The animals were sedated with 0.04 mg/kg BW romifidine (Sedivet®; Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim am Rhein, Germany) and 0.03 mg/kg BW butorphanol (Alvegesic®; CP-Pharma Handelsgesellschaft GmbH, Burgdorf, Germany) 15 h after the LPS challenge. Diazepam at a dose of 0.08 mg/kg BW (Diazepam-Lipuro®; Laboratoire TVM, Lempdes, France) and three mg/kg BW ketamine (Ursotamin®; Serumwerk Bernburg AG, Bernburg, Germany) were administered to induce general anesthesia. Inhalation anesthesia was maintained with isoflurane (CP-Pharma Handelsgesellschaft GmbH, Burgdorf, Germany). A 20-cm ventral midline incision was performed cranial to the umbilicus after aseptic preparation. Liver tissue (∼two g) was collected using biopsy forceps. Additionally, adipose tissue was collected from several locations for another part of the study. One part of each tissue biopsy specimen was immediately flash frozen in liquid nitrogen (−196 °C) and stored at −80 °C. The second part was stored in formalin. The animals were orally administered 0.55 mg/kg BW flunixin twice a day for 3 days after surgery (Flunidol®; CP-Pharma Handelsgesellschaft GmbH, Burgdorf, Germany).
Sedivet
Manufactured by Boehringer Ingelheim
Sourced in Germany
Sedivet is a lab equipment product developed by Boehringer Ingelheim. It is designed for the purpose of sediment analysis.
Automatically generated - may contain errors
Lab products found in correlation
Domosedan, by Zoetis (1 mentions)
Banamine, by Merck Group (1 mentions)
Detomidine, by Zoetis (1 mentions)
Dormosedan, by Zoetis (1 mentions)
Isoflurane, by CP-Pharma (1 mentions)
Butorphanol, by Zoetis (1 mentions)
Heparin sodium, by B. Braun (1 mentions)
Methanol, by Mallinckrodt (1 mentions)
Acetonitrile, by Mallinckrodt (1 mentions)
Sodium hydroxide (naoh), by Carlo Erba (1 mentions)
Hydrochloric acid (hcl), by Carlo Erba (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Torbugesic, by Pfizer (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
10 protocols using sedivet
1
Liver and Adipose Tissue Biopsy Protocol
2
Sedation Protocols for Equine Procedures
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Horses were sedated using either 0.03‐0.04 mg/kg IV romifidine chlorhydrate (Sedivet, Boehringer Ingelheim, Ingelheim, Germany) or 0.01 mg/kg IV detomidine chlorhydrate (Domosedan, Zoetis, Louvain‐la‐Neuve, Belgium) based on withdrawal time considerations. In horses with more marked clinical signs or in horses that were difficult to handle, 0.01‐0.02 mg/kg IV butorphanol (Dolorex, Intervet) was added to the alpha 2‐agonist drug.
3
Cerebrospinal Fluid Collection in Horses
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Horses were restrained in stocks for CSF collection. The procedures were performed using standing sedation with romifidine (80 μg/kg, IV; Sedivet, Boehringer Ingelheim Vetmedica, Duluth, Georgia) or detomidine hydrochloride (10 μg/kg, IV; Dormosedan, Zoetis, Parsippany, New Jersey). Cervical and LS CSF collections were performed consecutively, and an online random number generator (https://www.randomizer.org/ ) was used to determine the order of sampling. Lidocaine hydrochloride (100 mg; 2%; VetOne, Boise, Idaho) was infiltrated SC and into the musculature at both C1‐C2 and LS sites before the final aseptic preparation of the skin to provide local anesthesia. Cervical and LS CSF collection was performed as previously described.4 , 7 , 8 An 8‐in. 18 gauge spinal needle (Mila International, Florence, Kentucky) was used for LS centesis, and a 3.5‐in. 18 gauge spinal needle (Mila International) was used for cervical centesis. A total of 5 mL of CSF were collected in 1 mL aliquots from each site. After collection of CSF, horses were administered flunixin meglumine (1.1 mg/kg IV; Banamine, Merck Animal Health, Madison, New Jersey). Whole blood (10 mL) was collected into a serum tube by venipuncture of the jugular vein. The blood was allowed to clot at room temperature, centrifuged at 3000 rpm for 15 minutes, and the serum separated and frozen at −80°C until analysis.
4
Equine Tissue Sampling Protocol
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Equines were sedated with 0.04 mg/kg BW romifidine (Sedivet, Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim am Rhein, Germany) and 0.03 mg/kg BW butorphanol (Alvegesic, CP-Pharma Handelsgesellschaft GmbH, Burgdorf, Germany). General anaesthesia was induced with 0.08 mg/kg BW diazepam (Ziapam, Laboratoire TVM, Lempdes, France) and 3 mg/kg BW ketamine (Ursotamin, Serumwerk Bernburg AG, Bernburg, Germany). Animals were orotracheally intubated, and anaesthesia was maintained with isoflurane (CP-Pharma). The rp and mc AT (~ 5 g at each depot) samples were collected in dorsal recumbency 15 ± 1 h after LPS infusion. Liver tissue was also sampled for another part of this study at the same time. Both sc AT samples (~ 5 g at each depot) were taken in lateral recumbency 15.5 ± 1 h after LPS infusion. In subsequent evaluations, to avoid scar tissue, sc AT was collected on the other body side (t1) or at least with 5 cm distance from the first sampling point (t2). Each biopsy specimen was immediately flash-frozen in liquid nitrogen (− 196 °C) and stored at − 80 °C until analysis. After surgery, all animals were treated orally with 0.55 mg/kg BW flunixin (Flunidol, CP-Pharma) twice a day for 3 days to reduce post-surgical pain and were assessed for pain on base of a modified pain score [31 (link)].
5
Experimental Infection of Foals with R. equi
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Foals from Group 1 and 2 were experimentally infected at age 21 days with 1 x 106 CFU of live R. equi (strain EIDL 5–331, the same strain used for the vaccine). Prior to experimental infection with R. equi, each foal’s lungs were evaluated by auscultation and thoracic ultrasonography to document absence of pre-existing lung disease. Foals were sedated using intravenous injection of romifidine (0.8 mg/kg; Sedivet, Boehringer-Ingelheim Vetmedica, Inc., St. Joseph, MO, USA) and butorphanol (0.02 mg/kg; Zoetis, Florham Park, New Jersey, USA) to facilitate endoscopy. An aseptically-prepared, videoendoscope with outer diameter of 9-mm was inserted via the nares into the trachea and passed to the bifurcation of the main-stem bronchi. A 40-mL suspension of virulent EIDL 5–331 R. equi containing approximately 1 x 106 viable bacteria was administered transendoscopically, with 20 ml infused into the right mainstem bronchus and 20 ml into the left mainstem bronchus. The channel was flushed twice with 20 ml of air after each 10 ml bacterial infusion.
6
Romifidine Administration for Threshold Determination
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After determination of the baseline NWR threshold (at least 10 minutes of a stable NWR threshold and not less than 20 minutes after starting stimulation), romifidine 0.08 mg kg -1 IV (Sedivet 10 mg mL -1 ; Boehringer Ingelheim, **, **) was administered by hand over 1 minute.
Immediately thereafter, the romifidine infusion (diluted to 1 mg mL -1 with NaCl solution) was
Immediately thereafter, the romifidine infusion (diluted to 1 mg mL -1 with NaCl solution) was
7
Intraocular Pressure and Respiratory Effects of Romifidine in Buffalo
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The experimental protocol of the current study was approved by the Animal Care Committee of Mansoura University, in accordance with Egyptian ethical codes for studies on experimental animals. Buffalo were randomly allocated into three groups (six buffalo each). Group 1 received an IM romifidine (Sedivet; Boehringer Ingelheim, Vetmedica GmbH, Ingelheim/Rhein, Germany) at a dose of 40 μg/kg. While the second group received romifidine at a dose of 50 μg/kg. Animals in the third group received normal saline (0.9% NaCl; 0.4 ml/100 kg IM; El Nasr Pharmaceutical Company, Cairo, Egypt).
In all groups, the IOP of both eyes was measured and recorded by the same investigator and during the same work session. The measurements were recorded immediately before the administration of romifidine or saline (T0), and then at 5 (T5), 15 (T15), 30 (T30), 45 (T45), 60 (T60), 90 (T90), 120 (T120), 150 (T150) and 180 (T180) minutes post-administration. Heart rate (HR, beat/min) and respiratory rate (RR, breath/min) were assessed for each buffalo at each time point. Ocular rotation was also evaluated according to the method described in the literature (Greene, 2003 (link)).
In all groups, the IOP of both eyes was measured and recorded by the same investigator and during the same work session. The measurements were recorded immediately before the administration of romifidine or saline (T0), and then at 5 (T5), 15 (T15), 30 (T30), 45 (T45), 60 (T60), 90 (T90), 120 (T120), 150 (T150) and 180 (T180) minutes post-administration. Heart rate (HR, beat/min) and respiratory rate (RR, breath/min) were assessed for each buffalo at each time point. Ocular rotation was also evaluated according to the method described in the literature (Greene, 2003 (link)).
8
Equine Bone Marrow Aspiration Protocol
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All experiments were approved by the State Animal Care Committee (V12/09, Landesdirektion Leipzig, Free State of Saxony, Germany). Bone marrow was harvested from the sternum of 11 Warmblood geldings. Seven horses were sampled twice and used for two parts of the study (over six months between the aspirations). The horses were aged between 15–16 years and the procedure was performed as previously described (Delling et al., 2012 (link)). Briefly, after intravenous sedation with 0.06 mg/kg romifidine (Sedivet; Boehringer Ingelheim, Ingelheim, Germany) and 0.02 mg/kg butorphanol (Torbugesic; Fort Dodge Veterinär, Würselen, Germany), the area of the sternum was aseptically prepared and locally anesthetized. A BM aspiration needle (Bone Marrow Harvest Needle; Angiotech, Gainesville, FL, USA) was advanced into the bone and 2 × 20 ml marrow was aspirated into heparinized syringes (10,000 IU heparin-sodium (B. Braun, Melsungen, Germany)/20 ml syringe). Further, 2 × 20 ml venous blood samples were obtained from the jugular vein for the production of autologous plasma using heparinized syringes as described above.
9
Quantification of Romifidine in Equine Plasma
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Water, acetonitrile, methanol and diethyl ether (HPLC‐grade) were obtained from Mallinckrodt Baker (Milan, Italy). Hydrochloric acid (37%) and sodium hydroxide (analytical reagent grade) were purchased from Carlo Erba (Milan‐Italy).
Since romifidine pure standard was not available, the registered and commercial preparation Sedivet® (Boehringer Ingelheim Vetmedica, GmbH) was used as a reference standard, accepting the EU regulation that, concerning the veterinary medicinal products, establishes that the maximum acceptable deviation in the active substance content of the finished product shall not exceed ± 5% (Directive2001 /82/EC, 2001). Sedivet® is an aqueous solution containing romifidine hydrochloride (10 mg/mL). A bottle of Sedivet® was used to administer four horses the dosage of romifidine (100 μg/kg), and to prepare the reference and the calibration curves to be used for the analysis of samples from the same four horses. A second bottle of Sedivet®, from the same batch of the first, was used to administer the additional four horses, and to prepare the reference and the calibration curves to be used for the analysis of samples from these last four horses.
Since romifidine pure standard was not available, the registered and commercial preparation Sedivet® (Boehringer Ingelheim Vetmedica, GmbH) was used as a reference standard, accepting the EU regulation that, concerning the veterinary medicinal products, establishes that the maximum acceptable deviation in the active substance content of the finished product shall not exceed ± 5% (Directive
10
Isolation and Characterization of Equine BM-MSCs
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Animals from control group (n = 4) were sedated with 0.04 mg/kg IV romifidine (Sedivet, Boehringer-Ingelheim) and 0.02mg/kg IV butorphanol (Torbugesic, Pfizer) and 20–30 mL of bone marrow were collected from sternum in heparinized syringes with a Jamshidi needle. Procedures for MSC isolation, characterization and culture were performed as previously described [17 (link)]. Briefly, nucleated cells were harvested by density gradient centrifugation (Lymphoprep, Atom) and cultured in basal medium (low glucose Dulbecco’s Modified Eagle’s Medium [DMEM] with 10% fetal bovine serum [FBS], 2 mM L-glutamine, 0.1 mg/mL streptomycin and 100 U/mL penicillin [all from Sigma-Aldrich]) at 37 °C, 5% CO2. At passage 3, cells were characterized by flow cytometry and real time quantitative polymerase chain reaction (RT-qPCR) for surface markers expression and by tri-lineage differentiation potential according to methodology previously described [37 (link)]. Details about characterization of equine BM-MSCs can be found in Additional file 2 . Subsequently, BM-MSCs were frozen in medium FBS with 10% dimethyl-sulfoxide (DMSO) (Sigma-Aldrich) (− 80 °C).
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