Methods The surgical and experimental protocols were approved by the Danish Animal Research Committee, Copenhagen, Denmark according to license number 2007/561-1311 and followed the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health. Twenty-eight adult male Wistar rats weighing 300-350 g (M&B Taconic, Eiby, Denmark) were used for the experiment. Animals were housed in standard animal laboratories with a temperature maintained at 23°C and an artificial 12-hour light-dark cycle, with food and water ad libitum, until the time of the

experiment. The rats were randomly divided into five groups as follows: sham operated control (CG) (n = 4); pure ischemia and reperfusion (IRI) (n = 6); IPC (n = 6); IPO (n = 6); and IPC+IPO (n = 6) (Figure 1). All animals were anaesthetized with 0.75 ml/kg Hypnorm s.c. XL184 (Fentanyl/Fluanisone, Jansen Pharma, Birkerød, Denmark) and 4 mg/kg Midazolam s.c. (Dormicum, La Roche, Basel, Switzerland) and placed on a heated pad. A midline laparotomy was performed and total hepatic ischemia was accomplished selleck chemical using a microvascular clamp placed on the hepatoduodenal ligament, i.e., performing the Pringle maneuver. Reflow was initiated by removal of the clamp. Discoloration of the liver was used as a positive marker for hepatic ischemia. Reperfusion was ascertained by the return of the normal brown/reddish color of the

Dichloromethane dehalogenase liver. The experimental protocol was performed as described in Figure 1. At the end of each experiment after 30 min of reperfusion, a biopsy was taken from the right liver lobe, immediately frozen in liquid nitrogen and stored at -80°C for further analysis. Blood samples were collected from the common iliac artery in tubes for measurement of alanine aminotransferase (ALAT), alkaline phosphates and bilirubin, and analyzed immediately hereafter. All rats were subsequently killed with an overdose of pentobarbital. Figure 1 Experimental protocol of the five groups. Black areas represent periods of hepatic ischemia; white areas represent periods of normal hepatic

blood perfusion. Liver biopsies were collected at the end of each experiment. CG, Control group. IRI, 30 min of ischemia. IPC, ischemic preconditioning + 30 min of ischemia. IPO, 30 min ischemia + ischemic postconditioning. IPC+IPO, ischemic preconditioning + 30 min of ischemia + ischemic postconditioning. Quantitative Real-Time PCR (RT-PCR) After homogenization of liver tissue by the use of a MM301 Mixer Mill (Retsch, Haan, Germany), total cellular RNA was extracted from the liver tissue using a 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA). The quality of rRNA was estimated by agarose gel electrophoresis by the appearance of two distinct bands visible by fluorescence of ethide bromide representing intact rRNA.