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1

Myelographic Evaluation of Canine IVDE

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All dogs presenting with IVDE were pre-medicated with diazepam (0.2 mg/kg IV, Valium, Roche products, Illovo, Johannesburg) and morphine (0.5 mg/kg IV Pharma-Q-Morphine, Pharma-Q Holdings, Industria West, Johannesburg). Induction was performed with propofol (6.6 mg/kg IV, Fresenius Propoven, Fresenius Kabi, Midrand, Johannesburg). Patients were intubated and maintained on isoflurane (Isofor 100 mL, Safeline pharmaceuticals, Roodepoort, Johannesburg) in 100% oxygen.
Right lateral, left lateral and ventro-dorsal survey radiographs were taken. An area over the dorsal lumbar spine was clipped and surgically prepared for lumbar myelogram, which was performed by injecting Iohexal (Ominpaque, 0.3 mL/kg, 140 mg/mL, GE Healthcare, Midrand, Johannesburg) into the sub-arachnoid space. Ventro-dorsal, left lateral, left ventro-dorsal oblique and right ventro-dorsal oblique radiographs were taken to complete the myelographic study.
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2

Induction and Treatment of Pilocarpine-Induced Status Epilepticus in Rodents

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Lithium chloride (LiCl, 127 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO, USA) was administered to the animals 19 h before the injection of pilocarpine (25 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO, USA) to cause status epilepticus (SE) [43 (link)]. Scopolamine (2 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO, USA) was injected 30 min before the injection of pilocarpine. SE has been reported to occur 20–30 min after pilocarpine injection [44 (link)]. Animals were housed one per cage for individual observation. According to Racine’s method, animals exhibited up to five symptoms: (1) mouth and facial movements, (2) head nodding, (3) forelimb clonus, (4) rearing with forelimb clonus, and (5) rearing and falling with forelimb clonus. When the fifth symptom occurred, we judged that the seizure had occurred completely [45 (link)]. Two hours after the start of SE, diazepam (Valium, 10 mg/kg, i.p., Hoffman la Roche, Neuilly sur-Seine) was administered. Even after the diazepam treatment, repeated seizure behavior was observed in some animals [46 (link)]. When recurrent severe seizure was observed, additional diazepam was injected (2 mg/kg, i.p.) to stop the remaining seizure activity [47 (link)] (Figure S1).
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3

Pharmacological Interventions for Alcohol Withdrawal

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Two weeks before the pharmacological treatments, mice were housed in individual cages (331 × 159 × 132 mm; 335 cm2) with continuous access to alcohol. Diazepam (Valium®, Roche) and Baclofen (Baclofen®, Mylan) were diluted in a vehicle solution (0.9% NaCl) and injected intraperitoneally (10 mL/kg, i.p., 1 injection/day). Drug doses were based on pilot experiments and previous studies (46 (link), 47 (link)). In all experiments, drugs were administered over the 15 final days of the withdrawal phase while mice were still under a 12% ethanol (v/v) regimen at the beginning of the pharmacological treatments (Figure 3) according to previous studies (11 (link), 39 (link)). Doses were progressively decreased from Day-15 to Day-1, to avoid potential negative effects of an abrupt cessation of drug administrations. Behavioral testing began either 1- or 4-weeks after the last injection. Our previous biological analyses showed that, at the time of memory testing, both diazepam and Baclofen compounds were no longer detectable in the blood of treated animals and their controls (39 (link)).
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4

Pilocarpine-Induced Epilepsy Model in Rats

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Three days after surgery, rats were treated with pilocarpine (380 mg/kg, i.p.) as previously described [17 (link),18 (link),19 (link)]. Atropine methylbromide (5 mg/kg, i.p.) was injected 20 min before a single dose of pilocarpine in order to avoid the peripheral muscarinic effect. To terminate SE, diazepam (Valium; Hoffman la Roche, Neuilly sur-Seine, France; 10 mg/kg, i.p.) was administered 2 h after onset of SE and repeated as needed. As controls, age-matched normal rats were treated with saline instead of pilocarpine.
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5

Canine Knee Arthroscopy and ACL Transection

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Dogs underwent general anesthesia induced by acepromazine (Vedco; Saint Joseph, Missouri; intravenously (IV), 0.2 mg/kg), ketamine (Ketaset; Fort Dodge Animal Health, Overland Park, Kansas; IV, 6 mg/kg), and diazepam (Valium; Roche, Madison, Wisconsin; IV, 0.35 mg/kg) and maintained by isoflurane (IsoFlo; Abbott, Parsippany, New Jersey; infusion, 2%-4%). Bilateral knee arthroscopy using standard portals was performed to evaluate intra-articular structures. Using randomization, one knee had the anterior cruciate ligament transected, while the ACL in the contralateral knee was left intact (uninjured). The contralateral knee arthroscopy was performed to evaluate knee structures and to balance possible effects of the arthroscopy procedure itself, such as swelling or effusion, on postoperative knee imaging.
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6

Donepezil and Diazepam Pharmacological Analysis

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Drugs used in this study included donepezil and diazepam as Valium (10 mg/2 mL) from Roche Pharma, Karachi, Pakistan. These drugs and chemicals utilized in the biochemical analysis were of research grade.
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7

Surgical Approach for Equine Cervical Vertebral Stenosis

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All horses were pre-medicated with systemic antibiotic medications and non-steroidal anti-inflammatory drugs. The horses were sedated with medetomidine (Dorbene ad us. vet.; Dr. E. Graeub AG, Bern, Switzerland) (7 µg/kg, IV), and then induced with a mixture of ketamine (Ketanarkon 100 ad us. vet.; Streuli Pharma AG, Uznach, Switzerland) (2.2 mg/kg, IV) and diazepam (Valium; Roche Pharma AG, Reinach, Switzerland) (0.02 mg/kg, IV) and placed in dorsal recumbency with the neck positioned as straight as possible.
Anaesthesia was maintained with isoflurane (Isoflurane ad us. vet.; Provet AG, Lyssach, Switzerland) in oxygen and a medetomidine (Dorbene ad us. vet.) constant rate infusion (3.5 µg/kg/h, IV). The correct position of the neck and especially the CV was ensured with padding, air filled bags and controlled after and using fluoroscopy (Siremobil C3D; Siemens Medical Solutions, Erlangen, Germany). The ventral aspect of the neck was clipped and prepared aseptically. A standard surgical ventral approach to the affected CV was performed. 11 The ventral spine of the CV was flattened using an oscillating saw or osteotome. The intervertebral disc was partially removed using a Ruskin Rongeur, spoon curette or 3.2 to 4.5 mm drills depending on surgeon's preference.
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8

Inducing Status Epilepticus in Rodents

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To induce SE, animals were pretreated with an intraperitoneal injection of LiCl (127 mg/kg, i.p.) and atropine methylbromide (5 mg/kg, i.p.) 24 h and 20 min before pilocarpine (30 mg/kg, i.p.) treatment, respectively. Control animals received an equal volume of normal saline instead of pilocarpine. Two hours after SE, animals received diazepam (Valium; Roche, Neuilly sur-Seine, France; 10 mg/kg, i.p.) to terminate SE. Three days after SE, animals were used for immunohistochemistry and Western blot.
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9

SARS-CoV-2 Infection in Young and Aged Hamsters

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Male golden (Syrian) hamsters (Mesocricetus auratus), 2-months-of-age (the “young adults” group) and 22-months-of-age (the “aged adults” group), were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Hamsters were fed a standard rodent chow (SAFE® A04, Augy, France) and were given water ad libitium. The hCoV-19_IPL_France strain of SARS-CoV-2 (NCBI MW575140) was isolated on TMPRSS2 expressing Vero-81 cells and passaged 4 times on these cells before usage. For infection, hamsters were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) (Boehringer-Ingelheim, Lyon, France), atropine (0.75 mg/kg) (Agettant, Lyon, France) and valium (2.5 mg/kg) (Roche, Boulogne-Billancourt, France), and intranasally infected with 100 µl of DMEM containing (or not, for mock-treated control animals) 2 × 104 TCID50 (50% Tissue Culture Infectious Dose) of SARS-CoV-2.
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10

BALB/c Mouse Model for Disease

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Eight-week-old male or female BALB/c mice (Charles River Laboratories, Barcelona, Spain) were used in experiments. This study was carried out in accordance with the recommendations of European Community Directive 2010/63/EU and Spanish law (Real Decreto 53/2013) on the use of laboratory animals, and their housing and the experimental procedures were approved by the Junta de Andalucía animal care committee and adhered to animal welfare guidelines of the National Committee for Animal Experiments. The animals were anesthetized with 0.04 mL diazepam (Valium, Roche, Basel, Switzerland) and 0.1 mL ketamine (Ketolar, Pfizer, Kent, UK) When clear signs of disease were observed, the animals were anesthetized and euthanized by cervical dislocation, followed by complete necropsy.
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