PG-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum

Abstract

Small bowel epithelium is at the frontline of intestinal barrier function. Restitution is considered to be the major determinant of epithelial repair, because function recovers in parallel with restitution after acute injury. As such, studies of intact mucosa have largely been replaced by migration assays of cultured epithelia. These latter studies fail to account for the simultaneous roles played by villous contraction and paracellular permeability in recovery of barrier function. NSAIDs result in increased intestinal permeability and disease exacerbation in patients with inflammatory bowel disease (IBD). Thus we examined the reparative attributes of endogenous PGs after injury of ileal mucosa by deoxycholate (6 mM) in Ussing chambers. Recovery of transepithelial electrical resistance (TER) from 20-40 Omega.cm2 was abolished by indomethacin (Indo), whereas restitution of 40-100% of the villous surface was unaffected despite concurrent arrest of villous contraction. In the presence of PG, resident crypt and migrating epithelial cells were tightly apposed. In tissues treated with Indo, crypt epithelial cells had dilated intercellular spaces that were accentuated in the migrating epithelium. TER was fully rescued from the effects of Indo by osmotic-driven collapse of the paracellular space, and PG-mediated recovery was significantly impaired by blockade of Cl- secretion. These studies are the first to clearly distinguish the relative contribution of paracellular resistance vs. restitution to acute recovery of epithelial barrier function. Restitution was ineffective in the absence of PG-mediated paracellular space closure. Failure of PG-mediated repair mechanisms may underlie barrier failure resulting from NSAID use in patients with underlying enteropathy.

Figure 1

Transepithelial electrical resistance and mucosal-to-serosal flux (J_m→s) of ³H-labeled mannitol across porcine ileal mucosa mounted in Ussing chambers. Mucosae were acclimated for 15-min followed by a 15-min luminal exposure to 6 mM deoxycholate (black bar). Deoxycholate was then replaced by fresh Ringer’s solution (time = 30-min) and recovery of resistance and permeability were recorded over the following 180-min period (✱✱✱p<0.001; two-way repeated measures ANOVA)(✱✱ p<0.01 compared to injured mucosa at flux period 1 [values above bars indicate raw data]; one-way ANOVA). Values represent mean ± SE; n = number of pigs.

Figure 2

Restitution and villous contraction of deoxycholate injured porcine ileal mucosa mounted in Ussing chambers. (A) Percent villous epithelialization of mucosae removed from the Ussing chamber at specified time periods after deoxycholate injury. Bars represent mean ± SE; n = 4 pigs at 45 and 150-min; n = 8 pigs at 0, 75, and 210-min. (✱p<0.05,✱✱ p<0.01 –vs- time-matched, Ussing-chambered and uninjured control tissue (n = 4 each) (one-way ANOVA). (B) Histologic appearance of villi after removal from the Ussing chamber. Commensurate with deoxycholate injury (t = 30-min) there were epithelial losses from the tips of villi. Epithelial losses continued for the first 45-min of recovery culminating in peak injury at time = 75-min. Between 75 and 210-min there was partial restitution of the injured villi by flattened to cuboidal migrating enterocytes. Chamber-mounted, uninjured control tissue maintained epithelial continuity throughout the study period (chamber control). Magnification, 314X. (C) Villus and crypt measurements performed at specified time periods after deoxycholate injury. Bars represent mean ± SE. For injured tissue, n = 4 pigs at 45 and 150-min; n = 8 pigs at 30, 75, and 210-min. ✱ p<0.05, ✱✱ p<0.01, ✱✱✱ p<0.001–vs- time-matched, Ussing-chambered and uninjured control tissue (n = 18 at 0-min and n = 4 at each subsequent time period)(one-way ANOVA). There was a significant decrease in villous height of injured tissue between 45 and 210-min (II), ✱ p<0.05 (one-way ANOVA).

Figure 3

Recovery of transepithelial electrical resistance (A) and restitution (B) of Ussing chambered porcine ileal mucosa after deoxycholate injury and treatment with exogenous prostaglandins (PG) or indomethacin (INDO). PGE₂ (10⁻⁶ M) and prostacyclin (10⁻⁶ M) or INDO (5×10⁻⁶M) were added immediately after injury and prior to onset of repair (time = 30-min). (A) INDO abolished recovery of TER while addition of exogenous PG resulted in an early elevation in TER that did not exceed baseline recovery at 210-min (✱ p<0.05 ✱✱ p<0.01 and ✱✱✱ p<0.001 –vs- injured control; one-way ANOVA). (B) Percent epithelialization of villi are shown at timepoints corresponding to maximal effects of exogenous PG (75-min) and INDO (210-min) on recovery of TER. Values represent mean ± SE; n= number of pigs; n.s. = not statistically significant.

Figure 4

Transepithelial electrical resistance and degree of restitution attained by porcine ileal mucosa at peak repair (time = 210-min) after injury by deoxycholate in Ussing chambers. Although restitution was equivalent in the presence or absence of INDO (5 × 10⁻⁶M), there was no correlation between TER and restitution among mucosae treated with INDO (p>0.10; Pearson correlation coefficient).

Figure 5

Relative contributions of epithelialized and denuded villus to total villous surface area of deoxycholate-injured mucosae at peak injury (time = 75-min) and maximal repair (time = 210-min). Indomethacin (INDO; 5×10⁻⁶M) arrested villous contraction during repair, resulting in a significant increase in total villous surface area compared to injured control or prostaglandin-treated (PG) tissue at 210-min (✱ p<0.05; one-way ANOVA). Restitution (% villous epithelialization) was unaffected by either treatment. Values represent mean ± SE; n = number of pigs.

Figure 6

Effects of enhanced restitution on recovery of barrier function after deoxycholate injury to porcine ileal mucosa mounted in Ussing chambers. Restitution was stimulated by addition of L-arginine (ARG; 5 mM) and fetal bovine serum (FBS; 1%) to the serosal and mucosal reservoir immediately after injury and prior to onset of repair. Results are shown for mucosae at peak repair (A, B). Indomethacin (INDO; 5×10⁻⁶M) did not attenuate baseline or stimulated restitution despite arrest of villus contraction during recovery. Magnification, 250X. Barrier function, as determined by measures of transepithelial electrical resistance (TER)(C) and mucosal to serosal flux of ³H-mannitol (J_m→s) (D), was abolished in the absence of endogenous prostaglandin (PG) synthesis, despite nearly complete restitution of villi. (✱p<0.05, ✱✱p<0.01, ✱✱✱p<0.001; one-way ANOVA). Values represent mean ± SE; n = number of pigs. Bar = bile salt exposure.

Figure 7

Transepithelial electrical resistance (TER) of porcine ileal mucosa at peak repair (time = 210-min) after injury by deoxycholate in Ussing chambers. A mucosal to serosal osmotic gradient of urea did not attenuate PG-mediated recovery of TER. Conversely, a serosal to mucosal osmotic gradient of urea could reproduce the effect of endogenous PG on recovery of TER after deoxycholate injury. Values represent mean ± SE; n = number of pigs. (✱✱✱p<0.001; one-way ANOVA).

Figure 8

Transmission electron micrographs of crypt (A&B) and migrating villous enterocytes (C&D) from porcine ileal mucosa at peak repair after deoxycholate injury (time = 210-min). Mucosae shown in B & D were treated with indomethacin (INDO; 5×10⁻⁶M) immediately after injury and prior to onset of repair. White arrows indicate intercellular space. Black arrows indicate sites of lateral membrane attachment between adjacent, migrating enterocytes.

Figure 9

Immunofluorescence localization of the junctional complex protein ZO-1 in porcine ileal mucosa. (A) Uninjured villous mucosa. (B) Villous mucosa at peak repair after deoxycholate injury (time = 210-min). (C) Villous mucosa treated with indomethacin (INDO; 5×10⁻⁶M) immediately after deoxycholate injury and examined at peak repair (time = 210-min). Magnification, 314X.