All animals were ototoxically deafened. Under gaseous anaesthesia (1–2% isoflurane in oxygen, 1L/min; Isoflo; Zoetis, UK) the right jugular vein was exposed and cannulated by an intravenous injection of frusemide (130 mg/kg; Ilium, Australia), followed by kanamycin sulfate (420 mg/kg, s.c.; Sigma-Aldrich, USA) within 2 minutes of frusemide administration (29 (link)). This deafening technique produces a highly symmetrical bilateral sensorineural hearing loss (28 (link), 29 (link)). Animal weight and behaviour were closely monitored following deafening. Seven days following this deafening procedure the hearing status of each animal was re-tested. Only animals that exhibited a 50 dB increase in their click-evoked ABR threshold were included in this study.
Isoflo
Manufactured by Zoetis
Sourced in United Kingdom, United States, Japan, France, Belgium, Italy
IsoFlo is a laboratory equipment product manufactured by Zoetis. It is designed to provide precise and controlled administration of isoflurane, a commonly used inhalation anesthetic, in a laboratory setting.
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Lab products found in correlation
Diprivan, by AstraZeneca (2 mentions)
Dormicum, by Astellas Pharma (2 mentions)
Rimadyl, by Zoetis (2 mentions)
Kanamycin sulfate, by Merck Group (1 mentions)
Proposure, by Boehringer Ingelheim (1 mentions)
Onsior, by Novartis (1 mentions)
Asteion super 4, by Toshiba (1 mentions)
Propoflo, by Zoetis (1 mentions)
Periflux 5000, by Perimed (1 mentions)
Ultiva, by Johnson & Johnson (1 mentions)
Atropine sulphate, by Mitsubishi (1 mentions)
Xylovet, by Ceva (1 mentions)
Rompun, by Bayer (1 mentions)
Nanoject 3 injector, by Drummond (1 mentions)
Edta tube, by Sarstedt (1 mentions)
Gelatin from porcine skin, by Merck Group (1 mentions)
Hydroxypropyl methylcellulose hpmc, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Atropine sulfate, by Mitsubishi (1 mentions)
Cerenia, by Zoetis (1 mentions)
34 protocols using isoflo
1
Ototoxic Deafening of Animals for Hearing Research
2
Perioperative Anesthesia and Analgesia Protocol
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Premedication was provided by acepromazine (Prequillan; ATI, Italy) (10 µg/kg, intramuscularly [IM]) and methadone hydrochloride (Semfortan; Eurovet Animal Health B.V., The Netherlands) (0.3 mg/kg, IM), followed by fentanyl (Fentadon; Dechra Pharmaceuticals, UK) (2 mcg/kg, intravenously [IV]) and propofol (Proposure; Boehringer Ingelheim, Germany) (1 mg/kg, IV) for the induction. During surgery, anesthesia was maintained with isoflurane (IsoFlo; Zoetis, USA) in oxygen and air. All dogs received cefazolin sodium (Cefazolina Teva; Teva, Italy) (30 mg/kg, IV) at the time of anesthetic induction and every 90 min during surgery.
Postoperative analgesia was obtained using methadone (0.1–0.2 mg/kg, IM every 4h) or buprenorphine (Buprenodale; Dechra Pharmaceuticals) (10–15 µg/kg IM every 8h) for the first 24h, followed by tramadol hydrochloride (Altadol; Formevet, Italy) (2–4 mg/kg, orally, every 8h) for 2 day. All dogs received cephalexin (Rilexine; Virbac,France) (30 mg/kg, orally, every 12 h for 10 day) and robenacoxib (Onsior; Novartis,Switzerland) (1 mg/kg, orally, once a day) for 7 day.
The owners were instructed to limit physical activity for 20 day before allowing the dogs to resume normal levels of activity.
Postoperative analgesia was obtained using methadone (0.1–0.2 mg/kg, IM every 4h) or buprenorphine (Buprenodale; Dechra Pharmaceuticals) (10–15 µg/kg IM every 8h) for the first 24h, followed by tramadol hydrochloride (Altadol; Formevet, Italy) (2–4 mg/kg, orally, every 8h) for 2 day. All dogs received cephalexin (Rilexine; Virbac,France) (30 mg/kg, orally, every 12 h for 10 day) and robenacoxib (Onsior; Novartis,Switzerland) (1 mg/kg, orally, once a day) for 7 day.
The owners were instructed to limit physical activity for 20 day before allowing the dogs to resume normal levels of activity.
3
Evaluating GC-Induced Muscle Atrophy in Dogs
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We evaluated the skeletal muscle cross-sectional area using CT before and after GC
administration for 4 weeks. After 12-hr fasting, the dogs were anesthetized with 7 mg/kg
intravenous propofol (PropoFlo; Zoetis Japan, Tokyo, Japan) and maintained with isoflurane
(IsoFlo; Zoetis Japan) in oxygen. All CT scans were performed using a multidetector-row CT
scanner (Asteion Super 4; Toshiba Medical Systems, Tochigi, Japan) at 120 kV, 100 mA, and
2-mm slice thickness. In humans, the cross-sectional area of the paraspinal muscle at the
third lumbar (L3) vertebra correlates with the entire body muscle mass [13 (link), 15 (link)]. In
addition, the muscles of the proximal lower limbs, including the thigh muscles, are
strongly affected in GC-induced muscle atrophy [25 (link)]. Therefore, we focused on the paraspinal muscle at L3 and the mid-femoral
muscle. The skeletal muscle cross-sectional areas were analyzed using the region of
interest tool of OsiriX image software (Newton Graphics, Sapporo, Japan).
administration for 4 weeks. After 12-hr fasting, the dogs were anesthetized with 7 mg/kg
intravenous propofol (PropoFlo; Zoetis Japan, Tokyo, Japan) and maintained with isoflurane
(IsoFlo; Zoetis Japan) in oxygen. All CT scans were performed using a multidetector-row CT
scanner (Asteion Super 4; Toshiba Medical Systems, Tochigi, Japan) at 120 kV, 100 mA, and
2-mm slice thickness. In humans, the cross-sectional area of the paraspinal muscle at the
third lumbar (L3) vertebra correlates with the entire body muscle mass [13 (link), 15 (link)]. In
addition, the muscles of the proximal lower limbs, including the thigh muscles, are
strongly affected in GC-induced muscle atrophy [25 (link)]. Therefore, we focused on the paraspinal muscle at L3 and the mid-femoral
muscle. The skeletal muscle cross-sectional areas were analyzed using the region of
interest tool of OsiriX image software (Newton Graphics, Sapporo, Japan).
4
Anesthesia and Surgical Preparation Protocol for Canine Studies
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The dogs were fasted for food and water for 12 and 6 hours prior to the operation, respectively. A prophylactic antibiotic, cefotaxime (Cefotax, EIPICO, Egypt) at a dose of 50 mg/kg intramuscular (IM) was given prior to the surgery. Dogs were premedicated with an IM injection of chlorpromazine hydrochloride (Neurazine, Misr Co. Pharm., S.A.A.) at a dose of 1mg/kg, an IM atropine sulfate (Memphis Pharmaceutical, Egypt) at a dose of 0.04 mg/kg, and an IM injection of nalbuphine HCl (Nalufin®, Amoun pharmaceutical company, S.A.E.) at a dose of 1mg/kg (Clarke et al., 2014 (link)). General anesthesia was then induced by intravenous (IV) injection of Propofol at a dose of 2 mg/kg (Diprivan®, AstraZeneca) and maintained by 2% isoflurane (IsoFlo®, Zoetis) and oxygen (Clarke et al., 2014 (link)). The ventral abdominal wall was clipped, shaved and prepared for aseptic surgery using Povidone Iodine 10% (Nile Company for pharmaceutical and chemical industries, Egypt).
5
Severe Spinal Cord Injury and Chondroitinase ABC
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Animals were anaesthetised with Isoflurane: 5% IsoFlo® (Zoetis, UK Ltd, London, UK) at 2 L.min -1 O2 flow induction, and maintained with 2.5% throughout surgery. An incision was made from ~T4 to ~S5 and cleared of fascia and muscle around the laminectomy site. Partial laminectomies were performed at vertebral levels of the contusion (T10), and epidural implant sites (T12/13 and L2). A severe midline contusion injury was made at T9/10 31 using the Infinite Horizon impactor at 250 kdyn (Precision Systems & Instrumentation, Lexington, KY, USA).
Immediately following the injury, chondroitinase ABC was injected into the spinal cord 1 mm rostral and caudal to the lesion through a lentiviral vector. The chondroitinase ABC gene was Microvascular remodeling in SCI 6 produce as previously described by Bartus et al. 32 and subcloned into a lentiviral vector by Penn Vector Core (University of Pennsylvania). The promotor was mouse phosphoglycerate kinase 1 (mPGK1) 32, 33 and the viral titre was 1.51x10 10 genome copies per millilitre (GC/ml) determined by real-time PCR. This was delivered to all groups to maximize plasticity and the effects of each treatment [34] [35] [36] 33 .
Immediately following the injury, chondroitinase ABC was injected into the spinal cord 1 mm rostral and caudal to the lesion through a lentiviral vector. The chondroitinase ABC gene was Microvascular remodeling in SCI 6 produce as previously described by Bartus et al. 32 and subcloned into a lentiviral vector by Penn Vector Core (University of Pennsylvania). The promotor was mouse phosphoglycerate kinase 1 (mPGK1) 32, 33 and the viral titre was 1.51x10 10 genome copies per millilitre (GC/ml) determined by real-time PCR. This was delivered to all groups to maximize plasticity and the effects of each treatment [34] [35] [36] 33 .
6
Detailed animal care protocol
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All animal work was conducted in accordance with the U.K. Animals (Scientific Procedures) Act of 1986 and approved by the University of Oxford Animal Care and Ethical Review Committee for use under project licenses 30/2889 and P9804B4F1. All experimental animal work conducted at the Leiden University Medical Center (LUMC) (Leiden, The Netherlands) was approved by the Animal Experiments Committee of the LUMC (DEC 12042). Animals were group housed in individually ventilated cages under specific-pathogen-free conditions with constant temperature and humidity and with a 12:12 light-dark cycle (8 a.m. to 8 p.m.). For induction of short-term anesthesia, animals were either injected intramuscularly (i.m.) with xylazine and ketamine or anesthetized using vaporized IsoFlo (Zoetis). All the animals were humanely sacrificed at the end of each experiment by an approved schedule 1 method. All efforts were made to minimize suffering.
7
Transient Focal Cerebral Ischemia in Mice
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Transient focal cerebral ischemia was induced using the intraluminal method (Lo et al., 2005; Li et al., 2012; Yang et al., 2012). In brief, mice were randomly assigned and were subjected to isoflurane (IsoFlo; 50019100, Zoetis Inc., Kalamazoo, MI, USA) inhalation anesthesia (2% isoflurane in 70% N2O/30% O2 for induction; 1.25% isoflurane in 70% N2O/30% O2 for maintenance). A filament coated with vinylpolysiloxane material (ESPE 7302, 3M Dental Products, St. Paul, MN, USA) was inserted into the right internal carotid artery and further advanced until reaching the middle cerebral artery. After 2 hours of ischemia, the filament was removed to commence reperfusion. Animals in the sham groups received the same procedures except for filament insertion. Relative cerebral blood flow in the middle cerebral artery territory was monitored by a laser Doppler flowmeter (Periflux 5000, Perimed AB, Järfälla, Sweden), with the reading at 5 minutes before ischemia set as 100%. Body temperature was maintained at 37 ± 0.5°C throughout the surgery. Mice were placed in an intensive care unit set at 30 ± 0.5°C during ischemia and for 4 hours after reperfusion commenced. Mice were sacrificed at either 2 hours (2 hours ischemia/2 hours reperfusion) or 22 hours (2 hours ischemia/22 hours reperfusion) of reperfusion (Figure 1
8
Canine Lung Mass Resection Protocol
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General anaesthesia was pre‐administered by subcutaneous injection of 0.04 mg/kg atropine sulphate (Mitsubishi Tanabe Pharma Co., Osaka, Japan), followed by intravenous injection of 0.1 mg/kg midazolam (Dormicum; Astellas Pharma Inc., Tokyo, Japan) and 0.1 ml/kg fentanyl citrate‒droperidol (Thalamonal; Daiichi‐Sankyo Propharma Co., Ltd., Tokyo, Japan). General anaesthesia was induced using propofol (Mylan; Mylan Seiyaku Ltd., Tokyo, Japan). Endotracheal intubation was subsequently performed. Each dog was mechanically ventilated with 2.0%–2.5% isoflurane (IsoFlo; Zoetis Japan, Tokyo, Japan) and oxygen. For analgesia, intra‐ and post‐operative continuous drip infusions of remifentanil (5–40 μg/kg/h) (Ultiva; Janssen Pharmaceutical K.K., Tokyo, Japan) and pre‐ and post‐operative intramuscular injections of morphine hydrochloride (0.3 mg/kg each dose) (Takeda Pharmaceutical Co. Ltd. Osaka, Japan) were used.
Prior to surgery, CT was performed, and the volume (cm3) of the lung mass was measured in terms of length, width, and height. All patients were approached through an intercostal or median sternotomy depending on the region and size of the individual tumours, and the tumours were resected by lung lobectomy. All resected masses were weighed.
Prior to surgery, CT was performed, and the volume (cm3) of the lung mass was measured in terms of length, width, and height. All patients were approached through an intercostal or median sternotomy depending on the region and size of the individual tumours, and the tumours were resected by lung lobectomy. All resected masses were weighed.
9
Central Venous Catheter Implantation in Pigs
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For pigs receiving at least one intravenous (IV) administration of CMS, central venous catheters were implanted. These animals were firstly sedated with an IM injection of ketamine at 20 mg/kg (Imalgène, Mérial, Lyon, France) and xylazine at 2 mg/kg (Rompun, Bayer, Loos, France). Then, they were intubated and kept anesthetized by inhalation with isoflurane 2.5% (IsoFlo, Zoetis, Malakoff, France) during all the surgical procedure. An incision was performed on the neck under local anaesthesia with xylocaine (Xylovet, CEVA, Libourne, France). After dilaceration of superficial tissues and muscles, two catheters were implanted in the jugular vein, one for drug administration and one for blood sampling.
After surgery, pigs rested 48 h alone in their box. Then, they were housed separately in metabolism cage in order to facilitate drug administration and blood/urine sampling.
After surgery, pigs rested 48 h alone in their box. Then, they were housed separately in metabolism cage in order to facilitate drug administration and blood/urine sampling.
10
Infrared Thermography for Interscapular Brown Adipose Tissue
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iBAT skin temperature was measured using infrared technology (camera FLIR E95 24°, Teledyne FLIR Systems, Wilsonville, OR, USA) and analyzed with its specific software, FLIR TOOLS Thermography Software. Videos were recorded on the first and the last day of treatment. As previously described, skin surrounding iBAT was shaved under isoflurane (IsoFlo®, Zoetis, London, UK) anesthesia 2 days before recordings [27 (link)]. The increment of iBAT temperature was calculated as the difference of temperature from last day to first day of treatment.
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