Twenty-four male IL10-/- and twenty-four male C57 mice, four weeks of age were used for the experiment. All mice were between 28 to 35 days of age at the start of the trial, and were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). Mice were individually housed in standard shoebox size cages (332 mm x 150 mm x 130 mm) containing untreated wood shavings and a plastic tube for environmental enrichment, and maintained under conventional conditions with a temperature of approximately 22°C, 50% relative humidity and a 12 hour light-dark cycle. After 4 days, all mice were inoculated orally with bacteria commonly found in the intestinal lumen to obtain a more consistent and reproducible intestinal inflammation. Briefly, a mixture of Enterococcus faecalis and E. faecium (EF) strains and complex intestinal flora (CIF) was administrated via oral gavage. Mice were randomly assigned to one of four AIN-76A based diets made in-house with six Il10-/- and six C57 mice in each group. Mice were randomly assigned to four treatment groups of 12 animals each. Each treatment group received one of the following diets: 1) AIN-76A diet 2) AIN-76A (fat-free) + 1% corn oil + 3.7% oleic acid (OA, control) 3) AIN-76A (fat-free) + 1% corn oil + 3.7% eicosapentaenoic acid (EPA,n-3) 4) AIN-76A (fat-free) + 1% corn oil + 3.7% arachidonic acid (AA, n-6). Corn oil supplemented with purified linoleic and alpha-linolenic acid to meet the nutritional requirements of mice for these essential fatty acids. Diets fed for 6 weeks. The diets were isocaloric, contained equal amounts of protein and varied only in lipid composition, containing 5% fat (w/w) as in the AIN-76A base diet. The experimental diets met the nutritional requirements of laboratory mice. The extraction and analysis of fatty acids in the EPA and OA diets were performed before the start of the experiment to confirm fatty acid composition. The diets were aliquoted in daily portions and stored at -85°C until used. Fresh diet (3.5 to 4.0 g/mouse) was provided daily to reduce lipid oxidation. EPA (98% purity, ethyl ester form) was obtained from Brainfats Biotechnology Limited, (Auckland, NZ). The AA, OA, linoleic and alpha-linolenic acid (all 99% purity, ethyl ester form) were purchased from NuCheck, Inc. (Elysian, MN, USA).
Growth protocol
Throughout the experimental period, food intake was estimated by collecting and weighing uneaten food. Animals were not fed ad libitum; the food given was adjusted daily to equal the mean amount of feed consumed by Il10-/- mice on the previous day. Water was provided ad libitum and refreshed twice a week. All mice were weighed three times a week and carefully monitored for disease symptoms (weight loss, soft faeces, inactivity). After 77 days of age, all mice were randomly divided into two sampling days and three staggered groups of four mice within each day. This time was selected based on a previous study, where Il10-/- mice inoculated with EF and CIF showed an acceptable and consistent level of colon inflammation by 77 days of age. Randomisation of the mice into sampling days and groups was done to minimize any bias of food intake for the EPA or OA diet groups. On the last day, mice were fasted overnight for 14 hours, food was reoffered for 2 hours and again removed for the two hours immediately before sampling. This was done to minimize the variation in time between the last food intake and tissue sampling. Animals were euthanized by CO2 asphyxiation and cervical dislocation. Blood was sampled via cardiac puncture (0.5 to 1 ml) and centrifuged. The plasma was frozen in liquid nitrogen and stored at -85°C for serum amyloid A analysis. The intestine was quickly removed, cut open lengthwise, flushed with 0.9% NaCl to remove digesta and divided into duodenum, jejunum, ileum and colon. A piece of each section was stored at room temperature in 10% phosphate buffered formalin for histological analysis. Tissue samples were also frozen in liquid nitrogen and stored at -85°C for gene and protein profiling.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from colon tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer, with a subsequent purification step using RNeasy columns (Qiagen, San Diego, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was examined using a RNA 6000 LabChip Kit and a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). An equimolar pool of colon RNA extracts from 2 to 3 mice per treatment was made to minimize individual variation. A reference RNA was used for microarray hybridization.
Label
Cy3
Label protocol
cDNA and cRNA were synthesised using the Low RNA Input Linear Amplification kit (Agilent Technologies, Santa Clara, CA, USA), following the manufacturer’s instructions. Briefly, 500 ng of purified total RNA from each pool was amplified and reverse transcribed in vitro to cDNA using T7-polymerase, which was subsequently labelled with either cyanine 3-CTP (sample) or cyanine 5-CTP (reference pool) dyes. A reference design was used. The Nanodrop ND-1000 was used to monitor dye incorporation; sample and reference were between 9 and 22 pmol/μg cRNA.
reference: Pooled RNA from small intestine, colon, kidney, liver and fetuses
Biomaterial provider
Crop & Food Research (NZ) Small Animal Unit
Treatment protocol
Reference
Growth protocol
Reference
Extracted molecule
total RNA
Extraction protocol
as for Channel 1
Label
Cy5
Label protocol
As for channel 1 (but Cy5)
Hybridization protocol
Labeled cRNAs were hybridised using an In Situ Hybridization Plus Kit (Agilent Technologies, Santa Clara, CA, USA), following the manufacturer’s protocols, using 750 ng of sample cRNA and 750 ng of reference cRNA onto 44k mouse oligonucleotide arrays (Agilent Technologies, Santa Clara, CA, USA). After hybridisation, slides were washed in solutions I, II and III (Agilent Technologies, Santa Clara, CA, USA) and air-dried.
Scan protocol
Slides were scanned after washing using a GenePix Professional 4200A scanner (Molecular Devices, Sunnyvale, CA, USA), at photomultiplier tube voltages of 585 (red) and 600 (green). Spot identification and quantification was performed using GenePix 6.0 software (Molecular Devices). All slides were individually checked and manually flagged for abnormalities.
Description
The histological scoring of the inflammation was done with no prior knowledge of the treatments. Scoring of stained sections was based on: 1) inflammatory lesions (mononuclear cell, neutrophil, eosinophil and plasmocyte infiltration, fibrin exudation and lymphangiectasis); 2) tissue destruction (enterocyte loss, ballooning degeneration, oedema and mucosal atrophy); 3) tissue repair (hyperplasia, angiogenesis, granulomas and fibrosis). A score between 0 (no change from normal tissue) and 3 (lesions present in most areas and all layers of the intestinal section including mucosa, muscle and omental fat) was given for each aspect of inflammatory lesions, tissue destruction and tissue repair. The histological injury score (HIS) was calculated as: HIS = [(inflammatory lesions score) x 2] + tissue destruction score + tissue repair score. The concentrations of serum amyloid A in the plasma were measured to gain information on systemic inflammation levels and to complement HIS. Fifty μl of diluted plasma was used to assess the plasma concentration of SAA using the serum amyloid A kit (Tridelta Development Limited, Maynooth, County Kildare, Ireland), as described by the manufacturer. Total RNA was extracted from colon tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer, with a subsequent purification step using RNeasy columns (Qiagen, San Diego, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was examined using a RNA 6000 LabChip Kit and a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA).
Data processing
Statistical analysis was performed using Linear models for microarray analysis (Limma) from the Bioconductor project. Data with bad flags (-50 = poor or irregular spots, automatically flagged by the GenePix 4200A scanner; and -100, flagged by hand after visual inspection of the images) were not included in the analysis. Control probes (eQC, Bright corners, E1A and Pro25G) were also removed before normalization. Data were log transformed before analysis and the mean difference between treatments calculated on this scale, resulting in a log ratio for each probe. The normalized values in the database consist of these log ratios. Quality of the microarray data was assessed on diagnostic plots (boxplots and density plots) and spatial images generated from the raw (non-processed) data. MA plots of the microarray data were drawn in order to check that there was no dependence of the log ratio on the intensity for any slide. Intensity ratio values for all microarray spots were normalized using a global loess smoothing procedure to remove the effect of systematic variation in the microarrays and no background correction was necessary due to homogeneous hybridisation. For each experimental comparison, a candidate list of differentially expressed probe sets was generated by calculating a moderated t-statistic for each probe set using the Limma package. The Limma library implements an empirical Bayes approach to assign differential gene expression. The significance of the log ratio for each probe was determined by calculating one modified t-statistic per probe. Probe sets that satisfied the criterion of ≥ 1.3 fold change (FC) with a moderated p < 0.01 were considered to be significantly different. This fold change was chosen because recent studies show that a FC above 1.6 can underestimate the number of differentially expressed genes regulated by dietary treatment.
