Animals were premedicated with an intramuscular injection of ketamine (20 mg/kg) and atropin (1mg). Anaesthesia was induced with an intravenous bolus of pentobarbital (10 mg/kg) and fentanyl (10 µg/kg) and later maintained with continuous infusions of pentobarbital (4 mg/kg), fentanyl (0,02 mg/kg) and midazolam (0,3 mg/kg). All animals received a preoperative intravenous volume load consisting of 500mL 0,9 % NaCl containing 625 mg of glucose. Volume infusion was continued thereafter with 20mL/kg/hr 0,9% NaCl and 10% glucose plus 20% human albumin at 3mL/kg/hr. The animals were ventilated via a tracheotomy on a Servo Ventilator 900 (Elema-Schønander, Sweden). PCO2 was kept in the range 4,0 - 4,5 kPa by tidal volume adjustment. Core body temperature was maintained at 38,5 ± 1ºC with a heating blanket. A 16G central venous catheter (CVK, Secalon® T) was placed in the left external jugular vein for administration of anaesthesia and infusions. A 5 French Swan-Ganz catheter (Edwards Lifesciences) was floated via the right external jugular vein to the pulmonary artery for cardiac output (CO) measurements. A 16G CVK (Secalon® T) was placed in the left femoral artery for continuous arterial blood pressure monitoring. A 7 French 110 cm angiographic catheter (external diameter 2.3 mm, internal diameter 1.2 mm, 12 sideholes) (Cordis®, Johnson&Johnson) was placed in the hepatic vein draining segments V and VIII via the right internal jugular vein for blood pressure monitoring and blood sampling. Correct placement was ensured by direct palpation after laparotomy and later controlled by post-mortem dissection. A 5 French Swan-Ganz catheter (Edwards Lifesciences™) was placed in the hepatic vein draining segments II and III by direct transhepatic placement for pressure monitoring and blood sampling. A 4 French, 2 lumen 13 cm paediatric CVK (Arrow® International) was placed in the portal vein with the tip approximately 5 cm from the liver hilus. This was used for pressure monitoring and blood sampling. The bladder was drained via a cystostomy. Calibrated transducers (Transpac 3™, Abbott Critical Care Systems, Chicago, IL, USA) were used for continuous pressure registration. The transducers were connected to an amplifier (Gould, 2800S, Ohio, USA). Pulsatile signals were displayed on a monitor, digitalised and stored electronically (Advantech, Industrial Computer). All recordings were logged every fourth second. For graphic presentation the data was processed by Labview version 6.0™ (National instruments, Austin, TX, USA). Perivascular ultrasonic flow probes (CardioMed Systems, Medistim A/S, Oslo, Norway) were placed around the portal vein (12 mm probe), right hepatic artery (2 mm probe), left hepatic artery (3 mm probe) and around the aortoportal shunt (5 mm probe). Signals were displayed on a monitor and stored electronically (Advantech, Industrial Computer). Cardiac output was measured by the thermo dilution technique using 5 ml 0,9 % NaCl chilled to < 5 ºC, via a 5F Swan-Ganz catheter (Vigilance™ Volumetrics, Edwards Lifesciences™). Measurements were made in triplicate and documented as the average value. The same investigator made all measurements. A rectal probe recorded core body temperature. Three pigs (2048, 2055 and 2056) underwent a 62% liver resection (low-pressure resection, LPR) and three (2045, 2047 and 2049) underwent a 75% resection (high-pressure resection, HPR). Operative procedures: after a midline laparotomy and placement of all catheters as described above, the hepatic artery supplying segments II and III (left lateral lobe) together with these segments' portal branch were ligated using an absorbable polyfilament suture on a large needle. Thereafter the lobe was strangulated with a 0,5 cm wide cotton ribbon and then removed and weighed. Segments IV, V and VIII were removed in a similar manner in the LPR series leaving segments VI, VIII and I in place. In the HPR series, the resection was continued removing segments VI and VII as well. Biopsies were sampled from the remaining segments VI, VII in the LPR series and from segment I in the HPR series and placed immediately in RNALater (Ambion®). Sampling time points were 1, 5, 10, 30, 60, 90 minutes, 2, 3, 4, 5 and 6 hours after completion of resection. All biopsies were stored overnight in 4ºC and thereafter in - 70 ºC until further processing. The pigs were sacrificed with an intravenous overdose of 100 mg pentobarbital and 20mmol KCl intracardially on completion of the experiment.
Growth protocol
Animals were acclimatized in the animal department for at least 48 hours prior to experiments. Conditions were set to room temperature of 20±1ºC, relative humidity of 55±10%, and a 12-hour light/dark cycle. The animals were fed Combi Fri™ chow (Felleskjøpet, Trondheim, Norway), but were fasted overnight prior to the experiment with free access to water.
Extracted molecule
total RNA
Extraction protocol
RNeasy Maxi Kit with DNase treatment following the enclosed protocol (Qiagen)
Label
Alexa-594
Label protocol
20 µg total RNA was labelled by using the Superscript Indirect cDNA Labeling System (Invitrogen) in combination with ARES cDNA labeling kits (Molecular Probes/Invitrogen) following the enclosed protocols. Spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the cDNA reactions. ""Green"" spike-in RNA was added to the common reference samples and ""red"" spike-in RNA was added to the samples.
RNeasy Maxi Kit with DNase treatment following the enclosed protocol (Qiagen)
Label
Alexa-488
Label protocol
20 µg total RNA was labelled by using the Superscript Indirect cDNA Labeling System (Invitrogen) in combination with ARES cDNA labeling kits (Molecular Probes/Invitrogen) following the enclosed protocols. Spike-in RNA from the Lucidea Universal ScoreCard (Amersham Biosciences) was added to the cDNA reactions. ""Green"" spike-in RNA was added to the common reference samples and ""red"" spike-in RNA was added to the samples.
Hybridization protocol
The slides were hybridized in a Discovery XT hybridization station (Ventana Discovery Systems, Tucson, AZ, USA). Transfer Chip Prep-2 from 4 ºC to room temperature 1 hour before use. Prepare ChipSpread by mixing equal volumes of ChipSpread A (20 mg/mL BSA, 4x SSC, 0.5 mg/mL sodium azide) and B (formamide; 2 mg/mL SDS) and incubate at room temperature for 1 hour before use. A total of 2.5 mL is needed per slide. Print labels, trim them and place them on the slides. Mix the Chip Map reagents (Chip Prep-1, -2 and - 3) by inversion, remove the cap and place the reagents in the Discovery. Place the slides in the machine and initiate the run. Cover slide with 2.5 mL ChipSpread when the message appears (after few minutes). The machine now runs for app. 1.5 hours to pre-hybridize the slides. Heat a waterbath to 90°C or use a PCR machine. Mix the Chiphybe80, add 200 µL to the sample (<20 µL) and mix carefully. Heat the sample mixture at 90°C for 3 minutes and mix carefully by pipetting. Press ""button"" on the machine which then prepares the slides for hybridization. When the message appears apply the samples onto the slides and press ""button"" and the machine hybridizes at 48 ºC for 6 hours. Wipe oil from backside of slides using a clean-room napkin and place slides in the slide-holder from the High Throughput Wash Station (Telechem, cat.no. HTW) placed in a mTub filled with RiboWash. If processing more than 20 slides, place equal number of slides in two slide-holders and continue in parallel. Transfer the slide-holder to a HTW filled with RiboWash and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with RiboWash and repeat the wash. Dip the slide-holder in 2x SSC filled in a mTub (200 mL 20x SSC, Elga H2O + 1800 mL water). Transfer the slide-holder to a HTW filled with 2x SSC and wash for 2 min with magnetic stirring at 700 rpm. Refill the HTW with 2x SSC and repeat the wash. Dip the slide-holder 10 times in 0.1x SSC filled in a mTub (5 mL 20x SSC, Elga H2O + 995 mL water) and leave the holder submerged in 0.1x SSC. Transfer the slides to a mBox slide holder placed in a mTub filled with Elga H2O. Dry arrays by centrifugation (at 300 x g for 4 min placed in a mBox)
Scan protocol
Scanner: ScanArray Express HT system (Perkin Elmer), 5 µm resolution, 100 % laser power and PMT adjusted individually for each channel. Image analysis software: GenePix Pro (version 6.0.1.22, Molecular Devices) using FeatureType = Irregular Filled and LocalFeature as background measurement.
Description
The differential expression of immediate-early and delayed genes in the liver remnant was studied under two predefined pressure situations; low portal venous pressure and thus low sinusoidal pressure (62 % resection) and high portal venous pressure and high sinusoidal pressure (75 % resection). Gene expression profiles were obtained from biopsies sampled in the liver remnant at time points 1, 30, 90, minutes and 3, 4 and 6 hours after resection.
Data processing
Statistical analysis was carried out in the R computing environment (version 2.3.1 for Windows) using the package Linear Models for Microarray Analysis (Limma, version 2.7.10) which is part of the Bioconductor project. The median intensities were background corrected by using the ""normexp"" function on the median background intensities. Following background correction, the log2-transformed ratios of Alexa-594 to Alexa-488 were normalized within-slide using printtip-loess with default parameters as implemented in Limma. The set of normalized log-ratios were then analyzed in Limma to identify genes being significantly differentially expressed between time points within treatment as well as between treatments. Time point contrasts were formed referring to the sample taken at time point 1 min. followed by multiple testing across genes and contrasts with p-values below 0.001 considered as significant. The features of the arrays were mapped to a LocusLink identifier and an annotation package was built using the Bioconductor package AnnBuilder (version 1.9.14). Tests for significantly (P < 0.05) overrepresentation of gene ontology (GO) terms were conducted using the GOHyperG function of the Bioconductor package GOstats (ver 1.5.5). The sets of overrepresented GO terms were further analysed by utilising information from Online Mendelian Inheritance in Man (OMIM) to group the genes by function.
