The study was approved by the Institutional Animal Care and Use Committees (Committees for IAC & U) of the University of Defence Faculty of Military Health Sciences and the Charles University Faculty of Medicine in Hradec Králové, Czech Republic. All procedures were conducted in accordance with Act No 246/1992 Coll. on the Protection of Animals Against Cruelty. The study used 15 female domestic pigs weighing 31–42 kg each. All experimental animals were owned by the University of Defence Faculty of Military Health Sciences and the Charles University Faculty of Medicine in Hradec Králové, Czech Republic, which issued written informed consent to use the animals in this study. The manuscript adheres to the ARRIVE guidelines for the reporting of animal experiments.
After a 24 hour fast with free access to water, general anaesthesia was induced using an intramuscular administration of 20 mg/kg of ketamine (Narkamon, Zentiva Group, a.s., Prague, Czech Republic), 4 mg/kg of azaperone (Stresnil, Janssen Pharmaceutica, Beerse, Belgium), and 0.05 mg/kg of atropine (Atropin, Hoechst-Biotika, Martin, Slovakia), followed by cannula insertion into the peripheral vein to ensure venous access. The pigs were orotracheally intubated with a Müller long blade laryngoscope and the lungs were ventilated with a Servo 900C ventilator (Siemens-Elema AB, Solna, Sweden) at a tidal volume of 10–15 ml/kg and with an inspired oxygen fraction (FiO2) of 40%.
During the surgical part of the experiment, anaesthesia and analgesia were maintained with continuous intravenous (IV) infusions of midazolam at 0.3 mg/kg/h and fentanyl at 30 μg/kg/h. Postoperatively, the dosage of fentanyl was reduced to 5 μg/kg/h and neuromuscular blockade was induced by continuous IV infusion of pancuronium bromide at 0.2 mg/kg/h. The right femoral artery was cannulated for blood sampling and invasive blood pressure monitoring. The right jugular vein was cannulated for pulmonary artery catheterization and insertion of a central venous catheter (Pressure transducer, Gabarith PMSET 1DT-XX, Becton Dickenson, Singapore; Datex-Ohmeda S/5 monitoring system, Instrumentarium Corp, Helsinki, Finland) [Fig1]. Electrodes were attached to measure electrocardiogram signals, oxygen saturation, and rectal body temperature. Cystostomy and urinary catheterization were performed perioperatively through a middle laparotomy.
Preoperative blood samples were taken from all pigs to determine serum lactate concentrations, pH, base excess, and PaO2. In addition, a Swan-Ganz catheter was used to measure cardiac output.
The initial step in the procedure involved irrigating the rectum with an antiseptic Betadine solution (10% Povidone-iodine, Egis Pharmaceuticals Ltd., Budapest, Hungary) and evacuating residual stool from the rectum.
The abdominal cavity was accessed through a lower middle laparotomy, and a laser Doppler electrode was attached to the distal region of the sigmoid colon. The electrodes were then attached to the rectal wall using 4–0 monofilament sutures (Premilene, B. Braun Melsungen AG, Melsungen, Germany) [Fig2]. The electrode was then connected to a tissue perfusion monitor (Moor Instruments Ltd, Devon, UK) and tissue microperfusion values (baseline) were read and recorded. The inferior mesenteric artery (IMA) and inferior mesenteric vein (IMV))[Fig3] were then ligated, and tissue microperfusion values were recorded (starting 30 min after ligation); values were recorded at 30 min intervals until 120 min, and then at 60 min intervals until 240 min of the experiment.
The rectum and mesorectum were mobilized enbloc down to the pelvic floor, at the level where the rectum was ligated, using a Contour® curved stapler (Johnson & Johnson Inc, Cornelia, GA, USA). The rectal stump was irrigated with an antiseptic Betadine solution, during which the staple line was monitored for leakage. The ligated rectum was then extracted, followed by ligation of the large bowel in the distal region of the sigmoid colon. A circular stapler cap was inserted into the ligated lumen of the large bowel and the lumen was closed with a circular, monofilament, suture.
An LDF electrode was attached 1 cm proximal to the expected anastomosis site. An end-to-end stapled anastomosis was performed using a 25 mm circular stapler (Johnson & Johnson Inc., Cornelia, GA, USA). Following anastomosis, LDF values were recorded. Saline was instilled in the abdominal cavity and air was introduced into the rectum to test for anastomotic air leakage and verify anastomotic integrity. After saline aspiration, a laparotomy was used to remove the electrodes and the laparotomy was closed with a continuous suture.
Throughout the entire operation and post-operative period, the pigs received IV crystalloids (Infusio Hartmanni, Medicamenta, Vysoké Mýto, Czech Republic) at a constant rate of 5, 10, or 20 ml/kg/h depending on group. After 6 hours, the electrodes were removed and the animals were euthanized by the T61 Euthanasia Solution (Merck Animal Health, Canada). Each mL of T–61 contains 200 mg embutramide which produces a strong narcotic action and concurrently paralyses the respiratory centre, 50 mg mebezonium iodide which produces a curariform paralytic action on striated skeletal and respiratory muscles and rapidly induces circulatory collapse and 5 mg tetracaine hydrochloride, in aqueous solution.