Computational protocol: Distinct intestinal adaptation for vitamin B12 and bile acid absorption revealed in a new mouse model of massive ileocecal resection

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[…] Survival analysis was performed for two cohorts of mice: mice that underwent 75% ICR (n=6) or sham operation (n=6). Mice were excluded if they died during surgery or within 48 h post-surgery. As the Kaplan–Meier method and log-rank test showed statistically significant difference between the two cohorts (see B), we did not repeat the same survival analysis. Instead, by using the body weight data on 14 POD in this analysis (n=6 for sham and n=4 for ICR) as a pilot data, we performed a power analysis retrospectively by using G*Power 3.1.9.2. (Heinrich-Heine-Universitat Dusseldorf, Germany). With the given effect size value of 5.41 and an alpha value of 0.05, a power value of 1-beta >0.999 was obtained, which indicated the sample size of 4 to 6 to be large enough. Thus, we repeated mouse experiments on two more separate occasions (independent experiments), randomly assigning more than six mice into sham group or 75% ICR group. All mice were weighed and monitored daily for survival and clinical signs. Mice with severe weight loss (≥30% of body weight before operation), hunched posture, ruffled fur, or bleeding from any orifice were sacrificed by cervical dislocation, and these events were considered lethal endpoints.For tissue and blood sample analyses, mice were harvested at 14 POD. Blood samples were obtained by cardiac puncture during anesthesia with isoflurane (n=6 for each of the 75% ICR, and sham and un-operated groups), and outsourced to Oriental Yeast Co., Ltd. (Tokyo, Japan) for analysis of serum vitamin B12 levels. Tissues were harvested immediately after mice were sacrificed by cervical dislocation. Images of the removed intestines were obtained with an EOS Kiss X7 digital camera (Canon, Tokyo, Japan) equipped with a macro lens EFS (60 mm). Then, the entire lengths of intestines were measured on those digital images. As depicted in A, we designated in this study the first and second halves of the jejunum (J), ileum (I) and colon (C) as J1, J2, I1, I2, C1, and C2 (A). The remnant intestine in 75% ICR mice thus consisted of the entire duodenum (designated as D), the remaining portion of J1, and entire C1 and C2. We used the middle 1 cm of these four portions of the remnant intestine in the 75% ICR mice and the corresponding intestinal segments of sham operation mice for the following mRNA analyses. We also harvested the portion immediately proximal to each intestinal segment used for mRNA analysis for histological assays. RNA isolation and analysis were also performed with all seven segments (from D to C2 segments) of un-operated control mice. Then number of biological replicates in each analysis is indicated in the following sections. [...] Intestinal tissues were fixed overnight at 4°C in 4% paraformaldehyde, sequentially dehydrated in 10, 15 and 20% sucrose in PBS, and embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan). Frozen sections were stained with hematoxylin and eosin (H&E) or subjected to immunofluorescent staining by using antibodies specific for Ki67 (M7248, Dako Agilent Technologies Japan Ltd. Tokyo, Japan; used at 1:50 dilution), SLC10A2 (sc-27494, Santa Cruz Biotechnology Inc. TX, USA; 1:50 dilution), or CUBN (sc-20609, Santa Cruz Biotechnology Inc.; 1:500 dilution). In all immunofluorescent experiments, nuclei were counterstained with DAPI. For quantification of the caliber, five different perpendicular H&E sections were randomly chosen from a series of sections originating from each segment of a mouse (n=3 or 5 for each of the 75% ICR, sham and un-operated group per experiment), and the values were averaged and presented. We used the entire circumference of the intestinal submucosa as a measure of intestinal caliber as previously described (). Intestinal calibers were measured by using ImageJ (NIH Image, MD, USA). Villus height and crypt depth were analyzed by randomly choosing well-oriented, full-length crypt-villus units (D and J1) or crypt units (C1 and C2) on H&E sections. For comparison, the values of 60 or 100 villi (D and J1) and crypts (D, J1, C1, and C2) were obtained in total by examining 20 different structural units in sections originating from individual mice (n=3 or 5 for each of the 75% ICR, sham and un-operated group per experiment). Quantification of Ki67+ cells on fluorescently labeled sections was conducted in the same way as for crypt depth analysis by obtaining 60 or 100 values from individual mice (n=3 or 5 for each of 75% ICR, sham and un-operated group per experiment). All images were acquired by using a microscope system BZ-X710 with 2× or 20× objectives (KEYENCE, Osaka, Japan). If necessary, image processing was carried out using Photoshop CS4 software (Adobe, CA, USA). Each quantitative analysis was repeated two times and thus the number of total biological replicates is eight. Statistical significance was determined by the Mann–Whitney U test (P<0.05). […]

Pipeline specifications

Software tools G*Power, ImageJ
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Chemicals Vitamin B 12