Enteral nutrient deprivation in patients leads to a loss of intestinal epithelial barrier function

Abstract

Objective: To investigate the effect of nutrient withdrawal on human intestinal epithelial barrier function (EBF). We hypothesized that unfed mucosa results in decreased EBF. This was tested in a series of surgical small intestinal resection specimens.

Design: Small bowel specifically excluding inflamed tissue, was obtained from pediatric patients (aged 2 days to 19 years) undergoing intestinal resection. EBF was assessed in Ussing chambers for transepithelial resistance (TER) and passage of fluorescein isothiocyanate (FITC)-dextran (4 kD). Tight junction and adherence junction proteins were imaged with immunofluorescence staining. Expression of Toll-like receptors (TLR) and inflammatory cytokines were measured in loop ileostomy takedowns in a second group of patients.

Results: Because TER increased with patient age (P < .01), results were stratified into infant versus teenage groups. Fed bowel had significantly greater TER versus unfed bowel (P < .05) in both age populations. Loss of EBF was also observed by an increase in FITC-dextran permeation in enteral nutrient-deprived segments (P < .05). Immunofluorescence staining showed marked declines in intensity of ZO-1, occludin, E-cadherin, and claudin-4 in unfed intestinal segments, as well as a loss of structural formation of tight junctions. Analysis of cytokine and TLR expression showed significant increases in tumor necrosis factor (TNF)-α and TLR4 in unfed segments of bowel compared with fed segments from the same individual.

Conclusion: EBF declined in unfed segments of human small bowel. This work represents the first direct examination of EBF from small bowel derived from nutrient-deprived humans and may explain the increased incidence of infectious complications seen in patients not receiving enteral feeds.

Figure 1

Physiologic measurement of distal small bowel barrier function was performed in Ussing chambers. Mounted mucosa and submucosa specimens (post-dissection of the muscularis propria) were studied in triplicate, and a minimum of N=4 humans were studied for each group. A. Measurements of transepithelial resistance (Ω*cm²) of epithelial barrier function in Fed (dark gray upper line) vs. Unfed (light gray lower line) small bowel groups. Regression lines show a significantly increased resistance in fed patients with increasing age, and a similar trend toward increased resistance in unfed specimens from older patients. B. Age-stratified transepithelial resistance in Fed vs. Unfed small bowel (*P<0.05 by t test). C. FITC-Dextran-4000 paracellular permeability was assessed by placement of the tracer molecule on the mucosal side with interval sampling from the submucosal side (muscularis and serosa were stripped). Results are shown in subgroups (N=6/group) from fed and unfed segments of matched patients. Note the greater permeation (P<0.05) in unfed segments at each time point measured.

Figure 2

Representative immunofluorescence images from samples taken from Fed and Unfed portions of the small bowel for ZO-1 and Occludin, E-Cadherin, and Claudin-4, with DAPI nuclear counterstain. Note the loss of the tight lattice formation in Unfed segments, along with an internalization of ZO-1 into the cytoplasm. Note also the relative increase in goblet cells within the epithelial lining (some of which are marked with white asterisks).

Figure 3

Representative histologic sections of Fed and Unfed sections of bowel. Hematoxylin and eosin (H&E) staining demonstrates villus flattening in unfed intestine, while periodic acid-Schiff (PAS) staining shows an increase in goblet cells. Scale = 50 µm.

Figure 4

mRNA levels of Toll-like receptors (TLRs) from 12 individuals from whom fed and unfed specimens were collected. When grouped analysis was performed, no statistical significance was achieved between patient, however, paired analysis, as shown, demonstrated a significant increase in TLR4 expression in unfed bowel.

Figure 5

mRNA levels of inflammatory cytokines from 12 patients from whom fed and unfed specimens were collected. Substantial inter-individual variability is apparent. Unfed bowel segments demonstrated a significant increase in TNF-α expression versus patient-matched fed segments. IL-1β and IFN-γ expression was increased in 10 of the 12 patients, but not in 2 patients (patients 6 and 8). See Table 2 for individual patient characteristics.