During recent years, several studies have shown that ischemic preconditioning (IPC), defined as a brief period of ischemia followed by a short interval of reperfusion before a prolonged ischemic period, confers a state of protection in several organs, including the liver, resulting in improved organ tolerance to longer subsequent episodes of ischemia [5–7]. The exact mechanisms by which IPC confers protection is not fully understood [8, 9].

Microdialysis is a method that provides the opportunity for continuously monitoring metabolic changes in the liver and other tissues [10–13]. A number of different metabolites can be measured to monitor hepatic metabolism. In particular, glucose, lactate, pyruvate, and glycerol have been evaluated. In a previous study, we described profound metabolic changes in the pig liver during and after warm ischemia measured by microdialysis [14].

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. 1).

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. 1).

The main purpose of vascular occlusion is to decrease blood loss during liver resection. However, portal triad clamping by the PM not only produces ischemia but also leads to I/R injury when the blood flow to the liver is re-established. I/R injury can in some cases lead to either an uneventful postoperative course or postoperative death [15–17]. To avoid this situation, strategies for early detection of biochemical changes and potential minimization of the degree of I/R injury are important. We considered microdialysis to be an appropriate method to continuously obtain information about metabolic changes in the liver during ischemia before preconditioning. By monitoring glucose, lactate, pyruvate, and glycerol levels, we achieved an insight in molecules reflecting oxygen supply as well as cell membrane damage.

Glycerol is an integrate component of the cell membrane and, hence, an indicator of cell membrane disintegration [12, 18]. From the time of preconditioning, we observed a significant increase in glycerol levels before a decrease at the end of the ischemic period. This variation could be an expression of cellular disintegration because we observed similar variations in glycerol levels in a previous study on warm ischemia without preconditioning [14]. This is also in agreement with a study by Nowak et al. [12] on cold ischemia.

When total ischemia is performed and the oxygen supply is decreased, the organism changes to anaerobic metabolism and lactate is produced from pyruvate to maintain anaerobic glycolysis. In the aforementioned study [14], we observed a significant increase in lactate levels immediately after initiating ischemia. The same significant increase was observed in this study after preconditioning. In agreement with the change to anaerobic metabolism during ischemia, we had expected a decrease in pyruvate levels in the ischemic period. However, we did not see a decrease from the time of ischemia, but a steady state was observed during the ischemic period before a slight increase from time of reperfusion. This deviation could not be explained by collateral blood flow because all ligaments were transected to avoid this. Furthermore, the lactate–pyruvate ratio, which is a sensible marker for a low oxygen supply [19], increased significantly immediately after initiating ischemia as well as decreased rapidly after reperfusion.

The impaired blood flow to the liver and tissues is normally associated with a decreased delivery of glucose [20, 21]. However, we observed a significant increase in glucose levels from the start of ischemia. This increase could be explained by gluconeogenesis as part of anaerobic metabolism, but this is unlikely in the present study because we demonstrated an increase in lactate levels and a steady state in pyruvate levels at the same time. More likely, the increase in glucose levels results from the hepatic cells responding to activation from nucleotides as shown by Guinzberg et al. [22].

Transaminase levels are sensitive markers of hepatocyte injury [23]. In a study by Clavien et al. [24], IPC was found to confer protection to the liver as demonstrated by a decrease in AST and ALT levels 24 h after patients underwent surgery compared with patients who were not sustained to IPC. In our study, the hepatic injury was minimal as evaluated by changes in transaminase levels. Despite this, we demonstrated profound metabolic changes during and after warm ischemia preceded by IPC, changes that were similar to the ones we demonstrated in a previous study before, under, and after ischemia without IPC [14].