Pancreas perfusions were performed as previously described [14] (link). Briefly described, mice were anaesthetised with i.p. injection of ketamine (90 mg/kg; Ketaminol Vet.; MSD Animal Health, USA) and xylazine (10 mg/kg; Rompun Vet; Bayer Animal Health, Germany). The intestine, spleen, stomach and kidneys were tied off. The aorta was ligated proximally to the coeliac artery, and a catheter was inserted into the abdominal aorta allowing arterial perfusion with a modified Krebs-Ringer bicarbonate buffer (0.1% BSA, 5% (wt/vol.) dextran in addition). Venous effluent was collected via a catheter in the portal vein. The perfusion system (UP-100 universal perfusion system, Hugo Sachs Elektronik, Germany) maintained constant flow of 1 ml/min, perfusion buffer was heated and oxygenated (95% O 2 , 5% CO 2 ) and pressure was monitored throughout the experiment (40-50 mmHg). Experiments exhibiting significant changes in pressure or flow (>20%) were terminated and discarded.
Ketaminol vet
Manufactured by MSD animal health
Sourced in United States
Ketaminol Vet is a veterinary anesthetic agent containing the active ingredient ketamine. It is used for the induction and maintenance of general anesthesia in animals. The product comes in a sterile solution for injection.
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5 protocols using ketaminol vet
1
Perfusion of Murine Pancreas
2
Pancreas Perfusion for GLP-1R Assessment
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Pancreas perfusions were performed as previously described (46 (link)). In short, the mice were anesthetized with intraperitoneal injection of ketamine (90 mg/kg Ketaminol vet, MSD Animal Health) and xylazine (10 mg/ml, Rompun vet, Bayer Animal Health).
The stomach, kidney, and spleen were tied off. Proximally to the celiac artery, the aorta was ligated, and a catheter was inserted in the aorta thereby providing arterial perfusion with a modified Krebs-Ringer bicarbonate buffer (in mM: 118.3 NaCl, 3.0 KCL, 2.6 CaCl2*2H2O, 1.2 KH2PO4, 1.2 MgSO*2H2O, 25.0 NaHCO3, 10 glucose, 0.1% bovine serum albumin, 5% dextran) (Pharmacosmos). Effluent samples were collected through a portal vein catheter every minute. The perfusion system (UP-100 universal perfusion system, Hugo Sachs Electronic) had a constant flow of 1 mL/min, perfusion buffer was maintained at 37°C, oxygenated with 95% O2 to 5% CO2, and perfusion pressure (40-50 mmHg) was monitored throughout the experiment. GLP-1R KO mice or WT littermates (n = 8) were stimulated for 10 minutes with 0.1 nM and 1.0 nM GLP-1 7-36 amide (Bachem) at 15 and 40 minutes, respectively. At the end of the experiments, L-arginine was added as a positive control (10 mM).
Insulin concentrations in venous effluents were quantified by use of an in-house radioimmunoassay, employing ab code 2006-3 (47 , 48 (link)).
The stomach, kidney, and spleen were tied off. Proximally to the celiac artery, the aorta was ligated, and a catheter was inserted in the aorta thereby providing arterial perfusion with a modified Krebs-Ringer bicarbonate buffer (in mM: 118.3 NaCl, 3.0 KCL, 2.6 CaCl2*2H2O, 1.2 KH2PO4, 1.2 MgSO*2H2O, 25.0 NaHCO3, 10 glucose, 0.1% bovine serum albumin, 5% dextran) (Pharmacosmos). Effluent samples were collected through a portal vein catheter every minute. The perfusion system (UP-100 universal perfusion system, Hugo Sachs Electronic) had a constant flow of 1 mL/min, perfusion buffer was maintained at 37°C, oxygenated with 95% O2 to 5% CO2, and perfusion pressure (40-50 mmHg) was monitored throughout the experiment. GLP-1R KO mice or WT littermates (n = 8) were stimulated for 10 minutes with 0.1 nM and 1.0 nM GLP-1 7-36 amide (Bachem) at 15 and 40 minutes, respectively. At the end of the experiments, L-arginine was added as a positive control (10 mM).
Insulin concentrations in venous effluents were quantified by use of an in-house radioimmunoassay, employing ab code 2006-3 (47 , 48 (link)).
3
Intestinal Tissue Sampling Protocol
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Weight was measured daily between 8:00 a.m. and 11:00 a.m. Doxo-treated animals experiencing a weight loss <5% were excluded from the study due to presumed injection error (28 (link), 29 (link)). The mice were anesthetized by IP injection of ketamine 100 mg/kg (Ketaminol® Vet, MSD Animal Health, Boxmeer, The Netherlands) and xylazine 10 mg/kg (Rompun® vet, Bayer Animal Health GmbH, Leverkusen, Germany) and subsequently exsanguinated by collecting a blood sample by cardiac puncture. Animals were perfused with phosphate-buffered saline and a tail biopsy was collected. The small intestine was removed from the pyloric sphincter to the ileocecal junction and divided into three equally sized pieces representing duodenum, jejunum and ileum for use in histopathological analysis, quantitative real time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The length of these segments was measured, and the segments were flushed with phosphate-buffered saline. Samples for histopathological analysis were fixated in 4% formaldehyde (VWR Chemicals, Leuven, Belgium) for 48 h, transferred to phosphate-buffered saline with 0.05% NaN3, embedded in paraffin, mounted on glass slides, and stained with haematoxylin and eosin (HE) at the Department of Pathology, Odense University Hospital, Odense, Denmark.
4
Analgesic Drug Combination Protocol
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The selective 5HT1B/1D receptor agonist sumatriptan (GSK Denmark, Imigran 12 mg/ml; 1 mg/kg) was diluted in 0.9% saline and administered s.c. The CGRP receptor antagonist olcegepant (HCl salt; 1 mg/kg) was dissolved in 0.9% saline and administered i.p. The partial μ-opioid receptor agonist buprenorphine (Indivior UK, Temgesic 0.3 mg/ml; 0.1 mg/kg) was diluted in 0.9% saline and administered s.c. The GABAA receptor positive allosteric modulator midazolam (Midazolam Hamelm Pharma Plus Germany, 1 mg/ml; 0.3 mg/kg) was diluted in 0.9% saline and administered i.p. The NMDA receptor antagonist ketamine (MSD Animal Health Ketaminol Vet. 100 mg/ml; 30 mg/kg) was diluted in 0.9% saline and administered i.p. All drugs were administered in a dosing volume of 1 ml/kg, and obtained from Glostrup Hospital Pharmacy except olcegepant HCl which was obtained from MedChemExpress (MedChemTronica AB, Sweden).
5
Embryonic Intestine Transplantation in Mice
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EYFP male mice were mated overnight with C57BL/6Nrj females and the following morning was defined as gestational day 0.5 (E0.5). Pregnant dams were sacrificed at E12.5 and small and large intestine were dissected from embryos under a stereo microscope (VWR). Adult WT mice were anaesthetized by i.p injection of Ketaminol Vet. (100 mg/kg, MSD animal health) and Rompun Vet. (10 mg/kg, Bayer) and were injected subcutaneously with Bupaq (0.1 mg/kg, Richter Pharma). Washed embryonic intestine was transplanted under the kidney capsule of anesthetized recipients as described previously42 . Recipients were sacrificed at the time points indicated and grafts were dissected and cut into pieces prior to cell isolation as described below.
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