The study was performed using male Wistar rats (6 for each group) weighing 250–300 g. All animals (including controls) were anaesthetised by intraperitoneal administration of 2.7 ml/kg of a mixture of one part midazolam + one part Hypnorm ((1 ml of Hypnorm contains 10 mg fluanisone and 0.315 mg fentanyl citrate)) + two parts sterile water. The rats were placed supine on a heating pad to maintain body temperature between 36.5°C and 37.5°C throughout the procedure. The carotid artery was cannulated using an intravenous cannula (Portex, Hythe, UK, 20 G/32 mm) to allow for continuous blood pressure monitoring (Device Mx2, Lectromed UK, Letchworth, UK) and the infusion of normal saline at 0.5 ml/h. All animals underwent laparotomy. The left lateral and median lobes of the liver were mobilised and delivered through the incision after dividing their suspensory ligaments. An avascular plane was developed between the ventral surface of the liver and the portal vein and hepatic artery supplying these lobes. To induce hepatic ischemia, an atraumatic microvascular clip was applied to occlude the portal vein and hepatic artery just distal to the branches supplying the right lateral lobe for 45 min. After 6 h of reperfusion in the I-R groups, and in the control groups a time period equivalent to the duration of ischemia and reperfusion, blood (0.5 ml) was withdrawn from the arterial cannula for the measurement of liver enzymes. The left lateral and median lobes of the liver were harvested and divided for histological paraffin wax examination and immunohistochemical staining. All animal experimental procedures were carried out in accordance with UK Government legislation (Project Licence Number: 70/4393).
To study whether adenosine could modulate NOS expression in hepatic I-R, the following experimental groups were set up:
Group 1 animals subjected to anaesthesia and sham laparotomy only;
Group 2 animals subjected to 45 minutes of partial hepatic ischemia (as described above), followed by 6 h of reperfusion;
Group 3 animals subjected to hepatic ischemia (as described in Group 2), were pre-treated with adenosine (1 mg/kg) dissolved in bicarbonate-buffered saline (pH 7.4) administered via the carotid artery cannula over a 20 minute period. (Animals enrolled in a group not randomised to receive adenosine were given an equivalent volume of buffer solution at the corresponding time period). Hepatic ischemia was induced immediately following the termination of the adenosine infusion.
Group 4 animals treated with adenosine and subjected to hepatic ischemia (as described in Group 3) were also treated with the relative eNOS inhibitor NG-Nitro-L-arginine (L-NA). This was administered via the carotid cannula at a dose of 10 mg/kg, 3 minutes prior to the administration of adenosine. (Animals in all the other groups received an equivalent volume of buffer solution at the corresponding time period).
Heparinised blood samples from each rat were immediately centrifuged at 2,000 g for 10 minutes and the supernatant plasma was stored at -20°C. Aspartate and alanine transaminase levels in plasma were later measured in a clinical chemistry laboratory at Hammersmith Hospital, London, UK, using an Olympus AU600 Analyser (Olympus Optical Co Ltd, Tokyo, Japan), as markers of hepatocellular injury.
Liver sections from each animal were fixed in 10% neutral buffered formalin, dehydrated by passage through graded ethanol series, cleared in xylene and embedded in paraffin blocks. The blocks were 4-μm sectioned and stained with hematoxylin and eosin, according to standard protocols. Sections were evaluated using light microscopy by a blinded member of our research team.
Liver sections from each animal were fixed in 1% paraformaldehyde in phosphate buffered saline (PBS) for four hours and then transferred into a storage buffer, PBS/sucrose, at 4°C until the cryostat blocks were prepared. Tissue sections (4 μm) were cut on a cryostat and applied to slides. The avidin-biotin complex immunoperoxidase staining system was used. The primary antibody, anti-eNOS monoclonal, was produced in mice and was obtained from Affiniti Research Products Ltd, Exeter UK. The presence of positive staining with the specific antibody was indicated by the development of a reddish-brown stain in the section, as a consequence of using the 3,3'-diamino-benzidine substrate. Each section was reviewed by a blinded member of the research team who noted the presence or absence of staining and its cellular location.
The liver enzyme data were log transformed to satisfy the assumptions required to perform parametric tests and are therefore presented as geometric mean and 95% confidence intervals of the mean. Orthogonal contrasts in ANOVA were then used to analyse statistically the difference between any two specified groups.