Isoflurane

To investigate the impact of pressure overload-induced cardiac hypertrophy, transverse aortic constriction (TAC) surgery was performed in mice as described previously (17 (link), 18 (link)). Briefly, mice were anesthetized with 2% isoflurane (Baxter Healthcare Corporation, New Providence, NJ, USA) and then maintained on 1.5% isoflurane. Mice were ventilated by tracheal intubation using a rodent ventilator (Alcbio Corporation, Shanghai, China) with a respiratory rate of 120 breaths/min and a tidal volume of 0.3 mL. Then, the chest was opened at the suprasternal fossa along the midsternal line, and the thymus glands were superiorly reflected. The transverse thoracic aorta between the innominate artery and the left common carotid artery was dissected, and a 6-0 silk suture was tied around the aorta, which was pressed against a 26-gauge needle. Then, the needle was removed. A sham operation involving thoracotomy and aortic dissection without constriction of the aorta was performed in both the WT and SKO sham operation groups.

Pre-vascularized dermo-epidermal skin substitutes were transplanted onto full-thickness skin defects created surgically on the back of 10-week-old female, athymic Nu/Nu immune-compromised rats (Envigo, Horst, The Netherlands) (n = 3). Animals were anesthetized by inhalation of 5% Isoflurane (Baxter, Volketswil, Switzerland) and maintained by inhalation of 2.5% Isoflurane via mask. Full-thickness skin wounds were created in the middle of the back of the animals with a surgical scissor. A custom-made steel ring (diameter 26 mm) was then placed into the created full-thickness skin defect and the skin was sutured using non-absorbable polyester sutures (Ethibond®, Ethicon, Raritan, NJ, USA), on the outside of the ring to prevent wound closure by the rat skin. The skin substitutes were placed into the metal ring onto the animal tissue. The transplants were then subsequentially covered with a silicone foil (Silon-SES, BMS, New York, NY, USA), a polyurethane sponge (Ligasano, Ligamed, Innsbruck, Austria), a cohesive conforming bandage (Sincohaft, Theo Frey AG, Bern, Switzerland), and tape as a wound dressing. After 1 week animals were sacrificed and transplants were excised and processed for histological and immunofluorescence analysis.

We followed a previously described protocol to sedate adult zebrafish for ECG recording^{3 (link)}. Tricaine (Sigma, USA) was dissolved in distilled water to a final concentration of 2,000 parts-per-million (ppm) as a stock, and the pH value was adjusted to 7.2 with sodium hydroxide (Sigma-Aldrich). Isoflurane (Baxter, USA) was dissolved in absolute ethanol to create a stock solution of 100,000 ppm (Isoflurane: ethanol = 1:9) and maintained in a brown glass bottle. Stocks were all stored at 4 °C. For combined use of Tricaine and Isoflurane, each stock solution was added to the fish tank to the prescribed final concentration immediately before use.

Experimental laparotomy was performed as previously described with minor modifications [23 (link)]. Briefly, mice were anesthetized with 2.0% isoflurane (Baxter Healthcare, Puerto Rico, USA) for 2 min, and anesthesia was maintained with 1.4% isoflurane. Mice were placed on a heating pad to maintain body temperature between 36.5–37.0 °C. After the hair was shaved and the surgical field was disinfected, an approximately 1-cm median incision was made 0.5–1 cm below the xiphoid, and approximately 5 cm of the small intestine was gently pulled out and then exposed to sterile gauze presoaked with normal saline. After being rubbed for 10 min, the intestine was returned to the abdomen. The muscle and skin were closed layer-by-layer with 5–0 absorbable sutures (Polysorb, COVIDIEN, USA). Finally, 0.1 ml 0.2% lidocaine was injected subcutaneously for postoperative analgesia. The mice were allowed to recover in an incubator at 35 °C for 30 min before being returned to their home cages.

At the end of the experimental period, all the mice were anesthetized with isoflurane (Baxter, United States) isoflurane was administered in a mixture with oxygen at a constant flow of 0.5–0.7 L/min. The animals were placed in the induction chamber, a plastic container of 20 cm in diameter and 10 cm in height, connected with a polyurethane polyether tube to let in the anesthetic gas mixture. Induction of the anesthesia was achieved by using 4% vaporized isoflurane (Laboratorios Esteve S.A., Barcelona, Spain) in oxygen.
After sacrificed with a 12-h fast, blood samples were collected from the inferior vena cava into a heparin-coated tube for the measurement of plasma parameters. The blood was centrifuged at 1000× g for 15 min at 4 °C, and the plasma was separated. After blood collection, epididymal white adipose tissue (WAT), perirenal WAT, retroperitoneal WAT, mesentery WAT, subcutaneous WAT, and liver were promptly removed, rinsed with physiological saline, and weighed. Among them, epididymal WAT and liver were immediately frozen in liquid nitrogen and stored at −70 °C until the analyses of the enzyme activity and RNA.