Intestinal barrier dysfunction in human necrotizing enterocolitis

Abstract

Background: Intestinal barrier dysfunction has been implicated in necrotizing enterocolitis (NEC), but has not been directly measured in human NEC.

Methods: Small intestines removed during surgery were immediately mounted in an Ussing chamber. mRNA expression of tight junction (TJ) proteins was measured with RT-PCR.

Results: Fifteen infants were included, 5 with NEC and 10 with other diagnoses. Average transepithelial resistance (TER) was 11.61±1.65Ω/cm² in NEC specimens, 23.36±1.48Ω/cm² at resection margin, and 46.48±5.65Ω/cm² in controls. Average flux of permeability marker mannitol was 0.23±0.06μMol/cm² per h in NEC, 0.04±0.01 μMol/cm² per h at resection margin, and 0.017±0.004 μMol/cm² per h in control tissue (p<0.05). RT-PCR analysis showed marked decrease in mRNA expression of a TJ protein occludin in NEC affected tissue (p<0.03 vs. control). Additionally, mRNA expression of myosin light chain kinase (MLCK), an important regulator of TJ permeability, was increased in NEC specimens.

Conclusion: These studies show for the first time that NEC intestinal tissue have increased intestinal permeability, even at grossly healthy-appearing resection areas. The increase in intestinal permeability in NEC appeared to be related in part to a decrease in occludin and an increase in MLCK expression.

Level of evidence: Level 2.

Keywords: Intestinal barrier function; Necrotizing enterocolitis; Occludin; Tight junction.

Fig. 1

Intestinal epithelial barrier. Tight junctions form an extracellular seal between adjacent cells and provide gate function to the paracellular permeation.

Fig. 2

Tight junction proteins in intestinal epithelial cells. Cytoplasmic and transmembrane TJ proteins contribute to paracellular barrier function.

Fig. 3

Intestinal barrier function in NEC. The intestinal mucosal tissues were mounted on Ussing chambers as described in methods to measure transepithelial electrical resistance (TER). The NEC margin and NEC affected tissue had significantly lower TER compared to control tissue. Control groups were then stratified by weight (A) and corrected gestational age (B). Tissues from more preterm, lower birth-weight infants had lower TER compared to tissues from infants that were more mature (^{*, #}, p < 0.01 vs. control, n ≥ 3).

Fig. 4

Intestinal paracellular permeability in NEC. The intestinal mucosal tissues were mounted on Ussing chambers as described in methods to measure paracellular flux of [³H]mannitol. The NEC margin and NEC affected tissue showed significantly increased [³H]mannitol flux compared to control tissues. When stratified by weight (A) and corrected gestational age (B), control tissues from more premature, lower birth-weight infants showed increased [³H]mannitol flux. (^{*, #}, p < 0.01 vs. control, n ≥ 3).

Fig. 5

Occludin mRNA expression in NEC. RT-PCR analysis of intestinal mucosal tissues showed significantly decreased mRNA expression of occludin in NEC tissue compared to control tissue. The mRNA expression of occludin was normalized to mRNA expression of GAPDH (^*, p < 0.01 vs. control, n ≥ 3).

Fig. 6

Myosin light chain kinase (MLCK) mRNA expression in NEC. RT-PCR analysis of intestinal mucosal tissues showed significantly increased mRNA expression of MLCK in NEC margin and NEC affected tissue compared to control tissue. The mRNA expression of MLCK was normalized to mRNA expression of GAPDH (^{#, *}p < 0.01 vs control, n ≥ 3).