Zoletil

Experiments with mice (C57Bl/6J strain, The Jackson Laboratory, Bar Harbor, MN) were approved by the Animal Care and Use Committee (Inst. Molecular Genetics, ASCR) in accordance with the European Union Directive 2010/63/EU for animal experiments, U.K. Animals (Scientific Procedures) Act, 1986, and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and the ARRIVE guidelines. Mice were anesthetized using a mixed solution of Zoletil (40 mg/kg, Virbac SA, Carros, France) and 2% Rometar (10 mg/kg, Spofa, Czech Republic). Pancreases were perfused with collagenase IX (Sigma-Aldrich) solution in HBSS buffer and trimmed with surgical scissors. The pancreases were subsequently digested with collagenase for 10 min at 37°C. To remove exogenous tissue, samples were washed with HBSS two times. The tissue was then filtered through a 500 μm cell strainer, and islets were separated on a Ficoll gradient (Sigma-Aldrich) by centrifugation. The islets were placed in CMRL medium (PAN-Biotech, Aidenbach, Germany) and kept overnight at 37°C. The next day, the islets were seeded on wells coated with Biolaminin (BioLamina, Sundyberg, Sweden) and incubated overnight at 37°C. Experiments were carried out on the third day.

Mice (CTL, n = 5; M, n = 3; KA, n = 5; KA + M, n = 5) were anesthetized with zoletil (5 mg/kg; Virbac Laboratories, Carros, France), perfused transcardially with heparinized saline, and their brains were fixed using 4 % paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS). For regular immunostaining, the brains were post-fixed for 6 h in 4 % PFA fixative and then sequentially immersed in 0.1 M PBS containing 15 % sucrose and PBS containing 30 % sucrose at 4 °C until the tissues sank to the bottom of the container. After being frozen in OCT compound (Sakura Finetek USA Inc., Torrance, CA, USA), the brains were cut into 40-µm thick coronal sections. The sections were stained with cresyl violet, and visualized with a BX51 microscope (Olympus, Tokyo, Japan). Digital images were captured.

IT injection was performed as previously described (3 (link), 4 (link)). The animals were anesthetized with 33 mg/kg zoletil (Virbac, Westlake, TX, USA) and 8 mg/kg xylazine (Bayer AG, Leverkusen, Germany). The tympanic membrane was evaluated using a surgical microscope (Opmi Pico, Zeiss, Oberkochen, Germany). An air hole was made in the anterior inferior quadrant of the tympanic membrane with a 24-gauge intravenous catheter needle (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). An average of 51.4 ± 10.9 μl of drug/vehicle was injected into the middle ear (bulla) through the posterior inferior quadrant of the tympanic membrane.
The quality of drug/vehicle injection was classified according to the following three grades: poor, the middle ear cavity filled with ≤ 10 μl of drug/vehicle; fair, more than half of the middle ear cavity filled; and good, the middle ear cavity completely filled without air bubbles. Overall, we achieved good or fair injections in all animals in this study. The time interval between the first injection in the first ear and the second injection in the other ear was not more than 2 min. After injecting the drug/vehicle, the rats were maintained in a prone position without any tilting until the end of the experiment.

The study was approved by our Institutional Animal Care and Use Committee and all experiments were conducted in accordance with institutional guidelines. A total of 16 domestic male pigs (mean weight, 65 kg; range 60–70 kg) were used in our in vivo studies.
Each animal was sedated with an intramuscular injection of zolazepam (5 mg/kg, Zoletil; Virbac, Carroscedex, France) and xylazine (10 mg/ kg, Rompun; Bayer-Schering Pharma, Berlin, Germany), and the animals were intubated and ventilated during the procedures. Anesthesia was maintained by the inhalation of 1%-4% isofluorane (IsoFlo®; Abbott Laboratories, North Chicago, IL) in pure oxygen gas with mechanical ventilation. The pigs were placed in the supine position and a midline incision was made after sterile draping. One of the authors (W.C., with five years of experience in the RFA procedure and experiments) performed the ablation procedures through the midline incision under the guidance of ultrasonography (6–12 MHz linear transducer; Accuvix XQ; Medison, Seoul, Republic of Korea). Two to four ablation zones were generated in the liver of each animal; however, only one ablation was performed in each lobe of the liver. Therefore, a total of 56 ablation zones were created in 16 pigs. The animals’ vital signs, including pulse rate, electrocardiogram, and temperatures, were carefully monitored during the entire procedure.

Male C57BL/6 mice aged 8 weeks were purchased from The Orient Bio (Sungnam, Korea). All animal care procedures and experiments were conducted according to the guidelines stated in the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Ophthalmic and Vision Research. The Internal Review Board approved the study for Animal Experiments at the Asan Institute for Life Science (University of Ulsan, College of Medicine). Mice were anesthetized by an intraperitoneal injection of a mixture of Zoletil (40 mg·kg⁻¹ zolazepam and tiletamine) purchased from Virbac (Carros Cedex, France) and Rompun (5 mg·kg⁻¹ xylazine) purchased from Bayer Healthcare (Leverkusen, Germany), and their pupils were dilated using Mydrin‐P (0.5% tropicamide and 2.5% phenylephrine) acquired from Santen (Osaka, Japan). A 532‐nm neodymium‐doped yttrium aluminum garnet PASCAL diode ophthalmic laser system (Topcon Medical Laser Systems, Santa Clara, CA, USA) was used for laser photocoagulation (200 µm spot size, 0.02 s duration, 100 mW), wherein five or six laser spots were applied around the optic nerve of only the right eye. Gaseous bubble formation at the laser spot indicated the rupture of Bruch’s membrane.