Isothesia

The Tasmanian devils were housed in secured shelters in accordance with the guidelines of the Department of Primary Industries, Parks, Water and Environment of Tasmania (DPIPWE). All animal procedures were approved by the University of Tasmania Animal Ethics Committee under A009215, A0011436 and A0013685. All devils (6 females and 3 males) were adults with ages ranging from 5 to 7 years at commencement of experiments (Supplementary Table S5). All experimental methods were performed in accordance with the University of Tasmania guidelines.
Immunisation, blood collection, live cell challenge and therapy were performed under anaesthesia induced with 5% Isoflurane reducing to 3% via a mask (ISOTHESIA^® (Henry Schein, Northgate, Australia). 10 ml of blood was obtained from the jugular vein as previously described27 (link). Up to 4 ml of blood was placed into clot activating tubes (Greiner Bio-one) for serum analysis and the remainder into lithium heparin anticoagulant tubes for cell analyses. Tumour biopsies were collected using sterile 4 mm disposable biopsy punches (Kai Medical). Biopsies were divided with a scalpel blade and half placed into 1 ml 10% neutral buffered formalin and half into 1 ml RNAlater.

Animals were sedated with 10 mg/kg ketamine hydrochloride (Ketalar^® Parke-Davis, Detroit, Michigan) injected intramuscularly. Anesthesia was induced by isoflurane (Isothesia, Henry Schein, Melville, NY) at 5% initially and then maintained by using a gas flow rate of 1% to 3%. Vital signs were monitored with monitoring equipment (Dre Medical International, Louisville, KY). A liver biopsy was performed aseptically once on two animals prior to hepatitis E virus inoculation to observe baseline liver tissue for host response induced by hepatitis E virus infection using a one-port incisional abdominal entry method with a laparoscope (Biovision Veterinary Endoscopy, LLC., Denver, CO). The liver was visualized with the laparoscope, and a biopsy sample was obtained from a liver lobe with the laparoscopic biopsy instrument. Hemorrhage was assessed and controlled appropriately with applied pressure from biopsy instrument, bipolar cautery, or with sterile absorbable compressed sponge (Gelfoam^® Pfizer, New York, New York). Abdominal incision for port entry was closed with 3–0 polyglactin 910 (Ethicon, Somerville, NJ) and skin apposition with surgical glue (SutureVet VetClose, Henry Schein, Melville, NY) reinforced with simple interrupted sutures of 3–0 nylon (Ethicon, Somerville, NJ).

Osmotic minipumps (ALZET®, Model 1002, Charles River) were implanted subcutaneously into pregnant or non-pregnant mice to chronically administer test agents. Surgical implantation of osmotic minipumps was carried out on day 7 of pregnancy (or equivalent time interval for non-pregnant mice) under isoflurane anaesthesia (Isothesia®, Henry Schein®). Minipumps were loaded with physiological saline, non-specific CRHR antagonist (α-helical CRF_9–41, 1 mg/ml, Tocris) or receptor-specific CRHR antagonists, antalarmin hydrochloride (1 mg/mL, Tocris) or antisauvagine-30 (3 mg/mL, Tocris) for CRHR1 and R2, respectively. Test agents were delivered at a rate of 0.25 µL/h for a total period of 11 days. Assessment of glucose tolerance and insulin tolerance were conducted on gestational days 16 and 18, respectively.

C57BL/6J mice were used to study the tumor metastasis, the mice were anesthetized by Isothesia (HENRY SCHEIN, Cat#NDC11695-6776-2), and 5 × 10⁴ B16-F10 cells stable transfected with sh-Scramble or sh-Arf1 were injected into tail vein of each mouse. After 15 days, the mice were euthanized with CO₂ for 30 min and the metastasis tumors in different organs were analyzed .

Hamsters were infected intranasally by instilling 3 × 10¹⁰ plaque forming units (PFU) per kg of HAdV-C6 into the nostrils of the animals in 100 μL PBS under isoflurane (Isothesia, Henry Schein Animal Health, Dublin, OH, USA). Control animals were administered PBS. Hamsters were observed and weighed daily; moribund animals were sacrificed as needed. At necropsy, lungs were collected and the infectious virus burden was determined using a 50% Tissue Culture Infectious Dose (TCID₅₀) assay as described previously [14 (link)].