Experimental Design
Seven-week-old C57BL/6 male mice (Orient Bio Co., Ltd.®, Seongnam, Gyeonggi, Korea) were used in this study for IBS model induction. The mice were acclimated for 7 days at the Medical Research Center’s facility before experimentation. Mice were housed individually in standardized environmental conditions at 23 ± 2°C with 50–55% humidity and were allowed to feed food and drink ad libitum. The study protocol was approved by the Ethics Committee for Animal Research (EUM19-0456) and followed the ARRIVE 2.0 guidelines. Experimental procedures and results were conducted in accordance with regulations and guidelines.
The mice were divided into two groups to induce the post-inflammatory IBS modeling: an acute severe colitis recovery IBS model (acute model) and a chronic mild repeated colitis IBS model (chronic model) (Fig. 1). For acute model, mice (n = 10) were administered with 3% DSS (MP biochemical®, Irvine, CA, USA) for 5 days, followed by a 12-week recovery period. During the recovery period, mice were given free access to food and water. The traditional IBD model involved a chronic model in which mice (n = 10) were orally administered with DSS for 5 days, followed by a recovery period of drinking water for the next 5 days, repeated for 3 cycles. To induce low-grade inflammation without causing gross mucosal damage, preliminary experiments were conducted to find an appropriate concentration of DSS, including 0.5%, 1.0%, and 1.5% for chronic models. Body weight changes, stool consistency, and gross bleeding were measured by the same observer every 7 days in the acute model and 3 days in the chronic model to assess disease activity, and the disease activity index (DAI) was calculated by adding the changes in body weight, stool consistency, and gross bleeding (Supplementary Table S1).11
After completing the experiment in each model, all mice were euthanized through CO2 asphyxiation after overnight fasting. After euthanasia, the entire colon was dissected from the cecum, and the total length was measured. Whole colon tissue was divided into proximal and distal sections, and then tissue from each section was split and analyzed by real-time polymerase chain reaction (RT-PCR), histopathology, and immunohistochemistry (IHC).
Analysis of Intestinal Transit Time
We used the gastrointestinal transit time (GITT) and the bead expulsion test to determine the intestinal transit time. For both techniques, we measured transit time before DSS administration and then every 4 weeks for the acute model and 30 days for the chronic model, respectively. Mice were housed individually to measure the GITT. A 6% solution of carmine red (natural red 4; Sigma Aldrich®, St. Louis, MO, USA), a non-absorbable dye, was administered through a 21-gauge gavage in 0.3 mL of 0.5% methylcellulose solution (Sigma Aldrich®). Following administration, mice were placed in a cage, the floors of which were lined with white paper to facilitate the carmine-red coloration of their feces. T0 represented the time at which the carmine red solution was gavaged and the time it took for the expulsion of the first red fecal pellet was assessed.12 In GITT measurements, closed-circuit television (CCTV) was employed as an auxiliary tool for more objective and accurate measurement.
In the existing murine animal model, a bead expulsion test was a commonly used method to measure distal colonic transit time.13 The distal colonic transit time was defined as the time between the bead insertion into the distal colon and its expulsion. Briefly, similar to previous studies, we dipped a single 2mm bead into the lubricating gel, gently inserted it, and measured the time to expulsion.
RT-PCR analysis of Inflammatory Cytokines and Permeability Markers
The mRNA expression of inflammatory cytokines in the mice colon, including IL-1β, IL-6, IL-17, and TNF-α was measured. We assessed the mRNA expression of occludin, zonular occludens (ZO)-1, claudin-1, and claudin-4 using RT-PCR14 to assess the expression of the tight junction proteins in the IBS model
RT-PCR was performed on proximal and distal colon sections. The separated colon tissue was immediately frozen at -70°C in a cryogenic freezer. Total RNA was isolated from colon tissue using the TRIzol reagent (Ambion®, Waltham, MA, USA). After adding chloroform (Sigma Aldrich®), the extracted total RNA was centrifuged at 12,000 rpm for 10 min at 4°C. The supernatants were centrifuged for 10 min at 12,000 rpm at 4°C with isopropanol (Sigma Aldrich®). After 75% ethanol wash, the pellet was solubilized with nuclease-free water and quantified using nano drops. RNA (2 µg) was reverse-transcribed in the mixture of oligo dT primers (0.5 µg), 200 units of Molony-Murine leukemia virus reverse transcriptase (Promega®, Fitchburg, WI, USA), 5x RT buffer, dNTPs (2 nM), and 25 units of RNasin ribonuclease inhibitor (Promega®) at 42 ℃ for 60 min. The RT-PCR was conducted on 7000 Real-time PCR systems (Applied Biosystems®, Waltham, MA, USA) with 2X Power SYBR Green PCR Master mix (Applied Biosystems®), 0.1 µg cDNA, and each primer set (Macrogen®, Seoul, Korea; Supplementary Table S2) over 40 cycles (95 ℃ for 15 s and 60 ℃ for 60 s) following pre-denature at 95 ℃ for 10 min. The relative expression of the target genes was assessed using the comparative Ct method, and glyceraldehyde-3-phosphate dehydrogenase was used as an internal control.
Histopathological Analysis
Histopathological examination was conducted by a single pathologist, and colon tissues were fixed in 10% formalin, followed by paraffin sectioning and hematoxylin-eosin (H&E) staining. Each colon tissue was stained with H&E and analyzed in three categories for histological evaluations: (i) inflammatory cell infiltrate, (ii) epithelial changes, and (iii) mucosal architecture. According to the criteria in histomorphological scores for intestinal inflammation in mouse models (Supplementary Table S3), each category was calculated with a score value ranging from 1 to 5.15
Immunohistochemistry Analysis
IHC analysis was performed, focusing on neuroinflammatory markers. Tissue samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4), embedded in paraffin, and sectioned for IHC analysis. After deparaffinization and rehydration, antigen retrieval was performed by incubating the sections in citrate buffer (0.01 mmol/L, pH 6.0) and heating them in a microwave oven (720 W) for 15 min. Endogenous peroxidase was blocked by treating the sections in 0.3% hydrogen peroxide for 30 min at room temperature. Next, sections were incubated for 3 h at room temperature with specific antibodies: rabbit polyclonal transient receptor potential vanilloid 1 (TRPV 1) antibody (Invitrogen®, Waltham, USA), recombinant rabbit monoclonal tropomyosin receptor kinase A (TrkA) antibody (Invitrogen®), mouse monoclonal substance P antibody (Santa Cruz®, Dallas, TX, USA), rabbit monoclonal S-100 antibody (Leika®, Wetzlar, Germany), and mouse monoclonal neuron-specific enolase (NSE) antibody (Leika®) as visceral hypersensitivity markers. The sections were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies for 30 min. The sections were then stained with 3,3-diaminobenzidine solution, followed by counterstaining with hematoxylin for nuclei labeling.16 Immunopositive cells were counted using the Image J program (National Institutes of Health, Bethesda, MD, USA) in a 200 µm stretch of well-oriented entire colon epithelium sections at five randomly selective fields for each antibody. The average and standard deviation were subsequently calculated.10
Microbiome Analysis
We performed a microbiome analysis on the acute model selected as the IBS model based on comprehensive results. For microbiome analysis, intraluminal feces were collected from cecum of sacrificed mice and immediately frozen at -80 ℃. Microbial genomic DNA was extracted using QIAamp PowerFecal Pro DNA Kit (Qiagen®, Hilden, North Rhine-Westphalia, Germany), following the recommended protocols. The quality of all extracted bacterial genomic DNA was assessed using the Qubit 4 (ThermoFisher Scientific®, Waltham, MA, USA). The extracted mitochondrial DNA samples were stored at 4°C until further processing. The DNA sequencing library targeting the V3 and V4 hypervariable regions of 16S ribosomal RNA was constructed according to the sequencing library preparation protocol (Illumina®, San Diego, CA, USA) using specific universal primers (Illumina 16S forward 341F primer, 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG − 3' and reverse 805R primer, 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GACTACHVGGGTATCTAATCC-3').17 PCR was carried out using KAPA HiFi HotStart ReadyMix (Kapa Biosystems®, Wilmington, MA, USA), followed by purification of the PCR product with AMPure XP beads (Beckman Coulter Genomics®, Brea, CA, USA). An additional PCR amplification was performed to introduce the Illumina adapter and multiplex indices using the Nextera XD Index (Illumina®). The final PCR products were purified once again using the AMPure XP beads. The prepared library was then sequenced using the Miseq system (Illumina®) with 300 bp paired-end reads.
The gut microbiome data was analyzed using the QIIME2 pipeline plugin (2022.08).18 Before implementing DADA2 (Divisive Amplicon Denoising Algorithm 2) in QIIME2, Figaro was used to determine optimal options based on sequence quality.19
Amplicon sequence variants (ASVs) were obtained through the denoising step in the DADA2 algorithm. The ASVs were subjected to bacterial classification using a Naïve Bayes classifier based on targeted hypervariable reads extracted from the SILVA 138v 99% rRNA database to improve accuracy. Subsequently, various features annotated with Archaea, Eukaryotes, Mitochondria, or Chloroplasts were removed. The selected bacterial features were employed for the construction of a phylogeny tree using the align-to-tree-mafft-fasttree plugin, and taxonomy composition was determined. These features were rarefied at a specified depth and utilized for the analysis of alpha diversity, including Observed Features, Chao1 Index, Shannon's Index, Simpson's Index, and Pielou's Evenness, as well as beta diversity, encompassing Bray-Curtis and Unweighted UniFrac metrics.20
Statistical analysis
Statistical Package for the Social Sciences (SPSS) program, version 25.0 software (SPSS Inc.®, Chicago, IL, USA) was used for all statistical analysis. The mean ± standard deviation values were used for each analysis. Comparisons of values between the models were tested using the nonparametric Kruskal–Wallis test. Graphical representations were generated using GraphPad Prism software version 8 (GraphPad Software Inc.®, La Jolla, CA, USA). A P-value of < 0.050 was considered statistically significant.
For microbiome analysis, statistical significance in group comparisons for α-diversity and taxonomy composition was evaluated using the Mann-Whitney U-test (Wilcoxon Rank-sum test) in R through the 'ggpubr' package. To confirm the similarity between groups in distance matrices from β-diversity, principal coordinate analysis and permutational analysis of variance with 999 permutations were performed using the ‘Vegan’ and ‘Adonis’ packages in R. Additionally, linear discriminant analysis effect size (LEfSe) was calculated and features with a linear discriminant analysis score ≥ 2.0 were considered significant within each group.21