Animals

CFP mice were bred in house, and ICR mice were obtained from The Jackson Laboratory Japan, Inc. (Kanagawa, Japan). Because the assignment to experimental groups was determined based on mouse strain and sex, no grouping was conducted, and no mice were excluded before the experiments.

All mice were housed in flat-bottomed cages with bedding (Pulsoft; Oriental Yeast Co., Ltd., Tokyo, Japan) and nesting material (Rodent Nesting Sheets™ and Bio-Huts™; Bio-Serv, Flemington, NJ, USA) under specific pathogen-free conditions. The animal housing was maintained at a relative humidity of 30%–70%, temperature of 20 °C–26 °C, and a 12-h light/dark cycle with lights turned on at 8:00 am. The mice were provided free access to pelleted feed (F-2; Funabashi Farm Co., Ltd., Chiba, Japan) and water. No mice were fasted before the experiments. At the end of the experiments, all mice were euthanized under 3.5% isoflurane (isoflurane inhalation anesthetic solution; Pfizer Japan Inc., Tokyo, Japan) to collect GI tissues and contents and other materials for subsequent analyses as described below.

Study Design

As shown in Fig. 1, this study comprised five experiments involving a total of 24 female and 30 male CFP mice and an equal number of ICR mice. Each experiment included four groups based on strain and sex, with six mice per group; however, Experiment 4 involved six male CFP mice and six male ICR mice. In Experiment 1, whole GITT (WGTT) and mean retention time (MRT) as indices of total GI tract transit. In the same mice, gastric emptying rate and small intestinal (SI) geometric center were measured. Anatomically segmented GIT was also evaluated in Experiment 2 by gastric and cecal retention times and SI and colonic transit times. In Experiment 3, amounts of cecal and colonic contents and their respective water contents were measured, which could be affected by large intestinal transit. The fecal water content was measured using feces collected within 1 h of GITT and MRT measurements in Experiment 1 and at age 11–12 weeks in Experiment 3 and collectively analyzed. In Experiment 4, histopathological analysis of the colon was conducted and thickness of the colonic muscle layer was determined. In Experiment 5, cecal and fecal microbiota, which exhibit a bidirectional relationship with large intestinal transit, were analyzed. Body weight and intestinal length were measured in Experiments 1–3 and analyzed collectively.

Fig. 1

Study design. In Experiment 1, WGTT and MRT were measured as indices of total GI tract transit. In the same mice, gastric emptying rate and small intestinal geometric center were measured. In Experiment 2, anatomically segmented GI transit was evaluated using a mathematical model. In Experiment 3, cecal and colonic contents as well as their water contents were measured. In Experiment 4, histopathological analysis of the colon was conducted and thickness of the colonic muscle layer was determined. In Experiment 5, cecal and fecal microbiota were analyzed. Body weight, intestinal length, and fecal water content were measured in several experiments and analyzed collectively. GI gastrointestinal, WGTT whole GI transit time, MRT mean retention time, w.o. weeks old, p.o. peroral administration

Body Weight and Intestinal Length

The body weight and intestinal length of 11–12-week-old mice were measured. SI length was defined as the distance from the pylorus to the ileocecum, and the colonic length was defined as the distance from the cecocolic junction to the anus.

Whole-GIT

Mice were housed in same-strain and -sex pairs for 3 weeks before the experiment. To evaluate WGTT and MRT in mice housed in pairs instead of individually, two types of transit markers were used. Specifically, 5 mg/mL of 70-kDa dextran, labeled with tetramethylrhodamine isothiocyanate (TRITC-dextran; TdB Labs, Uppsala, Sweden), and fluorescein isothiocyanate (FITC-dextran; TdB Labs) were prepared in phosphate-buffered saline (PBS). At the age of 10–11 weeks, 0.1 mL of TRITC-dextran solution was orally administered to one mouse per cage, and 0.1 mL of FITC-dextran solution was administered to the other mouse between 8:15 and 8:50 am. Feces were collected at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 24, 26, 28, 30, 32, 34, and 48 h after marker administration. The fecal samples were freeze-dried, homogenized using 30 volumes of PBS (v/w), and left overnight at 4 °C. The fecal homogenates were centrifuged at 300×g for 3 min, and the supernatant was collected. Fluorescence intensity of the supernatant was measured in a microplate reader (Infinite® 200 PRO; Tecan Group Ltd., Männedorf, Switzerland) at 580-nm excitation and 547-nm emission for TRITC and at 522-nm excitation and 495-nm emission for FITC. The concentrations of TRITC-dextran and FITC-dextran were interpolated using a standard curve.

WGTT was defined as the time from the oral administration of the marker solution to the time of the first excretion of feces in which the marker was detected [19]. In cases where no marker was detected in the feces until 11 h after marker administration, the WGTT was recorded as 11 h. MRT was calculated using the formula Σ (xi ∙ ti)/Σ xi, where ti denotes the time to fecal excretion following marker administration and xi denotes the amount of the marker collected at time ti [20, 21]. The time of fecal excretion was defined as the interval between the fecal collection time and the previous collection time.

GI Segment-Specific TransitGastric Emptying Rate and SI Geometric Center

Gastric and SI transits were evaluated 2–6 days after measuring MRT. Mice were orally administered 0.5 mg/0.1 mL/mouse of 70-kDa FITC-dextran between 8:05 and 10:35 am. After 20 min, the mice were euthanized by cervical dislocation following cardiac blood sampling under anesthesia as described above. Immediately, the stomach, small intestine, cecum, and colon were collected. The small intestine was divided into 10 equal parts. Each GI segment and its contents were placed in 1 mL of PBS for each SI segment and 10 mL for others. The GI content samples were centrifuged as described above, and the supernatants were collected. The concentrations of FITC-dextran in the supernatant and diluted plasma were determined following the procedure described above.

Gastric emptying was calculated as the proportion of the amount of marker collected from the contents of the intestinal tract to the total amount of marker collected [22]. The SI geometric center, an index of SI transit, was calculated using the formula Σ (xj ∙ j)/Σ xj, where j denotes the segment number (0, stomach; 1–10, small intestine; 11, cecum; 12, colon), and xj denotes the amount of marker collected in segment j [22].

Gastric and Cecal Retention Times and SI and Colonic Transit Times

Mice were housed in same-strain and -sex pairs for at least 2 weeks before the experiment. At the age of 12 weeks, the mice were orally administered 0.5 mg/0.1 mL/mouse of 70-kDa TRITC-dextran and 70-kDa FITC-dextran. In each cage, one mouse received the TRITC-dextran dose first, followed by the FITC-dextran dose, and the other mouse received these doses in the reverse order. The dosing interval between the two markers was 4.5 h for CFP mice and 1.5 h for ICR mice to account for the difference in GITT between these strains, with the first dosing occurring between 8:05 and 9:45 am. After 1 h of the second dose, the mice were euthanized as described above. Immediately, the stomach, small intestine, cecum, and colon were collected. The colon was divided into four equal parts. Each GI segment and its contents were placed in 2 mL of PBS for the stomach and cecum, 5 mL for the small intestine, and 1 mL for each colonic segment. The sampled GI contents were centrifuged as described above, and the supernatants were collected. Additionally, all feces in the cages were collected, and marker extracts were obtained as described above. The concentrations of TRITC-dextran and FITC-dextran in the supernatants, fecal marker extracts, and diluted plasma were determined as described above. One female ICR mouse was excluded from the analysis owing to the suspicion of having ingested the excreted marker through feces, which was indicated by a higher proportion of the first dose marker than the second dose marker in the stomach.

Gastric, SI, cecal, and colonic transits were estimated using previously reported model equations [20]. Briefly, the stomach and cecum were treated as compartments and their contents were assumed to flow out with their own specific rate constants. The reciprocals of these rate constants were considered as the retention times of the stomach and cecum. The SI transit time was defined as the time taken for the marker excreted from the stomach to reach the cecum. In the colon, transit times were calculated for each of the four equal segments. The transit time of a segment was defined as the interval between marker excretion from the cecum or a previous segment and excretion into the next segment or feces. The colonic transit time was, therefore, the sum of the transit times of these four segments. These retention and transit times were estimated for each mouse using the nonlinear least squares method with the Excel Solver add-in (Microsoft® Excel® 2013; Microsoft Corporation, Seattle, WA, USA). Initial values were determined using the grid search method. However, for one male CFP mouse in which the most distal arrival site of the first dose marker was the third quarter colonic segment, the transit time of the third- and fourth-quarter colons and the entire colon could not be calculated.

Feed and Water Intake and Fecal Excretion

Feed and water intake were measured for each cage during the 48-h period of WGTT and MRT measurements, and the daily amounts of feed and water intake per mouse were determined. The dry weight of feces and the number of fecal pellets excreted per mouse per day were calculated for each cage using the feces collected during WGTT and MRT measurements.

Fecal Water Content

At the age of 10–12 weeks, fresh feces were collected between 8:15 and 9:10 am. Fecal water contents were calculated using the formula ((wff − wdf)/wff) × 100, where wff and wdf denote the weight of the fresh and freeze-dried feces, respectively.

Amount and Water Content of Cecal and Colonic Contents

Mice were housed in same-strain and -sex pairs for 4 weeks before the experiment. At the age of 12 weeks, the mice were euthanized between 8:00 and 10:30 am as described above, without blood collection. After rapidly measuring the distance of the colonic content from the cecocolic junction, cecal and colonic contents were collected. Colonic contents were collected every 2 cm from the cecocolic junction. The cecal and colonic water contents were calculated using the same method as the fecal water content described above.

Histopathological Analysis

Male mice were housed in same-strain pairs for at least 3 weeks before the experiment. At the age of 12 weeks, the mice were euthanized by exsanguination under anesthesia, as described above, between 9:00 and 11:30 am. The colon was collected and divided at half the length from the cecocolic junction to the anus. The proximal and distal sides from the center of the colon were then divided into 2-cm units. Each sample was fixed in 10% neutral-buffered formalin (Mildform® 10N; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for approximately 24 h and embedded in paraffin wax. The paraffin-embedded samples were sectioned longitudinally at about 4-μm thickness and stained with hematoxylin–eosin. Each section was examined under a light microscope by one pathologist in a blinded manner. Additionally, the thickness of the colonic muscle layer was measured at the first unit distal to the center of the colon using ImageJ (version 1.53c, Java 1.8.0_172) [23] at six points in the area that contained contents, and the average value was determined. Samples without contents were excluded from the evaluation.

Cecal and Fecal Microbiota AnalysisCollection of Feces and Cecal Contents

Mice were housed in same-strain and -sex groups of six per cage for 3 weeks before the collection of feces and cecal contents. At the age of 10 weeks, fresh feces were collected between 1:00 and 3:20 pm and stored at − 80 °C until use. The day after fecal collection, mice were euthanized by exsanguination under anesthesia, as described above, between 9:00 am and 12:00 pm. Cecal contents were immediately collected and stored at − 80 °C until use.

Bacterial DNA Extraction

For bacterial DNA extraction, cecal contents and fecal samples were homogenized with nine volumes of PBS (v/w). For 200 µL of homogenate, 250 µL of Tris–EDTA (TE) buffer solution, 50 µL of 10% sodium dodecyl sulfate solution, 0.3 g of glass beads (0.1 mm diameter), and 500 µL of TE-saturated phenol were added. The mixture was vigorously shaken for 15 min using ShakeMaster® (BioMedical Sciences, Tokyo, Japan). Following centrifugation at 20,630×g for 5 min, 400 µL of supernatant was collected. An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the supernatant. The mixture was vigorously shaken for 15 s. After centrifugation at 20,630×g for 5 min, 250 µL of supernatant was collected and purified via isopropanol precipitation. After rinsing with 80% ethanol, the DNA was suspended in 1 mL of TE buffer and stored at − 30 °C until use.

16S rRNA Gene Amplicon Sequencing

The V4 region of the bacterial 16S rRNA gene was amplified and sequenced according to a previously described method [24], with minor modifications [25]. Briefly, bar-coded amplicons were obtained through PCR using the primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), TB Green®Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio Inc., Shiga, Japan), and DNA extracted from each sample in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The thermal cycler program was set as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, 50 °C for 30 s, and 72 °C for 40 s. The reaction was immediately stopped before the amplification reached a plateau. Subsequently, the amplicons obtained were purified and quantified using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA) and QuantiFluor® dsDNA System (Promega, Madison, WI, USA). The purified amplicons were pooled in equimolar amounts and sequenced using the MiSeq Reagent Kit v2 (500 cycles) (Illumina, Inc., San Diego, CA, USA) on a MiSeq® System (Illumina, Inc.).

Meta 16S rRNA Gene Analysis

The raw sequence data were processed in QIIME2 [26] (version 2021.4) using the Silva database (version 138_1) as a reference to generate a feature table (Online Resource 1) and determine relative abundances at the bacterial phylum and family levels. Differences in microbiota between samples were assessed based on unweighted and weighted UniFrac distances and visualized using principal coordinate analysis (PCoA) conducted with QIIME2. Alpha diversity indices, including observed features, the Shannon index, and Faith’s phylogenetic diversity (Faith’s PD), were calculated using QIIME2.

Statistical Analysis

The data are presented as medians and interquartile ranges. The effects of mouse strain, sex, and their interactions were analyzed using an aligned rank transform (ART) [27] two-way analysis of variance (ANOVA). Additionally, the effects of mouse strain, sampling source, and their interactions were analyzed using ART two-way repeated measures ANOVA. For post hoc analysis, the Steel–Dwass test or Mann–Whitney U test was used for unpaired data and Wilcoxon signed-rank test was used for paired data. The Mann–Whitney U test and Wilcoxon signed-rank test underwent Bonferroni correction. Differences in muscle layer thickness were analyzed using Welch’s t test. Microbiota differences between CFP and ICR mice and between cecal and fecal contents were evaluated using permutational multivariate ANOVA (PERMANOVA). Statistical tests were performed using Bell Curve for Excel version 4.05 (Social Survey Research Information Co., Ltd., Tokyo, Japan), except for PERMANOVA, which was performed using QIIME2. A significance threshold of p < 0.05 was applied. Effect size was determined using partial epsilon squared (εp2) and r-squared (r2).