The study was approved by the Linköping Animal Ethics Committee (Linköping, Sweden, ID 105/10) and conducted in accordance with the guidelines of the European Union for the protection of animals used for scientific purposes (23). The study was undertaken during November 2014 at a research laboratory at the Örebro University Hospital, Örebro, Sweden. Twenty-five pigs (a crossbreed between Swedish country breed, Hampshire and Yorkshire; 3-4 months old; weight range 25-35 kg) of both sexes were used for the experiment. Consent to participate was obtained from the farmer. The animals had free access to food and water before experimentation.
The dataset used in this article are available from the corresponding author on request.
Premedication, general anesthesia, ventilation, antibiotic prophylaxis and euthanization at the end of the experiment were recently described (21). However, one difference was that intravenous bolus doses of pethidine (25-50 mg h-1) were used in this study instead of fentanyl infusion. In brief, the animals were premedicated with azaperone (intramuscular injection) at the farm and transported to the laboratory. General anesthesia was induced by an intramuscular injection of a mixture of azaperone, tiletamine and zolazepam, and maintained by continuous intravenous infusion of propofol and intermittent intravenous boluses of pethidine (25-50 mg h-1). Cefuroxime and atropine were also administered intravenously and intramuscularly, respectively. After endotracheal intubation, the animals were mechanically normoventilated with a positive end-expiratory pressure of 5 cmH2O. Fluid loss was substituted with continuous intravenous infusions of Ringer’s acetate and glucose solutions. Body temperature was kept between 37-39 ºC using forced-air warming blankets. At the end of the experiments, the animals were euthanized with fast intravenous injections of propofol and potassium chloride, and ECG confirmed asystole.
The basic surgical preparation and measurements have been recently described in detail (21). In short, a pulmonary arterial catheter was used for measurements of cardiac output (CO), pulmonary wedge pressure and central venous pressure, and for sampling of mixed venous blood. The common carotid artery catheter was used to measure systemic blood pressure and heart rate (HR), and for arterial blood sampling above the occlusion balloon. The right common femoral artery (CFA) was surgically exposed and used for REBOA introduction and arterial blood sampling below the balloon. The left CFA and superior mesenteric artery (SMA) were dissected, and transonic flow measurement probes were placed around them to measure distal blood flow. A catheter was placed in the superior mesentery vein for measurement of mesenteric venous pressure and for blood sampling. A urinary catheter was placed in the urinary bladder for measuring urinary output and taking urine samples. Microdialysis (mDialysis M62, Sweden) catheters (M70 catheter) were placed intraperitoneally, in the left lower side of the abdomen, and intrahepatically for analysis of extracellular concentrations of glucose, glycerol, lactate and pyruvate.
Before intervention, the animals were each randomized, using envelopes, into one of four groups: 15 min REBOA (REBOA15), 30 min REBOA (REBOA30), 60 min REBOA (REBOA60) in the thoracic descending aorta (Zone I) and a control group (no REBOA, C). The REBOA catheter (Rescue Balloon, 7 Fr, Tokai Medical Products Inc, Kasugai, Japan) was retrogradely advanced with fluoroscopic guidance. After the occlusion, the balloon was slowly deflated over a period of two minutes but not removed. The animals were observed for 3 hours after the intervention and hemodynamic variables, blood gases, serum for inflammatory markers, and end organ metabolic enzymes via arterial blood samples were examined. During reperfusion, the animals received only crystalloid fluids, and no vasopressors or inotropic drugs were used. So as not to interfere with the physiological response, the same amount of fluid was given to all groups.
Temperature, urinary output and blood gas analysis from the carotid artery and mesenteric vein were monitored. Intraperitoneal and intrahepatic metabolism was measured using microdialysis catheters, including analysis of glucose, lactate, pyruvate and lactate-pyruvate ratio, and also glycerol as a marker of cellular damage (21). The inflammatory response was measured by collecting arterial carotid blood in test tubes, allowing coagulation for 30 minutes followed by centrifugation to achieve serum. The serum was then stored at -80° C until analysis. Cytokine detection in serum was performed as previously described (21). Tissue samples from the small bowel (2 cm x 2 cm) were taken, placed in formalin solution and stained with hematoxylin and eosin stain (21). The pathologist was blind to the randomization of the animals, and analysis of the severity of the histological changes was assessed using a six-grade system with grade 0 representing normal tissue and grade 5 necrosis (24, 25).
IBM SPSS version 23 was used for statistical analysis. Data are presented as mean and 95% confidence interval. For analysis of variance, a linear mixed model analysis was performed where the repeated factor was time and the other factor was group. If values were significant, post-hoc multiple comparisons were performed using Bonferroni correction. Normal distribution was investigated with the Shapiro-Wilk test. For non-normal distribution, log10 transformation was used, mainly for inflammatory markers. Statistical significance was considered at p <0.05.