Animals and Surgical Procedure
Male Sprague-Dawley rats (200-250g) (Laboratory Animals Center, Jinling Hospital) were housed in a controlled environment (12 hr daylight cycles) in groups of two rats per cage, and were allowed free access to standard chow and water. The experiments were carried out under the supervision of Jinling Hospital Animal Care and Use Committee. Twenty-two rats were randomized into four groups of 6 animals each.
SAP model was created essentially according to the previously described procedure[8]. Briefly, All rats underwent intraperitoneal injection of ketamine (100 mg/kg) as anesthesia. 3.5% sodium taurocholate (dissolved in normal saline) was injected into pancreatic and bile duct retrogradely with micro-pump set at a speed of 0.1 ml/min and volume of 1 ml/kg body weight. The main pancreatic and bile duct was clamped just below the liver. This clamp remained in place throughout the intraductal infusion to prevent misdirected flow into the biliary system. The abdomen was then closed. For the sham operation group, the abdomen was opened and the pancreatic and bile duct were flipped and 1 ml/kg body weight normal saline was injected pancreatic and bile duct retrogradely before closing abdomen. A group of six rats were sacrificed 2, 6, and 12 h after operation to collect samples. All procedures and sample collections were under sterile techniques. Blood samples and tissues were harvested for further analysis. All samples were stored at -80°for further analysis. Colonic samples and its contents at 1 cm distal to the cecal-colonic junction were collected. All operations were performed under anesthesia. Rats were then killed by over-dosed anesthesia (pentobarbital :250 mg/kg).
DGGE of intestinal Microbiota
Luminal bacterial DNA was extracted from 200 mg of intestinal contents, using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions, and stored at -20 °C until use. The hypervariable V3 region of the 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) using forward primer (GC357f 5’-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGATTACCGCGGCTGCTGG-3’) and reverse primer (518r 5’-CCTACGGGAGGCAGCAG-3’). The 50 μl PCR reaction mixture were: 1 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), 40.5 μl sterile water. PCR was run in a 2720 thermal cycler (Applied Biosystems, Foster City, CA) as a touchdown PCR. The program was 94 °C (5 min), followed by 20 cycles of 94 °C for (30 sec), 65 °C(30 sec) decreased by 0.5 °C for each cycle, and 68 °C (30 sec), additional 10 cycles of 94 ºC (30 sec), 55 ºC (30 sec), and 68 ºC (30 sec), and a final extension at 68 °C (7 min).
DGGE gels were prepared using a DCode Universal Mutation Detection System (BIO-RAD, Hercules, CA) with 8 % (w/v) polyacrylamide (acrylamide: Bis 37.5:1) in 1 × TAE (20 mM Tris, 10 mM acetate, 0.5 M EDTA, pH 7.4) in denaturing gradients ranging from 35 to 50%. While 100% chemical denaturant solution consists of 7 M urea and 40% formamide. At both outer lanes of each gel, a common sample was loaded for gel normalization to allow comparison between gels. Electrophoresis was run in 1 × TAE at 60ºC, first at 200V for 10 minutes and then at 120V for 7.5 hours. All gels were stained with SYBR Green I (Invitrogen) for 30 min, and viewed under U.V illumination. Images were captured by ChemiDoc XRS Camera (BIO-RAD, Hercules, CA).
Analysis of DGGE Profile
Gel images were aligned using Adobe Photoshop CS4 by running common samples on both outer sides of each gel, to allow comparison of two gels in one profile. Digitizing the profile was done by Quantity One software (version 4.6). Individual bands in each sample lane were marked automatically by the software, followed by manual correction if necessary. Bands occupying the same position in different lanes were matched and identified as the same band type, using the match tolerance of 0.5%. The relative quantity of a given band was expressed as a fraction (%) of the sum of all defined bands in the same lane. The quantitative information derived from relative band quantities per band type per sample was exported as a data matrix. The method for calculating similarity between two lanes was the Dice Coefficient. Unweighted pair group method with arithmetic mean was used to compare the similarity of samples in a dendrogram. Principal component analysis (PCA) was performed using Canoco (version 4.5) based on the data matrix mentioned above. Species richness was calculated as the total number of individual lane’s bands. Shannon’s evenness index, Shannon’s diversity index were generated on quantitative information.
Sequencing of DGGE Bands
Bands of interest from DGGE gels were cut out with a sterile scalpel, placed into a single Eppendorf tube, and incubated in 20 μl of sterile water overnight at 4°C. Four microliters of the overnight solution was used as template for PCR re-amplification. Using the forward primer (without GC clamp) (357f 5’- ATTACCGCGGCTGCTGG -3’) and the reverse primer (518r 5’-CCTACGGGAGGCAGCAG-3’), the 50 μl PCR reaction mixture were: 4 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), 37.5 μl sterile water. The touchdown program was 94 °C (5 min), followed by 20 cycles of 94 °C for (30 sec), 65 °C(30 sec) decreased by 0.5 °C for each cycle, and 68 °C (30 sec), additional 15 cycles of 94 °C (30 sec), 55 °C (30 sec), and 68 °C (30 sec), and a final extension at 68 °C (7 min). After purification, PCR products were sequenced using the Sanger’s method on a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA). The obtained sequences were compared 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.
Real-time PCR
Bacterial species that characterize the predominant dysbiosis in rat model of severe acute pancreatitis injury derived from the DGGE comparative analyses were quantified by quantitative PCR using the 7300 Real-Time PCR System (Applied Biosystems, USA). The primer pairs for the bacteria were reported in previous studies (see Table, Supplemental Digital Content 1). The real-time PCR was carried out in a 20 μl total reaction mixture: 1 μl of template DNA, 0.4μl of each primer, 10 μl 2 × SYBR Green Realtime PCR Master Mix (TOYOBO, Japan) and 8.2 μl of sterile water. The amplification program consisted of one cycle at 95 °C for 5 min; 40 cycles at 95 °C for 15 sec, 55 °C for 60 sec. Specific bacteria 16S rRNA gene amount was normalized to total bacteria 16S rRNA gene amount using the 2-ΔCt method. Quantification values are represented by fold changes relative to control rats.
Histopathology
Colonic samples was fixed in 10% formalin. Paraffin sections were prepared and stained with hematoxylin and eosion. The degree of colonic tissue injury was evaluated by a pathologist in a blinded set-up using a grading system described previously by Chiu et al. (14) The scales were: grade 0, no damage; grade 1, subepithelial space development at the apex of the villus; grade 2, subepithelial space extension; grade 3, massive lifting of epithelia, including a few denuded tips; grade 4, denuded lamina propria with cellularity increase; grade 5, lamina propria disintegration and hemorrhage.
Immunofluorescence
One centimeter segments of colon was harvested, washed with normal saline (0.9%), snapped frozen in liquid nitrogen and stored at -80 °C until use. Tissues were embedded in OCT compound and cut into 5 μm frozen sections, followed by fixation in acetone at -20 °C for 5 minutes. After 15 minutes of blocking with 1% bovine serum albumin, sections were incubated with polyclonal anti-occludin (1:150) or polyclonal anti-ZO-1 (1:150) (Zymed Laboratories Inc, San Francisco, CA) as primary antibodies overnight at 4 °C. Sections were probed with Alexa Fluor 488-conjugated anti-rabbit secondary antibodies (1:150) (Invitrogen) for 30 minutes at 4 °C, followed by counterstained with DAPI (1:500) (Invitrogen). Images were obtained with Leica TCS SP5 confocal scanning microscope (Leica Microsystems, Heidelberg GmbH, Mannheim, Germany).
Immunohistochemistry
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded specimens. A standard immunohistochemical technique was applied. After blocking endogenous peroxides and proteins, specimens were exposed to primary antibody. Rabbit polyclonal anti- zonula occludens （ZO）-1 antibodies were purchased from Zymed Laboratories Inc (San Francisco,CA). Horseradish peroxidase-conjugated secondary antibody was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA).
Statistical Analyses
Data are presented as mean ± standard error of the mean. Statistical analysis was performed using one-way analysis of variance followed by post hoc Bonferroni’s multiple comparison test. For non-parametric data, Kruskal-Wallis followed by Dunn’s multiple comparison test was used. A p value of less than .05 was considered significant. The analyses were conducted using GraphPad Prism version 5.