved by the Institutional Animal Care and Use Committee of Dartmouth Medical School. Fourteen male Fisher 344 rats, 200–250 g (Charles River Laboratories, Wilmington, MA) were used and divided into two groups: (i) Skeletal muscle group, N = 8; (ii) 9L tumor group, N = 6.

MSCs were harvested from rat femora and tibiae of 6–8 week old Fisher 344 rats (Charles River Laboratories, Wilmington, MA) in accordance to approved protocols by the Rice Institutional Animal Care and Use Committee as previously described [10 (link)]. The MSCs were plated in 75 cm² tissue culture flasks at 37°C under humidified, 5% CO₂ atmosphere and cultured in osteogenic media without dexamethasone, which was replaced every 2–3 days.

A total of 115 male Fisher 344 rats (Charles River), ranging from 7 to 10 weeks of age were used in this study (n=46 for behavioral experiments, n=49 for imaging experiments, n=20 for gene expression experiments). Fischer 344 rats were used as evidence suggests they show an increased immunological response to inflammatory challenges - likely due to a hyperactive hypothalamic–pituitary–adrenal (HPA) axis - compared to other rat stains (Taylor et al., 2005 (link), Wu and Wang, 2010 (link)). Rats were singly housed, maintained in a pathogen-free facility on a 12-hr light/dark cycle, and had ad libitum access to regular chow and water. All experiments, with the exception of the sucrose preference test (conducted in the first 3hrs of the dark phase), were performed during the light phase. E. Coli lipopolysaccharide (LPS, Sigma Aldrich, batch 055:B5) was injected intraperitoneally (i.p.) at a dose of 0.83mg/kg (0.25mg/ml) in experimental (LPS) rats, while control animals received a similar volume of saline. This dose was chosen because it is sufficient to induce upregulation of gene expression and depressive-like behaviors in rodents (e.g., Jiang et al., 2017 (link), Sens et al., 2017 (link)).

For all experiments and procedures, anesthesia was induced with 0.5 mL/kg IP injection of a mixture of ketamine, 50 mg/kg (Ketaject; Phoenix Pharmaceutical, Inc, St. Joseph, Missouri), and xylazine, 5 mg/kg (Bayer Animal Health, Shawnee Mission, Kansas). Animals were sacrificed with an overdose of ketamine and xylazine—1.5 mL/kg. Experiments were performed in R3230 mammary adenocarcinoma tumors implanted in female Fisher 344 rats (150 g ± 20; 14–16 wk old; Charles River Laboratories, Wilmington, Massachusetts) (12 (link)). Tumor implantation, evaluation, and preparation techniques were performed as previously described (12 (link)). Briefly, one tumor was implanted into each animal by slowly injecting 0.3–0.4 mL of tumor suspension into the mammary fat pad of each animal via an 18-gauge needle. Tumors used were 1.3–1.5 cm solid nonnecrotic tumors. For experiments performed in normal liver, similarly sized female Fisher rats were used. Briefly, after achieving deep anesthesia, the rat’s abdomen was shaved and prepared using povidoneiodine (Betadine; Carefusion, San Diego, California) and alcohol. A minilaparotomy was achieved by a 1.5-cm subcostal incision exposing the right lobe of the liver. After therapy administration, the abdomen was closed in layers using interrupted sutures.