All experiments with zebrafish were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee of Model Animal Research Center, Nanjing University (MARC-AP#: QZ01), in accordance with the Guideline on the Humane Treatment of Laboratory Animals in China and the Regulations for the Administration of Affairs Concerning Experimental Animals.
Zebrafish maintenance and embryo collection
Wild-type (AB strain) zebrafish were reared at 28±0.5°C on a 14-h light/10-h dark cycle in a closed flow-through system in charcoal-filtered and fully aerated tap water according to standard procedures . The fish were fed with commercial flakes twice daily.
Zebrafish embryos were collected from spawning adults in groups of about 16 males and 8 females in tanks overnight. Spawning was induced in the morning shortly after the light was turned on. Collected embryos were maintained in embryo medium (13.7 mM NaCl, 0.54 mM KCl, 1.3 mM CaCl2, 1.0 mM MgSO4, 0.25 mM Na2H PO4, 0.44 mM KH2 PO4, 0.42 mM NaHCO3) at 28.5°C. At 4–5 hours post-fertilization (hpf), those embryos that had developed normally and reached the blastula stage were selected under a dissecting microscope for subsequent experiments.
Induction of IBD by TNBS exposure
A stock solution of 5% (w/v) 2, 4, 6-trinitrobenzenesulfonic acid (TNBS; Sigma, St Louis, USA) in embryo medium was used for the induction of IBD. Zebrafish from 3 days post fertilization (dpf) were randomly placed into groups of 15 larvae in 20 ml of exposure solution (embryo medium containing 0, 25, 50 and 75 μg/mL TNBS). The range of concentrations was selected based on previously ascertained range-finding studies and information from the available literatures [14, 15]. A 90% (v/v) water change was performed each day starting at 3 pdf when larvae hatch from their chorions. Samples were collected at 4, 6 and 8 days postfertilization (dpf).
Larval zebrafish from 4 dpf, 6 dpf and 8 dpf were anesthetized by immersion in 0.2 mg/ml 3-amino benzoic acid ethylester (MS222, Sigma). For histology, samples were fixed in Bouin’s Fixative overnight at 4°C and mounted in SeaPlaque 1% low-melting point agarose. Then samples were dehydrated through a standard series of alcohols and Histo-clear and embedded in paraffin. 5 μm sections were cut for staining with hematoxylin and eosin. Histological sections were imaged and photographed with an Olympus CX41 system microscope (Olympus USA, Center Valley, PA, USA) and the DS-5 M-L1 digital sight camera system (Nikon, Japan). The enterocolitis scores were quantified by an observer who was blinded to the prior treatment of the fish. And these data represent three independent experiments.
Detection of goblet cells using AB-PAS staining
For goblet cell quantification, 5-μm paraffin sections were prepared as described in the Methods and stained sequentially with 1% Alcian blue pH 2.5 for 15 min, 1% aqueous periodic acid for 10 min and Schiff’s reagent for 10–15 min. Using this method, goblet cells stain blue. The number of goblet cells was counted manually along the length of the gut from the intestinal bulb to the anus.
Larvae at 4 dpf, 6 dpf and 8 dpf were fixed in 4% paraformaldehyde overnight at 4°C. Fixed larvae were soaked in 30% sucrose until they sink, transferred to embedding chamber filled with OCT Compound (Sakura Finetek USA, Inc, Torrance, CA, USA), snapped frozen in liquid nitrogen and stored at −80°C.
For immunofluorescence, 5-μm frozen sections were cut and blocked with 1% bovine serum albumin prior to being incubated with anti-TNF-α(IN), Z-Fish™, Catalog No. 55383P (1:150, 100 μg/400 μl, AnaSpec, Fremont, CA) overnight at 4°C. Sections were washed in PBS and incubated with Alexa Fluor 488-conjugated anti-mouse secondary antibodies (1:150, Invitrogen, La Jolla, CA) for 30 minutes at 4°C, followed by counterstained with DAPI (1:500). Sections were imaged and photographed with Leica TCS SP5 confocal scanning microscope (Leica Microsystems, Heidelberg GmbH, Mannheim, Germany). The intensity of TNF-α immunofluorescence was quantified for each treatment group, with a minimum of 6 samples per group, using color threshold and area measurements with AnalySis software.
Microbial analysis by denaturing gradient gel electrophoresis (DGGE)
The DGGE analysis was carried out to identify the microbial community in the intestine and to study the potential changes between the different groups of zebrafish.
Extraction of DNA and PCR amplification
Bacterial DNA was extracted from pools of 20 zebrafish larvae using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol, and stored at −20°C until use.
PCR was performed on an Applied Biosysterm 2720 Thermal Cycler as a touchdown PCR. The hypervariable V3 region of the 16S ribosomal DNA gene was amplified using polymerase chain reaction (PCR) with forward primer (GC357f 5′CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGATTACCGCGGCTGCTGG3′) and reverse primer (518r 5′CCTACGGGAGGCAGCAG3′). The PCR reaction mixtures consisted of 2 μl of extracted bacterial DNA, 5 μl of 10×PCR buffer, 1 μl of dNTP mixture (2.5 mM each), 1 μl of each primer (10 pM), 0.5 μl of Taq-Polymerase (5 U/μl) and sterile water to final volume of 50 μl. The cycling program was as follows: predenaturation at 94°C for 5 min, followed by 20 cycles of 94°C for 30 s, 65°C for 30 s decreased by 0.5°C for each cycle, and 68°C for 30 s, after which 10 additional cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 30 s were carried out, and a final extension at 68°C for 7 min, soak at 4°C.
Integrity of PCR products was determined by running agarose gel electrophoresis, and the quantity was determined using QubitTM fluorometer (Invitrogen, NY, USA).
Denaturing gradient gel electrophoresis
DGGE was performed on the PCR products from DNA samples using 16 cm × 16 cm ×1 mm gels with a DCode Universal Mutation Detection System (Bio-Rad, Hercules, CA). A 35-50% urea and formamide denaturing gradient and 8% polyacrylamide gel (37.5:1 acrylamide-bisacrylamide) were used. The gradient was prepared using the gradient delivery system (Bio-Rad), following the manufacturer’s protocol. A 100% denaturant solution contained 7 M urea and 40% formamide. Gels were run in 1×TAE (20 mM Tris, 10 mM acetate, 0.5 M EDTA, pH 7.4) at 60°C, first at 200 V for 10 minutes and then at 120 V for 7.5 hours. The resulting gels were stained with SYBR Green I (Invitrogen) for 30 min, visualized and photographed using the Gel Doc EQ system (Bio-Rad, USA). All gels were normalized using a reference sample with bands distributed throughout the whole gel.
Analysis of DGGE profile
Gel images were aligned using Adobe Photoshop CS5 by running common samples on both outer sides of each gel, to allow comparison of two gels in one profile. DGGE profiles were analysed using Quantity One software (version 4.6; Bio-Rad Laboratories, Hercules, CA). The lanes were identified, and their background intensities were removed using the rolling disk method described in the program. Then bands were detected automatically by the software, followed by manual correction if necessary, and they were matched at 0.5% tolerance level. The tolerance level is the minimum spacing that the matching model expects to find between unique bands, and it is expressed as a percentage of lane height. The relative quantity of bands is expressed as a proportion (%) relative to the sum of the intensities of all of the bands in the same lane. A similarity matrix was computed by comparing the profiles of lanes, and the percentage similarity was expressed as the Dice coefficient. The presence or absence of a band in a lane was considered. Identical profiles have a percentage similarity of 100. Unweighted pair group method using arithmetic averages (UPGMA) was used to compare the similarity of samples in a dendrogram. The general diversity of bacterial communities was calculated by generating Shannon’s index of diversity on quantitative information .
Sequencing of DGGE bands
Bands of interest from DGGE gels were excised and immersed in 20 μl of sterile water and left overnight at 4°C. 2 μl of eluted DNA from each band was used as template for PCR re-amplification with the forward primer (without GC clamp) (357f 5′- ATTACCGCGGCTGCTGG -3′) and the reverse primer (518r 5′-CCTACGGGAGGCAGCAG-3′). PCR was performed in a 50 μl reaction mixture including 2 μl of template DNA, 5 μl of 10×PCR buffer, 1 μl of dNTP mixture (2.5 mM each), 1 μl of each primer (10 pM), 0.5 μl of Taq-Polymerase (5 U/μl) and 39.5 μl sterile water. Amplification was performed under the following conditions: 94°C for 5 min, 20 cycles of 94°C for 30s, 65°C for 30s decreased by 0.5°C for each cycle, and 68°C for 30 s, additional 15 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 30 s, with a final extension at 68°C for 7 min.
After the PCR products were purified (QIAquick PCR Purification Kit, QIAGEN) and quantified (Qubit fluorometer, Invitrogen), the sequence analysis of the products was carried out using the Sanger’s method on an ABI 3730 automated sequencing system. The sequences obtained were then aligned with NCBI GenBank databases using the BLAST tool. The phylogenetic tree was constructed using the MEGA 4.0 program in the method of neighbor-joining based on evolutionary distances.
Quantitative real-time PCR analysis
Bacterial species that characterize the predominant intestinal dysbiosis in zebrafish larvae with TNBS-induced enterocolities derived from the DGGE comparative analyses were quantified by quantitative PCR using the 7300 Real-Time PCR System (Applied Biosystems, USA). A reaction mixture (20 μl) consisted of 1 μl of DNA (10 ng), 0.4 μl of each primer, 10 μl 2×SYBR. The primers and probes based on 16S rRNA gene sequences were chosen to target total bacteria, Lactobacillus group, the dominant group of Firmicutes, Enterobacteriaceae family and Burkholderia species, the main Proteobacteria phylum in zebrafish gut. Total bacterial 16S rRNA gene copies were quantified with primers (Bact1369; 5′CGGTGAATACGTTCYCGG3′and Prok1492; 5′GGWTACCTTGTTACGACTT3′). PCR was performed with an initial denaturation step of 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 56°C for 30 s and 72°C for 30 s. Lactobacillus group were quantified using the combination of forward, (LAC1; 5′AGCAGTAGGGAATCTTCCA3′), and reverse primer, (Lab0677; 5′CACCGCTACACATGGAG3′) in a cycling program where after the initial denaturation 95°C for 3 min, 40 cycles were applied at 95°C for 30 s, and binding and extension at 60°C for 1 min. Primer (Eco1457F; 5′CATTGACGTTACCCGCAGAAGAAGC3′) combined with primer (Eco1652R; 5′CTCTACGAGACTCAAGCTTGC3′) were used for the quantification of Enterobacteriaceae family with the following conditions: an initial DNA denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, and primer annealing and extension at 72°C for 30 s. Burkholderia species were quantified using the forward primer (Burk3; 5′CTGCGAAAGCCGGAT3′) and the reverse primer (BurkR; 5′TGCCATACTCTAGCYYGC3′) with the following cycling conditions: predenaturation at 95°C for 4 min; 60 cycles of 94°C for 1 min, 62°C for 90 s decreased by 1°C for every fifth cycle, after which 25 additional cycles were carried out at 58°C, and 72°C for 2 min, and a final extension at 72°C for 10 min. Data analysis was proceeded with Sequence Detection Software version 1.6.3 ( Applied Biosystems). All reactions were performed in triplicate. Specific bacteria 16S rRNA gene amount was normalized to total bacteria 16S rRNA. Quantification values were represented as mean (SEM) log 16S rRNA gene copies per 10 ng of bacterial genomic DNA.
Biochemical measurements were performed at least in duplicate. Quantitative histological analyses were performed by a blinded scorer. Results are presented as mean ± standard error of the mean. Survival curve comparison calculations used the Gehan-Breslow-Wilcoxon test. Two-way anova was applied to analyze the data to understand the combined effect of the two factors - time and treatment. Bonferroni multiple comparison post hoc tests were used to find the significant differences between the means at a particular time point⁄treatment. Pearson correlation, α =0.05, was used to assess linear relationships between enterocolitis score/inflammatory cytokine expression level and intensity/diversity in gut microbiota. All statistical analyses were performed with Graph-Pad Prism version 5.0 (GraphPad Software, San Diego, CA), and the significant differences are reported at P < 0.05.
Nucleotide sequences accession number
The sequences of 16S rRNA gene obtained in this study have been deposited in the GenBank database (EMBL, U.K.) under accession numbers KF515539-KF515557.