All animal experiments were subject to local ethical approval and performed under the terms of a UK government Home Office license. Wbp2tm2a(EUCOMM)Wtsi mice (outbred on a C57BL6/CBA background) were provided by Karen P. Steel49 (link). Homozygous mutant mice are referred to as Wbp2−/−. Wild-type (Wbp2+/+) and heterozygous (Wbp2+/-) mice were used as controls and showed no apparent phenotype in skin49 (link) (Supplementary Fig. 9 ). Animals were genotyped using standard procedures, and with the recommended set of primers49 (link). In the developmental analysis time course both male and female mice were analysed, while for the wound healing experiment only 8-weeks-old adult female mice were used. Before wounding, back skin hair was clipped and mice were anaesthetized using isofluorane (CP-Pharma) and injected subcutaneously with the analgesic Vetergesic (diluted 1:20 in sterile PBS; 100 μl per 20 g body weight; Ceva Animal Health). A 5 mm diameter biopsy punch (Stiefel) was used to introduce a full-thickness wound in the central back skin, and wounds were excised at the indicated time points and embedded in optimal cutting temperature compound (OCT). All tested animals were included; no statistical method was used to predetermine sample size; no randomization or blinding was used.
Vetergesic
Manufactured by Ceva
Sourced in United Kingdom, Belgium
Vetergesic is a veterinary analgesic medication used to provide pain relief in animals. It contains the active ingredient buprenorphine, an opioid agonist-antagonist. Vetergesic is administered via injection or transdermal patch to manage moderate to severe pain in animals.
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Lab products found in correlation
Baytril, by Bayer (1 mentions)
Prolene, by Johnson & Johnson (1 mentions)
Alzet osmotic minipumps, by Charles River Laboratories (1 mentions)
Vicryl 5 0, by Johnson & Johnson (1 mentions)
Chirocaine, by Abbvie (1 mentions)
Baytril 2 5, by Bayer (1 mentions)
Monocryl 3 0, by Johnson & Johnson (1 mentions)
Depo provera, by Pfizer (1 mentions)
Buprenorphine, by Ceva (1 mentions)
Ritodrine hydrochloride, by Merck Group (1 mentions)
Cd 1 crl cd1 icr mice, by Charles River Laboratories (1 mentions)
Cuy650p3, by Nepa Gene (1 mentions)
Nepa21 super electroporator, by Nepa Gene (1 mentions)
Dolethal, by Vetoquinol (1 mentions)
Hypnomidate, by Johnson & Johnson (1 mentions)
Isoflo, by Abbott (1 mentions)
10 protocols using vetergesic
1
Wbp2 Mutant Mice Wound Healing Analysis
2
Surgical Induction of Muscle Hypertrophy
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Surgical anesthesia was induced and maintained using isoflurane (5% and 2%, respectively, in 100% O2; IsoFlo®). An incision was made along the distal two thirds of the TA, toward the lateral side of the muscle. Covering facia was cleared to expose the TA, allowing surgical release of the tendon and dissection from the lateral surface of the tibia. This procedure induces a functional overload leading to hypertrophy of the synergist EDL, an angiogenic response mediated via upregulation of VEGF and Flk‐1,18 , 19 and altered metabolic profile.21 , 24 Post‐operative analgesia (buprenorphine: 0.015 mg kg−1; Vetergesic®, Ceva, Amersham, UK) and antibiotic (Enrofloxacin: 2.5 mg kg−1; Baytril®, Bayer, Reading, UK) were provided for two days.
3
Osmotic Minipump Implantation for AngII Infusion in Mice
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Alzet osmotic minipumps (supplied by Charles River Laboratories) were used for continuous delivery of 0.8 mg/kg/d AngII or vehicle for 7 d. Mice were anaesthetised in an induction chamber using vaporised 5% isoflurane in a constant oxygen supply of 2 l/min. Anaesthesia was maintained at 2.5% isoflurane using a nose cone. Mice were positioned on a heated mat in the prone position. Buprenorphine (Vetergesic, Ceva Animal Health Ltd.) (0.05 mg/kg, diluted in sterile PBS) was administered subcutaneously for analgesia. The fur covering the mid-scapular region was removed using an electric razor and the area was sterilised with HIBISCRUB® (VioVet). Under aseptic conditions, a 2 cm incision was made at the mid-scapular region and blunt dissection generated a pocket towards the lower-left flank of the mouse for the minipump to be inserted. The wound was closed with two simple interrupted sutures using polypropylene 4-0 thread (Prolene, Ethicon) and then sterilised with HIBISCRUB®. Mice were recovered singly and returned to a clean cage once fully recovered.
4
Placental Vessel Ligation in Rabbit Model
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Briefly, rabbits were administered induction anesthesia with IM ketamine (35 mg/kg Nimatek® , Eurovet Animal Health BV, Bladel, The Netherlands) and xylazine (5 mg/kg XYL-M® 2%, VMD, Arendonk, Belgium), antibiotic prophylaxis (10 mg/kg enrofloxacin, Baytril® 2.5% SC, Bayer, Diegem, Belgium), tocolysis (10 mg/kg medroxyprogesterone, Depo-Provera® SC, Pfizer, Puurs, Belgium), and analgesia (0.03 mg/kg buprenorphine, Vetergesic® SC, Ceva Animal Health, Brussels, Belgium) prior to surgery. Anesthesia was maintained with a continuous IV infusion of ketamine (8–16 mg/kg/h) and xylazine (2.4–4.8 mg/kg/h) while monitoring vital signs. Following laparotomy, 33–50% of the vessels going to each placenta were ligated in one random horn with Vicryl® 5–0 (Ethicon® , Johnson & Johnson, Diegem, Belgium), leaving the contralateral horn as internal control. The abdomen was closed with Vicryl® 2–0 and Monocryl® 3–0 (Ethicon® , Johnson & Johnson) for fascia and skin, respectively. The surgical wound was infiltrated with levobupivacaine (2 mg/kg Chirocaine® , Abbvie, Wavre, Belgium) and sprayed with aluminum (Kela, Hoogstraten, Belgium).
5
Femur Fracture Repair in Mice
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Anaesthesia was induced with isoflurane and maintained with isoflurane throughout the surgery and post-surgery x-ray. Buprenorphine (100 μL subcutaneous; 3% Vetergesic, Ceva, UK) was administered before induction of anaesthesia. The fracture surgery was performed as previously described [25 (link), 26 (link)]; briefly, a 13-mm incision was made on the right thigh and a blunt dissection performed to visualise the femur. Care was taken to visualise and avoid manipulating the sciatic nerve. A local anaesthetic (0.1 mL mepivacaine hydrochloride, Intra-Epicaine 5 mg/mL, Dechra, UK) was applied to the femur and surrounding muscles. An external fixator made of plastic (MouseExFix Simple L, RISystem, Landquart, Switzerland) was fixed to the anterolateral side of the femur with four titanium pins. The fixator and pins combined weigh 0.20 g (± 0.01 g). A wire saw was passed underneath the femur, and a transverse osteotomy (0.4 mm) performed mid-way between the middle pins through the full thickness of the bone. Postoperative x-rays (HFX90v, 70 kV, 0.8 mAs/s) were acquired of all animals with external fixators whilst under anaesthesia from the surgery, to confirm correct placement of the fixator and pins. Postoperative analgesia was administered twice a day as Buprenorphine in jelly, for 3 days.
6
In Utero Electroporation of Cortical Neurons
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In utero electroporation (IUE) was performed as described before (Bartolini et al., 2017 (link)). CD-1 [Crl:CD1(ICR)] mice (Charles River, #022) were used for all IUE experiments. Timed-pregnant females were deeply anesthetized with isoflurane (Piramal Critical Care Limited). Buprenorphine (Vetergesic, Ceva Animal Health Ltd) was administered for analgesia via subcutaneous injection, and ritodrine hydrochloride (Sigma-Aldrich, Cat# R0758) was applied to the exposed uterine horns to relax the myometrium. DNA solution was mixed with Fast Green (Roche, Cat# 06402712001) and 1–2 μl of the solution was injected into the lateral ventricle of embryos at E14.5. Forceps-shaped electrodes (CUY650P3, Nepa Gene) connected to an electroporator (NEPA21 Super Electroporator, Nepa Gene) were used to deliver five electric pulses (45 V for 50 ms, with 950 ms intervals). The electrodes were positioned to target cortical pyramidal cell progenitors in the subventricular zone.
7
Rat Spinal Cord Transection Model
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Rats were anesthetized with inhalant isoflurane (4–5% with 2 L/min O2 in the induction chamber, followed by 1–2% for maintenance via a nose cone), prepped for surgery and administered buprenorphine (Vetergesic; 0.02 mg/kg, S.C., Ceva Animal Health Inc., Cambridge, ON, Canada). A detailed protocol for T3 complete transection SCI surgery has been published by our group previously7 (link). Briefly, a dorsal durotomy was performed via an incision at the dorsal midline from C8 to T2 vertebrae or from L1 to L2 vertebrae, and the dura was snipped between the T2 and T3 spinal levels or between the L1 and L2 spinal levels. Next, the animal underwent a complete transection of the T3 or L2 segment using micro scissors, and a vacuum-powered suction was used to remove ~0.5 cm of tissue. Using a dissecting microscope, complete transection was confirmed by ensuring no tissue remained between the rostral and caudal ends of the spinal cord. Hemostatic Gelfoam (Pharmacia & Upjohn Company, Pfizer Inc., New York, NY, USA) was placed in the space between the rostral and caudal ends. SHAM animals underwent a dorsal durotomy but no SCI.
8
Evaluation of DMD mdx Rat Model
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A total of 69 DMDmdx rats and 59 Sprague Dawley WT rats (littermates) were used in this study. They were obtained, handled and housed from the UTE IRS-UN (University de Nantes, France) and the Boisbonne Center for Gene Therapy (ONIRIS, Nantes, France). The Institutional Animal Care and Use Committee of the Région des Pays de la Loire (University of Angers, France) as well as the French Ministry for National Education, Higher Education and Research approved the protocol (authorizations #2016070618053653 and 2017040616371353). Before sacrifice, animals received a subcutaneous injection of buprenorphine (0.04 mg/kg, Vetergesic, Ceva Santé Animale, Libourne, France), after 30 min rats were anesthetized by intraperitoneal injection with etomidate (16 mg/kg, Hypnomidate, Janssen-Cilag, Issy Les Moulineaux, France), delivered in 2 or 3 injections separated by 3 min, and ketamine (20 mg/kg, Imalgene 1000, Merial, Lyon, France). Animals dedicated to ex vivo skeletal muscle contractility analysis and Ca2+ measurements were euthanized by heart excision. Other animals were euthanized by intravenous injection of pentobarbital sodium (Dolethal, Vetoquinol, Paris, France).
9
Subcutaneous Scaffold Implantation in Mice
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Mice were given subcutaneously analgesic buprenorphine (Vetergesic®, Ceva) diluted to 0.05 mg/mL in 0.9 % saline solution, used at a dose of 1 µg/Kg. Anaesthesia was induced in a chamber with 5 % isoflurane (IsoFlo®, Abbot Laboratories) and oxygen (2 L/min) and maintained at 1–3 % isoflurane in oxygen (2 L/min) using a nose cone. Surgical procedures were performed using an aseptic technique throughout. A small dorsal area was shaved and cleaned by swabbing with 1 % w/v iodinated povidone (Videne antiseptic solution, Ecolab) in water for injections (Hameln Pharmaceuticals Ltd.). To implant the scaffold, a small incision (< 1 cm) was made using a sterile scalpel (Swann-Morton) on the central dorsal surface. Autoclaved blunt Adson forceps (WPI) were used to create a pocket in the subcutaneous space for each scaffold. One sterilised disc-shaped 3D printed SiO2/PTHF/PCL-diCOOH hybrid scaffold was implanted into each mouse, ∼1 cm apart from the skin incision. After implantation, each wound was closed with an autoclaved 9 mm surgical Reflex clip with clip applier (WPI). Sham surgery was performed in the control group. After surgery, mice were removed from anaesthesia and transferred to a recovery chamber at 37 °C. Mice were carefully monitored by animal care services and housed for 1 or 7 days (n = 5 mice for each group). The experiment was repeated twice for each time point.
10
Anesthesia and Analgesia for Surgical Rats
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Animals were anesthetized by an intraperitoneal (I.P.) injection mix of 57.14 mg/kg body weight (BW) of ketamine (100 mg/mL, Nimatek, Eurovet, Bladel, The Netherlands) and 5.71 mg/kg BW of xylazine (Xyl-M 2%, Inovet, Arendonk, Belgium). Following animal welfare standards, rats were monitored at least 3 times daily and buprenorphine subcutaneous (0.016 mg/kg BW, 0.3 mg/mL, Vetergesic, Ceva, Brussel, Belgium) was used for analgesia, twice daily, during the first 3 days following the surgery [26 (link),40 (link)].
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