The first step consists in performing a sample size calculation. Once it is clear, the experimental procedure can start. All animals should be managed in compliance with the laws for animal use in the respective country and the directives of the European Community Council (2010/63/EU) and ARRIVE guidelines(5). Adult swine (Sus scrofa ssp. domesticus - midweight) should be housed and acclimatised for 48 hours in an enriched environment, with constant humidity and temperature conditions. Twenty four hours of fasting before surgery should be planned, with ad libitum access to water. The stress can be reduced via sedation (zolazepam + tiletamine 10mg/kg IM) 30 min before the procedure. Circadian cycles of light-darkness must be respected. The anesthesia can be performed intravenously (18 gauge IV catheter in ear vein) with Propofol (3mg/kg) and maintained with rocuronium 0.8mg/kg along with inhaled isoflurane 2%. Animals can be euthanised with a lethal dose of pentobarbital (40mg/kg) at the end of the procedure.
Once a midline laparotomy has been performed, different types of vascular occlusion can be planned to follow the models listed in the introduction. For the TVIO with the inclusion of the bile duct, it is possible to ligate the whole hepatic pedicle in the proximal section to the branch’s bifurcation and after the gastroduodenal artery. For the isolation of the bile duct and of any partial ligation, the dissection of the hepatic pedicle is mandatory. Once the ligation of the targeted vessel(s) has been performed, the ischemic phase can be held for 90 minutes, which is sufficient to induce liver damage in pig models, or for a specific amount of time depending on the aim of the study(6). The reperfusion phase can be observed at different timepoints considering that a period of 5 hours is necessary to observe a damage between the early stage and the beginning of the late stage (7). Data can be collected every 30 minutes during the ischemic phase and every hour during the reperfusion phase.
Blood analysis can be obtained by sampling blood from a catheter placed in the jugular vein (6 French IV catheter). Systemic and capillary lactate should be analysed to understand the stress of the organ in real time (EDGE, ApexBio, Taipei, Taiwan). Capillary lactate can be sampled on the dorsal surface of the liver with a needle. The surgical intervention should be monitored to rule out any bias during the ischemic phase using a blood gas analysis (BGA) with the epoc Blood Analysis System (Siemens Healthineers) to measure pO2, pCO2, pH, glucose, creatinine, urea, and blood urea nitrogen (BUN). Liver functionality should be assessed at each timepoint analysing aspartate aminotransferase (AST), alanine aminotransferase (ALT), prothrombin time (PT), gamma glutamyl-transferase (GGT), alkaline phosphatase (ALP), total protein (TP), and albumin analysis.
Tissue sampling can be used to make many different analyses (such as H&E, IHC, IF, RNA expression) depending on the aim of the study.