Villus Growth, Increased Intestinal Epithelial Sodium Selectivity, and Hyperaldosteronism Are Mechanisms of Adaptation in a Murine Model of Short Bowel Syndrome

Abstract

Background: Short bowel syndrome results from extensive small bowel resection and induces adaptation of the remaining intestine. Ileocecal resection (ICR) is the most frequent situation in humans. Villus hypertrophy is one hallmark of mucosal adaptation, but the functional mechanisms of mucosal adaptation are incompletely understood.

Aims: The aim of the study was to characterize a clinically relevant model of short bowel syndrome but not intestinal failure in mice and to identify outcome predictors and mechanisms of adaptation.

Methods: Male C57BL6/J mice underwent 40% ICR and were followed for 7 or 14 days. Small bowel transection served as control. All mice underwent autopsy. Survival, body weight, wellness score, stool water content, plasma aldosterone concentrations, and paracellular permeability were recorded.

Results: Unlike controls, resected mice developed significant diarrhea with increased stool water. This was accompanied by sustained weight loss throughout follow-up. Villus length increased but did not correlate positively with adaptation. Plasma aldosterone concentrations correlated inversely with body weight at day 14. After ICR, intestinal epithelial (i.e., tight junctional) sodium permeability was increased.

Conclusions: 40% ICR results in moderate to severe short bowel syndrome. Successful adaptation to the short bowel situation involves villus elongation but does not correlate with the degree of villus elongation alone. In addition, increased intestinal epithelial sodium permeability facilitates sodium-coupled solute transport. Hyperaldosteronism correlates with the severity of weight loss, indicates volume depletion, and counterregulates water loss.

Keywords: Clinical outcome; Ileocecal resection; Intestinal adaptation; Intestinal failure; Mice; Short bowel syndrome.

Fig. 1

Kaplan–Meier curves of mice who underwent ileocecal resection (ICR) or sham operation with defined experimental endpoint at day 7 (a) or day 14 (b) (POD). Mice that survived until the expected observation period were defined as “survivor,” whereas mice that died unexpectedly or were killed because of reaching a humane endpoint were defined as “dead.” Similar early survival of ICR and sham controls (POD 7, p = 0.813). b At later time points (POD 14), survival curves of sham and ICR mice separated although the difference did not become significant during the observation period (p = 0.061)

Fig. 2

Absolute frequency of complications in ICR and sham mice related to the days of occurrence. Each symbol represents one mouse that died spontaneously or was killed because of reaching a humane endpoint: black, resected mice (n = 28); gray symbols indicate sham-operated animals (n = 6). Median is shown as a horizontal line

Fig. 3

Gross morphology of the intestine after sham surgery (a) or ileocecal resection (b, c). Adapted small bowels displayed some dilatation over the entire length at 14 days after resection (b). Mechanical ileus with massive pre-anastomotic dilatation due to obstruction (arrow) at day 11 (c). sb: small bowel, lb: large bowel, a: anastomosis, c: cecum

Fig. 4

Age (a) and baseline body weight (b) of resected (ICR) and sham control mice that reached the expected end point (survivor) or died earlier (dead) at time of surgery. Each symbol represents one mouse. Medians are shown as horizontal line. a *p = 0.024, U test. c Small bowel lengths of survivors and dead ICR mice (mean ± SEM, **p = 0.008, U test). d Correlation between remaining small bowel length and body weight on day 14 as a percentage from day 0 in ICR mice that survived to the expected endpoint. Pearson r = 0.28, p = 0.18, n = 24

Fig. 5

Weight course (a) and wellness (b) of mice with ileocecal resection (ICR) or sham operation (sham) over time. a Body weight as percentage from day 0. b Wellness score of 11 indicates maximal wellbeing. Data only from mice that survived until the end of the experiment, those who that died earlier were excluded. All data are shown as mean + SEM. ICR (n = 44 d0–d7; n = 27–28 d8–d14) versus Sham (n = 30 d0–d7; n = 17–18 d8–d14) α_adjust = 0.004; *p < 0.004, **p < 0.001, ***p < 0.0001

Fig. 6

Wellness score change predicts premature death. In surviving mice, the day-to-day wellness score change from 48 h to 24 h prior to visit was calculated for all days. In premature dying mice, the wellness score change from 48 h to 24 h before death was calculated. Mean change during the first 4 days was similar between survivors (n = 132, black) and dead mice (n = 11, light gray, p = 0.827). Thereafter, mean wellness score change for dead mice (n = 13, white) was significantly reduced compared to survivors (n = 327, dark gray). Mean + SEM, ***p < 0. 001, U test

Fig. 7

Stool water content was measured before surgery (0 h) and at d2, d5, d7, d10, and d14 after ileocecal resection (ICR, n = 10) or sham operation (Sham, n = 3) in a separate group. Resected mice had increased stool water contents compared to controls. Mice that died before day 14 were excluded. Data are shown as mean + SEM. α_adjust < 0.01; *p = 0.007 d10

Fig. 8

Correlation between body weight and plasma aldosterone concentration. In a random group (n = 9) of ICR mice, plasma aldosterone concentration at day 14 was correlated with relative body weight at day 14 (compared to d0). Plasma aldosterone concentration at day 14 was inversely correlated with body weight at d14. Spearman r = − 0.6, p < 0.05

Fig. 9

Villus lengths (a) and crypt depths (b) at time of surgery (0 days), 7 days (n = 6 ICR, n = 7 sham), or 14 days (n = 9 ICR, n = 4 sham) after ileocecal resection (ICR) or sham surgery. Data are shown as mean + SEM. a **p = 0.0014 ICR 0d versus ICR 7d, n = 6 and ##p = 0.0011 ICR 0d versus ICR 14d, n = 9 paired t test. b ***p = 0.001 ICR 0d versus ICR 7d, n = 6 and ##p = 0.0013 0d ICR versus 14d ICR, n = 9

Fig. 10

Correlation between body weight and villus length. In a random group (n = 9) of ICR mice, relative villus length (compared to d0) was correlated with relative body weight on d14 (compared to d0). Relative villus length was inversely correlated with body weight on d14. Pearson r = − 0.67, p = 0.05 (a). Representative H and E images of resected segments at day 0 (b, c) and of the corresponding adapted intestines from mice with low (d) or high (e) body weight at day 14. Note that not only villi become longer but also that the subepithelial and intravillous tissue enlarges. Scale bar represents 100 µm

Fig. 11

Ussing chamber studies of mucosal barrier function in the jejunum after ileocecal resection. Jejunal tissues were stripped off the serosa and the muscularis. a Transmucosal electrical resistance (TMER) did not change in sham-operated mice (n = 4). At day 14, the jejunum of ICR mice displayed significantly higher TMER (n = 9; p < 0.05, Wilcoxon test). b Relative Na/Cl permeability, which is represented by epithelial tight junctions, was calculated from Na/Cl dilution potentials. Both after sham and after ICR, ion selectivity was unchanged (sham n = 6; ICR n = 9; p > 0.05, respectively) despite villus hypertrophy, which only occurred in ICR animals. c The paracellular leak pathway was studied by 4 kDa fluorescein isothiocyanate–dextran FITC 4-kDa dextran diffusion. At day 14, the permeability in sham-operated mice (n = 7) was not significantly changed. At day 14, permeability was numerically lower in ICR mice, although not statistically significant (n = 6; p = 0.16, Wilcoxon test)