A total of 8 female pigs (Danish Landrace/Yorkshire; Påskehøjgård Centre, Aarhus, Denmark) weighing approximately 60 kg were used for the experiments. The research procedure was conducted under a local project license (registration number: 2002-561-574) in accordance with the Danish regulations on animal experiments.
The animals were overnight fasted before the experiment. After premedication with an intramuscular injection of midazolam 0.4 mg/kg and ketamine 4 mg/kg, the pigs were intubated and mechanically ventilated with a mixture of air, oxygen, and 1.5% isoflurane. Fentanyl was administered intravenously at 10 ml/h, as well as saline at 12.5 ml/kg/h.
The left carotic artery and the left jugular vein were exposed and catheters were inserted. Samples for blood gas analysis were drawn hourly from the left carotic artery and analyzed for pO2, pCO2, pH, arterial base excess, and lactate concentration on an ABL615 apparatus (Radiometer, Copenhagen, Denmark); blood pressures were also measured in this vessel. Fluid and drugs were administered through the left jugular vein, and blood samples for measurements of alanine aminotransferase (ALT), alkaline phosphatase (AP), bilirubin, prothrombin time (PT), and total leukocytes (TL) were also drawn from this vessel. Animals were placed on a heating blanket and rectal temperature was taken and maintained at 37.5°C. Finally, a urinary catheter was inserted.
Pigs were humanely killed with an overdose of saturated potassium chloride while under anesthesia at the end of each experiment.
A midline laparotomy was performed and the liver was mobilized. Structures in the portal triad were exposed. Two microdialysis catheters (CMA 60 Microdialysis Catheter, Stockholm, Sweden) were inserted and fixed in the liver, one in the left lateral lobe and another in the right lateral lobe. A reference catheter was inserted in the right biceps femoris muscle. Each microdialysis catheter was connected to a microinfusion pump (CMA 107: CMA Microdialysis AB) and perfused with Ringer’s chloride at a flow rate of 0,3 μl/min. After insertion of the catheters, a “washout period” of 1 h was used to flush the dialyses probes and allow the liver tissue to recover from cellular damage due to the implantation procedure.
Microdialysis samples were collected every 30 min during the experiment. Ischemic preconditioning was performed by subjecting pigs to 10 min of hepatic ischemia, followed by 10 min of reperfusion. Ischemia was achieved by using the portal triad clamping, that is, the PM. Total ischemia for 60 min was followed by 3 h of reperfusion (Fig. ).
The experimental design schematically
A total of 12 samples were collected. The dialysate in collected samples was analyzed for metabolites of the carbohydrate and lipid metabolism (glucose, lactate, pyruvate, and glycerol), using a CMA 600 microdialysis analyzer (CMA Microdialysis AB), and the lactate–pyruvate ratio was calculated. Between events in the monitored tissue and collected dialysate, there was a time delay of approximately 30 min REF. Baseline was an average of sample points 1–5. Sample point 6 represents time of preconditioning. Sample points 7 and 8 express the ischemic period. Sample point 9 represents the reperfusion period, and an average of sample points 10–12 represents the postreperfusion period (Fig. ).
During the experiment ALT, AP, bilirubin, PT, and total leukocyte (TL) were measured three times, at the baseline, at the end of ischemia, and at the end of reperfusion.