For intravenous sedation, medetomidine (10-20 µg/kg i.v. Domitor, Pfizer) and butorphanol (0.2 mg/kg i.v. Butomidor, Richter Pharma AG, Austria) were used. Medetomidine (10-20 µg/kg i.v. Domitor, Pfizer), butorphanol (0.2 mg/kg i.v. Butomidor, Richter Pharma AG) and propofol (10 mg/kg i.v. Norofol, Norbrook, Norway) were employed for intravenous anaesthesia. Two orthogonal views of the entire caudal spine were obtained in all subjects, in laterolateral and ventrodorsal projection. All images were obtained using an X-ray machine (Proteus XR/a, GE Healthcare, Wisconsin, USA) with cassettes for computed radiography. Table-top technique was employed, without the use of a grid. The digital radiographs were stored in DICOM format using the Capsula XL (Fuji, Japan) computed radiography system. The resolution of the radiographs was 1760 × 2140 and 1576 × 1976.
Domitor
Manufactured by Pfizer
Sourced in United States, Germany, France, United Kingdom, Italy, New Zealand, Switzerland, Australia, Belgium
Domitor is a veterinary sedative and analgesic used to induce a state of calmness and pain relief in animals. It contains the active ingredient medetomidine hydrochloride. Domitor is an alpha-2 adrenergic agonist that acts on the central nervous system to produce its effects.
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Lab products found in correlation
Antisedan, by Pfizer (19 mentions)
Medetomidine, by Pfizer (5 mentions)
Vetalar, by Pfizer (4 mentions)
Tobramycin, by Alcon (4 mentions)
Tobrex, by Alcon (4 mentions)
Temgesic, by Merck & Co (4 mentions)
Dormicum, by Roche (3 mentions)
Ketaset, by Zoetis (3 mentions)
P0130, by Merck Group (2 mentions)
Envisu r2210, by Bioptigen (2 mentions)
Ketalar, by Pfizer (2 mentions)
Zoletil 100, by Virbac (2 mentions)
Rimadyl, by Pfizer (2 mentions)
Spc 320, by Millar (2 mentions)
Vetalar, by Boehringer Ingelheim (2 mentions)
5 μl syringe, by World Precision Instruments (2 mentions)
Tropicamide, by Bausch & Lomb (2 mentions)
Pancuroniumbromide, by Avantor (2 mentions)
Mr compatible small animal heating system, by SA Instruments (2 mentions)
Mr compatible small animal monitoring and gating system, by SA Instruments (2 mentions)
97 protocols using domitor
1
Intravenous Sedation and Anesthesia in Caudal Spine Imaging
2
Optogenetic Activation of Locus Coeruleus
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To allow optogenetic activation of LC neurons, a Canine-Adenoviral vector (CAV) was used (CAV-PRS-ChR2-mCherry) (Li et al., 2016 (link)). The prolyl tRNA synthetase (PRS) promoter in this vector has been previously demonstrated to achieve LC-specific expression by harnessing the Phox2B transcription factor, which drives endogenous dopamine-β-hydroxylase (the final enzyme in the synthesis pathway for NA).
Stereotaxic injection of the CAV vector was made into the dorsal pons in the region of the LC of PD 19 Wistar rats using an established protocol (Li et al., 2016 (link)). Rats were anesthetized (intraperitoneally) with ketamine (5 mg/100 g, Vetalar; Pfizer, New York, NY, United States) and medetomidine (30 μg/100 g, Domitor; Pfizer) until loss of paw withdrawal reflex. The animal was placed in a stereotaxic frame and core temperature was maintained at 37°C using a homeothermic blanket (Harvard Apparatus, Holliston, MA, United States). Following a craniotomy (from bregma: –1 mm AP, +1.1 mm ML), a microinjection pipette was advanced into the brain with a 10° rostral tilt. Two hundred nanoliters of vector was injected at 4.6, 4.9. 5.2, and 5.5 mm depth. The titre of vector used was 2.15 × 1011 physical particles/ml. Aseptic surgical techniques were used throughout.
Stereotaxic injection of the CAV vector was made into the dorsal pons in the region of the LC of PD 19 Wistar rats using an established protocol (Li et al., 2016 (link)). Rats were anesthetized (intraperitoneally) with ketamine (5 mg/100 g, Vetalar; Pfizer, New York, NY, United States) and medetomidine (30 μg/100 g, Domitor; Pfizer) until loss of paw withdrawal reflex. The animal was placed in a stereotaxic frame and core temperature was maintained at 37°C using a homeothermic blanket (Harvard Apparatus, Holliston, MA, United States). Following a craniotomy (from bregma: –1 mm AP, +1.1 mm ML), a microinjection pipette was advanced into the brain with a 10° rostral tilt. Two hundred nanoliters of vector was injected at 4.6, 4.9. 5.2, and 5.5 mm depth. The titre of vector used was 2.15 × 1011 physical particles/ml. Aseptic surgical techniques were used throughout.
3
Adoptive Transfer of Lymph Nodes
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mLN and pLN from B6-Il10-/- donor mice were isolated and disrupted. Under the combined anesthesia with ketamine (Gräub AG, Bern, Switzerland) and Domitor (Pfizer, Karlsruhe, Germany) all mLN of the small and large intestine from recipient B6-Il10-/- mice were excised and previously isolated mLN or pLN (axillary, brachial, popliteal, and inguinal LN) from donor mice were transplanted into the mesentery (12 (link)). The recipients were sacrificed 8 weeks after transplantation and the transplanted LN (LNtx) were analyzed.
4
Anesthesia and Euthanasia Protocol
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Surgical procedures were performed under general anesthesia (75 mg/kg ketamine, Anesketin, Eurovet; 1 mg/kg medetomidine, Domitor, Pfizer), which was reversed by atipamezole (1mg/kg atipamezole, Antisedan, Pfizer). In addition, a drop of local anesthesia (oxybuprocaïne 0.4%, Unicaïne, Théa) was given before the surgery and an antiobiotic ointment (tobramycin 0.3%, Tobrex, Alcon) was applied afterwards. All animals were sacrificed with an overdose of sodium pentobarbital (60 mg/kg, Dolethal, Vetoquinol), followed by cervical dislocation.
5
Fluorescein-Based Permeability Assay for Mouse Retina
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To measure the permeability of the BRB in the mouse retina, fluorescein concentration was measured within the eye using the Fluorotron Master TM fluorophotometer (Ocumetrics). Before anesthesia, tropicol (5 mg/ml, Théa) was administered to the eye, followed by a subcutaneous injection of 200 l 2.5% fluorescein (SERB laboratories, diluted in sterile saline). The mouse was kept in dark during 30 minutes and anesthetized (75 mg/kg ketamine, Anesketin, Eurovet; 1 mg/kg medetomidine, Domitor, Pfizer). Fluorotron measurements of the fluorescein concentration (ng/ml) were taken at 149 data points in the posterior to the anterior direction, representing different distances along the optical axis of the eye (Kadam, Williams, Tyagi, Edelhauser, & Kompella, 2013) . For the retina, a curve was constructed from 7 data points (corresponding to 7 locations in the retina) and the area under the curve (AUC) was measured for each WT and Mmp3 -/-mouse eye at baseline and at 2 dpi.
6
Ovariectomy and Estrogen Replacement Protocol
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Unless otherwise stated, females were ovariectomized in adulthood (>8 weeks of age) under general anesthesia after either subcutaneous (sc) injections of ketamine (80 mg kg−1 per mouse) and medetomidine (Domitor, Pfizer, 1 mg kg−1 per mouse) or under 5% isoflurane, in order to control for endogenous hormone concentrations and to prevent pregnancies upon repeated testing. At the same time, all females received a 5-mm-long silastic capsule (inner diameter: 1.57 mm; outer diameter: 2.41 mm) containing crystalline 17β-estradiol (diluted 1:1 with cholesterol) subcutaneously in the neck. The dose of E2 (E8875, Sigma) was based on a previous study61 (link) showing that this treatment leads to estradiol levels similar to mice in estrus. At the end of surgery, females under ketamine/medetomidine anesthesia received an sc injection of atipamezole (Antisedan, Pfizer, 4 mg kg−1 per mouse) to antagonize medetomidine-induced effects and accelerate recovery. In order to induce sexual receptivity at the day of testing, all females received a subcutaneous injection with progesterone (500 µg, P0130, Sigma) 3 h before the onset of the behavioral test, unless stated otherwise (for overview of all the different hormone treatments, see Supplementary Table 1 ).
7
Anesthesia and Sedation Protocol for MRI
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Anaesthesia was induced using 3% isoflurane in 60% air, 40% O2 at 1L/min. The isoflurane concentration was maintained at 2–2.5% during the preparation which took around 30 min. Each mouse was positioned on an MRI-compatible cradle (Bruker Biospin, Germany) with ear bars and bite bars to reduce head motion. A peritoneal catheter was inserted and fixed to the mouse for delivery of medetomidine (Domitor, Pfizer, USA). For sedation, a bolus of 0.05 mg/kg medetomidine was given intraperitoneally and then sedation was maintained with a continuous infusion of 0.1 mg/kg/h. Once the animal was inside the MRI scanner, isoflurane was then reduced gradually and kept at approximately 0.25% throughout the experiment. The total time under anaesthesia for each animal was approximately 2.5 h. At the end of the scanning session, 1.25 mg/kg atipemazole (medetomidine reversal) (Antisedan, Pfizer, USA) was given intraperitoneally.
8
Retinal Layer Thickness Analysis Using SD-OCT
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Thickness of the retinal layers was evaluated using a spectral domain optical coherence tomography (SD-OCT) system (Envisu R2210, Bioptigen) (Buys et al., 2013) . Upon general anesthesia (i.p. 75 mg/kg body weight ketamine, Anesketin, Eurovet; i.p. 1 mg/kg medetomidine, Domitor, Pfizer), pupils were dilated by topical application of 0.5% tropicamide (0.5% Tropicol, Thea). SD-OCT was performed using 100 consecutive B-scan lines composed of 1000 A-scans, in a 1.4x1.4mm field. After the procedure, anesthesia was reversed by means of atipamezol (i.p. 1 mg/kg, Antisedan, Pfizer) and antibiotic ointment was applied to the eye (tobramycin 3 mg/g, Tobrex, Alcon). Total retinal thickness and thickness of different retinal layers were analyzed using InVivoVue Diver 2.2 software (Bioptigen).
9
Murine Carotid Artery Ligation Model
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Mouse carotid artery ligation was performed as previously described [18] (link). Briefly, the left common carotid artery of 12–13 weeks old male wild-type and FHL2-KO mice was ligated just proximal to the carotid bifurcation. Before surgery, mice were anaesthetized with an intra-peritoneal injection of a mixed solution of 5 mg/kg midazolam (Dormicum, Roche), 0.5 mg/kg medetomidine hydrochloride (Domitor, Pfizer) and 0.05 mg/kg fentanyl (Bipharma). The right carotid artery served as an uninjured contralateral control. Mice were sacrificed at 0, 1, 2, and 4 weeks after carotid ligation (n = 7 for 1 and 2 weeks and n = 14 for 4 weeks). Prior to sacrifice, the body weight of all mice was measured followed by anaesthesia by intra-peritoneal injection with 80 mg/kg ketamine (Nimatek; Eurovet) and 5 mg/kg xylazine (Sedamun, Eurovet) and perfused with saline. The left and right carotid arteries were placed in Trizol (Invitrogen) for RNA isolation or embedded in paraffin for histological analysis. Morphometric analysis was performed on at least 5 sections per mouse at a fixed position proximal from the ligation site; 1 week at 1.7 mm, 2 weeks ligation at 2.0 mm and 4 weeks at 2.3 mm.
10
Sedation and Examination of Dingoes
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An intra-muscular injection of Domitor® (medetomidine hydrochloride, Pfizer Australia Pty. Ltd., West Ryde, NSW, Australia) was used to sedate the animals. To further reduce risk to field workers, dingoes were placed on a restraint board with straps over their neck and loins and fitted with a muzzle. Animals were examined for the presence of external parasites and faecal samples were collected. Age and body condition scores were estimated and animals were weighed (Shimano 45 kg stainless weighing scale). Five mL of blood was collected from the jugular vein of each animal and stored in ethylene diamine tetraacetic acid (EDTA) tubes and refrigerated at 4 °C for antigen testing within 24 h. Animals were implanted with a microchip (Trovan 956 ISO, Microchips Australia Pty. Ltd., Keysborough, Australia) on the dorsal midline between the shoulder blades using a 12-gauge implanter needle (Trovan Deluxe [IME] Implanter, Microchips Australia Pty. Ltd., Keysborough, Australia).
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