Catheters: In the acute series, a 16G central venous catheter (CVK, Secalon® T) was placed in the left external jugular vein for administration of anesthesia 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 (Cordis®, Johnson&Johnson™) was placed in the right hepatic vein draining segments V, VI, VII and VIII via the right internal jugular vein for blood pressure monitoring and blood sampling. 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. Inflation of the balloon allowed wedged hepatic pressure measurement. A pediatric CVK (Arrow® International) was placed in the portal vein for blood pressure monitoring and blood sampling. No catheters were placed in the pigs in the chronic series, as the main objective here was to anastomose the shunt from the aorta to the left portal vein branch with minimal damage to the hepatic hilus. Measurements: In the acute series, calibrated transducers (Transact 3™, Abbott Critical Care Systems, Chicago, IL, USA) were used for continuous pressure registration and signals were stored electronically (Macintosh Quadra 950, Apple Computers, CA, USA). Perivascular ultrasonic flow probes (CardioMed Systems, Medistim A/S, Oslo, Norway) were placed around the portal vein, right hepatic artery, left hepatic artery and around the aortoportal shunt. Cardiac output was measured by thermo dilution (Vigilance™ Volumetrics, Edwards Lifesciences™). Measurements were made in triplicate and averaged. The heart rate was monitored with an electrocardiogram (ECG). In the chronic series, the heart rate was monitored with an electrocardiogram (ECG). Flow in the aortoportal shunt was measured using an 8 mm perivascular ultrasonic flow probe (CardioMed Systems, Medistim A/S, Oslo, Norway). Surgery: In the acute series, after a midline laparotomy and placement of all catheters and flow probes as described above, we isolated and recorded the flow in the left portal vein branch (LPVB). When the activated clotting time (ACT) was above 250 seconds a 5mm Propaten Gore-Tex™ graft was anastomosed end-to-side from the aorta (between truncus coeliacus (TC) and the superior mesenteric artery (SMA)) to the LPVB. The LPVB was then ligated proximal to the bifurcation to prevent backflow to the main portal vein trunk (MPVT). The Bulldog was removed and time = 0 noted. Flow in the shunt was standardized to 1000 mL/minute by shunt constriction using a ligature and flow probe (fig. 1). In the chronic series, after a midline laparotomy, a similar shunt was placed from the aorta to the LPVB once the animal had received 5000 IE Heparin i.v. Only this time we used an interposed aorta graft from a donor pig (as the Gore-Tex grafts™ tended to become occluded). The LPVB was ligated proximal to the portal bifurcation to prevent backflow to the MPVT. Flow was standardized to 100mL/minute. Upon relaparatomy three weeks later, the shunt was isolated and flow measured. The flow in the MPVT (now supplying the right liver only) was recorded. Sampling: In the acute series, sequential biopsies were taken from the shunted segments II, III and IV at time points 1, 5, 10, 30, 60, 90 minutes and 2, 3, 4 and 6 hours after shunt opening (t = 0). Biopsies were placed immediately in RNAlater (Ambion®). In the chronic series, only peroperative arterial blood gas samples were taken (directly from the aorta) to monitor respiratory status.
Growth protocol
Eighteen castrate sus scrofa domesticus pigs were used for all experiments, conducted in compliance with the institutional animal care guidelines and the National Institute of Health’s Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985]. In the acute series, we followed the same anesthesia protocol as previously described (Mortensen et al. Am J Physiol Gastrointest Liver Physiol 294: G819–G830, 2008). In the chronic series, anesthesia was maintained with Isoflurane 1.5-2% mixed with 55% oxygen. Respiratory rate was adjusted to achieve an Et CO2 between 35 and 40 mmHg. Mean alveolar concentration of Isoflurane was maintained at 1.3 using a Capnomac (Nycomed Jean Mette). Analgesia was induced and maintained with Fentanyl 0.01 mg/kg. Before surgery, all animals received a single i.m. shot of antibiotic prophylaxis (Enrofloxacin, 2.5 mg/kg).
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 test 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 test 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 genes in the liver was studied under two predefined situations; portal arterialization of segments II, III and IV ( by aortoportal shunting) and sham. Gene expression profiles were obtained from biopsies sampled from the arterialized segments and from the same segments in sham animals. Sampling points were 1, 5, 10, 30, 60, 90 minutes and 2, 3, 4 and 6 hours after shunt opening
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.
