Extracorporeal membrane oxygenation causes loss of intestinal epithelial barrier in the newborn piglet

Abstract

Extracorporeal membrane oxygenation (ECMO) is an important life-support system used in neonates and young children with intractable cardiorespiratory failure. In this study, we used our porcine neonatal model of venoarterial ECMO to investigate whether ECMO causes gut barrier dysfunction. We subjected 3-wk-old previously healthy piglets to venoarterial ECMO for up to 8 h and evaluated gut mucosal permeability, bacterial translocation, plasma levels of bacterial products, and ultrastructural changes in gut epithelium. We also measured plasma lipopolysaccharide (LPS) levels in a small cohort of human neonates receiving ECMO. In our porcine model, ECMO caused a rapid increase in gut mucosal permeability within the first 2 h of treatment, leading to a 6- to 10-fold rise in circulating bacterial products. These changes in barrier function were associated with cytoskeletal condensation in epithelial cells, which was explained by phosphorylation of a myosin II regulatory light chain. In support of these findings, we also detected elevated plasma LPS levels in human neonates receiving ECMO, indicating a similar loss of gut barrier function in these infants. On the basis of these data, we conclude that ECMO is an independent cause of gut barrier dysfunction and bacterial translocation may be an important contributor to ECMO-related inflammation.

Fig.1. ECMO-mediated gut barrier dysfunction results in bacterial translocation

A. B. Plasma LPS (panel A) and D-lactate (panel B) levels increased rapidly during ECMO (n=5 animals/group). Line diagrams show means ± SEM values as a function of time. Black circles indicate data points from the ECMO group, while open circles depict the sham animals. * indicates p<0.05 when compared to sham at corresponding data points. C. Representative western blots show bacterial LTA in plasma. Bar graph of plasma lipoteichoic acid represents the sum of densities at each point (time after initiation of ECMO) for the 3 ECMO and 3 sham control animals. * indicates p<0.05 when compared to sham at corresponding data points. D. Brown and Brenn stain on intestinal sections (magnification 400x) reveals bacteria in the ECMO intestine. Inset (1500x) shows rods and cocci (arrows). Bar diagram shows means ± SE of the number of bacteria counted per 100 IECs in all 3 ECMO animals (2 hrs after initiation of ECMO) and sham controls. * indicates p<0.001 when compared to sham at corresponding data points.

Fig.2. ECMO increases gut mucosal permeability

ECMO increased the mucosal permeability in ex planted jejunal (panel A) and colonic (panel B) samples, measured as mucosal-to-basal penetration of FITC-dextran 4400 across tissue specimens in 60 min (n=3–4 tissue replicates per animal. N=3 animals per group; tissue replicates from each animal were averaged and used as a single data point. * indicates p<0.01, ** indicates p<0.001.

Fig.3. Increased gut mucosal permeability during ECMO is not due to decreased expression of genes involved in the assembly of the tight junctions

Bar diagram (means ± SEM) depicts the expression of 25 porcine genes involved in tight junctions after 2 hours of ECMO (black bars), shown as fold change in mRNA expression above sham (grey bars marked at fold change = 1); n= 3 animals/group. The expression of 20 of these 25 genes was increased (80%). * indicates p<0.05 when compared to sham. Analysis for reduced gene expression by calculating 95% confidence intervals did not show significant reductions. Claudin 10 mRNA was not detected in either sham or ECMO intestine. Abbreviations: OCLN = occludin, CLDN1-23 = claudin 1–23, ZO1/TJP1 = zonula occludens protein-1/tight junction protein-1, CAR = coxsackie-adenovirus-receptor homolog, CTNNA1 = catenin (cadherin-associated protein), alpha 1, CTNNB1 = catenin (cadherin-associated protein), beta 1.

Fig.4. Ultrastructural changes in IECs in the ECMO intestine

A. Loss of intercellular apposition in IECs in the ECMO intestine: Transmission electron micrographs (TEMs) of (a) sham and (b) ECMO intestine showing increased separation of IECs after 2 hours of ECMO (open arrows), except at desmosome sites (black arrows). Bar diagram (means ± standard error) on the lower left summarizes the distances between adjacent epithelial cells in sham and ECMO intestine. Distance bar represents 500 nm. Data include junctions in 2 villi (or colonic crypts) in 3 randomly chosen areas. * indicates p<0.01, ** indicates p<0.001. B. Increased junctional permeability in the ECMO intestine: TEMs of (a) sham and (b) ECMO intestine show increased penetration of the electron-dense dye ruthenium red in the ECMO intestine (arrows). Distance bar represents 500 nm.

Fig.5. ECMO causes cytoskeletal contraction in IECs

A. TEMs of (a) sham and (b1) ECMO intestine (magnification 15000x). Unlike the uniform appearance of the perijunctional IEC cytoskeleton in the sham intestine, IECs in the ECMO intestine show electron-dense areas of cytoskeletal condensation (arrows). High-magnification image b2 shows prominent cytoskeletal condensation near a tricellular junction. B. ECMO increases the phosphorylation of myosin II light chain (MLC) in IECs: Western blots show increased phosphorylation of MLC in IECs. Bar diagrams shows densitometric analysis (means ± SE densitometric units) of the three bands (each representing a different animal) from sham and ECMO groups. Data were confirmed by repeating the blots. * indicates p<0.01, ** indicates p<0.001.

Fig.6. Human neonatal ECMO is associated with increased plasma LPS concentrations

Scatter plots showing higher plasma LPS concentrations in neonates during the first 48 hours of ECMO therapy (n = 8) than in critically-ill neonates on mechanical ventilation but not treated with ECMO (n = 8). * indicates p<0.05.