124 Chapter 6 Animals and anesthesia Four mature female Landrace pigs (35-45kg) underwent general anesthesia ensured with appropriate analgesia using the following medication: intramuscular injection of Zolazepam/ Tiletamine (6 mg/kg, Virbac, Barneveld, The Netherlands) and Thiopental (10 mg/kg, Panpharma SA, Trittau, Germany), a combination of sufentanyl (0.01 mg/kg/h, Hameln Pharma GmbH, Hameln, Germany), Propofol (9 mg/kg/h, B. Braun Melsungen AG, Melsungen, Germany), and Midazolam (1 mg/kg/h, Aurobindo, Baarn, The Netherlands) intravenously. All pigs were intubated and mechanically ventilated. Alterations in vital parameters were monitored by an animal anesthesiologist, and whenever necessary, anesthesia and analgesia were intensified. At the end of the procedure, all animals were euthanized with a lethal dose of 200 mg/kg pentobarbital. Preparation of dyes The preparation of dyes was carried out as previously described 8. In short, MB was diluted in a sterile phosphate-buffered saline (PBS) solution to a concentration of 1 mg/mL and ICG in a sterile H2O solution to a concentration of 2.5 mg/mL. An IV dose of 0.25 or 0.50 mg/kg of MB (doses based on a previously published dose finding study 8) and 0.2 mg/kg of ICG was administered. This ICG dose is the current frequently clinically used dose range in patients, based on the analysis of 1,240 patients registered in the EURO-FIGS registry on fluorescence angiography 10. Surgical procedure and measurements After appropriate sedation and analgesia, a midline laparotomy was performed by an experienced surgeon. A small bowel loop with a length of approximately 15 cm, was measured at 250 cm from the gastric pylorus. To ensure optimal exposure, the loop was placed on a gauze. The mesenteric side with at least 8 vessels was transected to create a gradual ischemic loop, as described in an earlier study 11. After compromising the intestinal tissue perfusion (T = 0), 5 ROIs (Figure 1) were marked and defined as follows: 2 on the lateral sides of the loop (well-perfused), 1 in the exact middle of the loop (not perfused), and 2 between the lateral and middle ROIs (partly perfused = watershed area). This method has been previously explained by our group 12. Subsequently, a systemic lactate measurement was taken from the central ear vein. MB was injected after 10 minutes (T = 10) and bowel perfusion imaging in MB mode was performed for at least 60 seconds. Sixty minutes after ischemic loop creation, the camera system was switched to ICG mode, followed by ICG injection (T = 60) and fluorescence quantification analysis for 60 seconds. This procedure was directly followed by local capillary lactate sampling by puncturing the serosa at each of the 5 ROIs. The latter was done using a 23 Gauge needle and an EDGE lactate analyzer (ApexBio, Taipei, Taiwan, People’s Republic of China), which only requires a small drop of blood (3 μl) 12. As lactate is a marker of ischemia 13, it was used as the gold standard to correlate the fluorescence signal. All animals were followed for a minimum of 120 minutes (T = 120). A schematic overview of the surgical procedure is displayed in Figure 1.
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