@article{moeser_haskell_shifflett_little_schultz_blikslager_2004, title={CIC-2 chloride secretion mediates prostaglandin-induced recovery of barrier function in ischemia-injured porcine ileum}, volume={127}, ISSN={["1528-0012"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-4444353665&partnerID=MN8TOARS}, DOI={10.1053/j.gastro.2004.06.004}, abstractNote={Background & Aims Ischemia results in the breakdown of the intestinal barrier, predisposing patients to sepsis and multiple organ failure. Prostaglandins play a critical role in mediating recovery of barrier function in ischemia-injured intestine through a mechanism involving stimulation of Cl− secretion. In the present study, we investigated the contributory role of individual Cl− channels in the recovery of barrier function in ischemia-injured porcine ileum. Methods Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers. Short-circuit current (Isc) and transepithelial resistance (TER) were measured in response to prostaglandin E2 (PGE2) and pharmacologic inhibitors of epithelial Cl− channels. Immunoassays were used to assess the expression and localization of ion channels. Results Application of PGE2 to ischemia-injured ileal mucosa stimulated increases in Isc, an indicator of Cl− secretion, that was followed by marked increases in TER, an indicator of barrier function recovery. In vitro studies revealed that although PGE2 induced Cl− secretion via at least 3 distinct secretory pathways, recovery of barrier function was initiated by Cl− secretion via ClC-2 Cl− channels co-expressed with occludin and localized to tight junctions within restituting epithelium. Intravenous administration of furosemide to pigs subjected to 1 hour of ileal ischemia impaired recovery of barrier function, as evidenced by decreased TER and increased mucosal-to-serosal 3H-mannitol flux after a 2-hour reperfusion/recovery period, confirming an important role for Cl− secretory pathways in vivo. Conclusions ClC-2–mediated intestinal Cl− secretion restores TER in ischemia-injured intestine. These data may provide the basis for targeted pharmacologic therapy for diseases associated with impaired barrier function. Background & Aims Ischemia results in the breakdown of the intestinal barrier, predisposing patients to sepsis and multiple organ failure. Prostaglandins play a critical role in mediating recovery of barrier function in ischemia-injured intestine through a mechanism involving stimulation of Cl− secretion. In the present study, we investigated the contributory role of individual Cl− channels in the recovery of barrier function in ischemia-injured porcine ileum. Methods Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers. Short-circuit current (Isc) and transepithelial resistance (TER) were measured in response to prostaglandin E2 (PGE2) and pharmacologic inhibitors of epithelial Cl− channels. Immunoassays were used to assess the expression and localization of ion channels. Results Application of PGE2 to ischemia-injured ileal mucosa stimulated increases in Isc, an indicator of Cl− secretion, that was followed by marked increases in TER, an indicator of barrier function recovery. In vitro studies revealed that although PGE2 induced Cl− secretion via at least 3 distinct secretory pathways, recovery of barrier function was initiated by Cl− secretion via ClC-2 Cl− channels co-expressed with occludin and localized to tight junctions within restituting epithelium. Intravenous administration of furosemide to pigs subjected to 1 hour of ileal ischemia impaired recovery of barrier function, as evidenced by decreased TER and increased mucosal-to-serosal 3H-mannitol flux after a 2-hour reperfusion/recovery period, confirming an important role for Cl− secretory pathways in vivo. Conclusions ClC-2–mediated intestinal Cl− secretion restores TER in ischemia-injured intestine. These data may provide the basis for targeted pharmacologic therapy for diseases associated with impaired barrier function. The intestinal epithelium serves a divergent role in gastrointestinal function. On one hand, the intestinal epithelium facilitates the efficient transport of water and nutrients across the epithelium while on the other hand it forms a selective barrier that restricts potentially pathogenic luminal microorganisms and their products from traversing the epithelium and gaining entry into the subepithelial tissues and circulation.1Gerwitz A.T. Liu Y. Sitaraman S.V. Madara J.L. Intestinal epithelial pathobiology past, present, and future.Best Pract Res Gastroenterol. 2002; 16: 851-867Abstract Full Text PDF PubMed Scopus (50) Google Scholar Disruption of the intestinal barrier is a sequela to a number of gastrointestinal diseases including inflammatory bowel disease, ischemia-reperfusion injury, nonsteroidal anti-inflammatory drug-induced gastropathy, and assault by enteric pathogens.2Berkes J. Viswanathan V.K. Savkovic S.D. Hecht G. Intestinal epithelial responses to enteric pathogens effects on the tight junction barrier, ion transport, and inflammation.Gut. 2000; 52: 439-451Crossref Scopus (461) Google Scholar, 3DeMeo M.T. Mutlu E.A. Keshavarzian A. Tobin M.C. Intestinal permeation and gastrointestinal disease.J Clin Gastroenterol. 2002; 34: 385-396Crossref PubMed Scopus (234) Google Scholar, 4Hawkey C.J. 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Prostaglandin biology in inflammatory bowel disease.Gastroenterol Clin North Am. 2001; 30: 971-980Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar The latter effect has been shown by the ability of PGs to induce Cl− secretion and secretory diarrhea in both in vitro and in vivo models.16Gaginella T.S. Eicosanoid-mediated intestinal secretion.in: Lenethal E. Duffy M. Textbook of secretory diarrhea. Raven, New York1990: 15-30Google Scholar, 17Eberhart C.E. DuBois R.N. Eicosanoids and the gastrointestinal tract.Gastroenterology. 1995; 109: 285-301Abstract Full Text PDF PubMed Scopus (406) Google Scholar, 18Lanza F.L. Kochman R.L. Geis G.S. Rack E.M. Deysach L.G. A double-blind, placebo-controlled, 6-day evaluation of two doses of misoprostol in gastroduodenal mucosal protection against damage from aspirin and effects on bowel habits.Am J Gastroenterol. 1991; 86: 1743-1748PubMed Google Scholar Despite the negative implications associated with excessive PG production, a low level of PG synthesis is required for normal gut function. This apparent PG tone is involved in the maintenance of gastrointestinal protective functions such as stimulation of epithelial mucus and bicarbonate secretion, regulation of mucosal blood flow, and induction of epithelial cell proliferation.17Eberhart C.E. DuBois R.N. Eicosanoids and the gastrointestinal tract.Gastroenterology. 1995; 109: 285-301Abstract Full Text PDF PubMed Scopus (406) Google Scholar, 19Wallace J.L. Bell C.J. Gastromucosal defense.Curr Opin Gastroenterol. 1996; 12: 503-511Crossref Scopus (23) Google Scholar The cytoprotective role of PGs is evidenced clearly by the gastrointestinal tract injury induced by chronic nonsteroidal anti-inflammatory drug therapy.20Bjarnason I. Williams P. Smethurst P. Peters T.J. Levi A.J. The effect of NSAIDs and prostaglandins on the permeability of the human small intestine.Gut. 1986; 27: 1292-1297Crossref PubMed Scopus (240) Google Scholar, 21Graumlich J.F. Preventing gastrointestinal complications of NSAIDs risk factors, recent advances, and latest strategies.Postgrad Med. 2001; 109: 117-128Crossref PubMed Scopus (29) Google Scholar We previously have shown a critical role of PGs in the recovery of barrier function in injured intestine.22Blikslager A.T. Roberts M.C. Rhoads M.J. Argenzio R.A. Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum.J Clin Invest. 1997; 100: 1928-1933Crossref PubMed Scopus (112) Google Scholar, 23Blikslager A.T. Roberts M.C. Argenzio R.A. Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion.Am J Physiol. 1999; 276: G28-G36PubMed Google Scholar, 24Little D. Dean R.A. Young K.M. McKane S.A. Martin L.D. Jones S.L. Blikslager A.T. PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum.Am J Physiol. 2003; 284: G46-G56Google Scholar, 25Gookin J.L. Galanko J.A. Blikslager A.T. Argenzio R.A. Prostaglandin-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum.Am J Physiol. 2003; 285: G967-G979Google Scholar This is based on evidence that treatment of acutely injured porcine ileum with the nonsteroidal anti-inflammatory drug indomethacin significantly impaired the ability of the intestine to recover barrier function, as measured by the recovery of transepithelial electrical resistance (TER). Furthermore, application of exogenous PGs to injured mucosa rapidly restored TER, an effect associated with the closure of the paracellular space. PGs stimulate cyclic adenosine monophosphate-dependent Cl− secretion and inhibit electroneutral Na+ absorption,7Argenzio R.A. Lecce J. Powell D.W. Prostanoids inhibit intestinal NaCl absorption in experimental porcine cryptosporidiosis.Gastroenterology. 1993; 104: 440-471PubMed Google Scholar, 26Halm D.R. Rechkemmer G.R. Schoumacher R.A. Frizzell R.A. Apical membrane chloride channels in a colonic cell line activated by secretory agonists.Am J Physiol. 1998; 254: C505-C611Google Scholar both of which represent critical components of the resealing process of the tight junctions after injury.23Blikslager A.T. Roberts M.C. Argenzio R.A. Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion.Am J Physiol. 1999; 276: G28-G36PubMed Google Scholar We hypothesize that increases in TER induced by PGs are a result of a signaling pathway involving an osmotic gradient triggered by luminal accumulation of Cl− and Na+. The detailed mechanisms by which PGs alter electrolyte transport and trigger recovery of barrier function have not been characterized fully. Therefore, the objective of the present experiments was to define further the mechanisms through which PGs alter electrolyte transport in the ischemia-injured intestine, specifically assessing the role of individual Cl− channels in restoration of barrier function. Our data indicate that although PGE2-induced Cl− secretion is mediated through at least 3 epithelial Cl− channels in ischemia-injured porcine ileum, restoration of barrier function involves a distinct Cl− secretory event mediated through ClC-2 Cl− channels, expressed in the interepithelial tight junctions. All studies were approved by the North Carolina State University Institutional Animal Care and Use Committee. Six- to 8-week-old Yorkshire cross-bred pigs of either sex were housed individually, and maintained on a commercial pelleted feed. Pigs were held off feed for 24 hours before experimental surgery. General anesthesia was induced with xylazine (1.5 mg/kg, intramuscularly), ketamine (11 mg/kg, intramuscularly), and thiopental (15 mg/kg, intravenously), and maintained with intermittent infusion of thiopental (6–8 mg/kg/h), as previously described.22Blikslager A.T. Roberts M.C. Rhoads M.J. Argenzio R.A. Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum.J Clin Invest. 1997; 100: 1928-1933Crossref PubMed Scopus (112) Google Scholar, 24Little D. Dean R.A. Young K.M. McKane S.A. Martin L.D. Jones S.L. Blikslager A.T. PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum.Am J Physiol. 2003; 284: G46-G56Google Scholar The ileum was approached via a ventral midline incision. Ileal segments were delineated by ligating the intestine at 10-cm intervals, and subjected to ischemia by occluding the local mesenteric blood supply for 45 minutes. After the 45-minute ischemic period, tissues were harvested from the pig and the mucosa was stripped from the seromuscular layer in oxygenated (95% O2/5% CO2) Ringer’s solution. Tissues then were mounted in 3.14-cm2 aperture Ussing chambers, as described in previous studies.27Argenzio R.A. Liacos J.A. Endogenous prostanoids control ion transport across neonatal porcine ileum in vitro.Am J Vet Res. 1990; 51: 747-751PubMed Google Scholar For each Ussing chamber experiment, ileal tissues from one pig were mounted on multiple Ussing chambers. Tissue segments were mounted randomly in Ussing chambers without reference to their location along the length of the ileum. Each tissue was subjected to a different in vitro treatment. Tissues were bathed on the serosal and mucosal sides with 10 mL of Ringer’s solution. The serosal bathing solution contained 10 mmol/L glucose, and was balanced osmotically on the mucosal side with 10 mmol/L mannitol. Bathing solutions were oxygenated (95% O2/5% CO2) and circulated in water-jacketed reservoirs. The spontaneous potential difference was measured using Ringer-agar bridges connected to calomel electrodes, and the potential difference was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. TER (Ω · cm2) was calculated from the spontaneous potential difference and short-circuit current (Isc), as previously described.22Blikslager A.T. Roberts M.C. Rhoads M.J. Argenzio R.A. Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum.J Clin Invest. 1997; 100: 1928-1933Crossref PubMed Scopus (112) Google Scholar, 24Little D. Dean R.A. Young K.M. McKane S.A. Martin L.D. Jones S.L. Blikslager A.T. PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum.Am J Physiol. 2003; 284: G46-G56Google Scholar For experiments assessing the role of Cl− channels in the PGE2-induced recovery of barrier function, tissues were pretreated (t = 0 min) with pharmacologic Cl− channel inhibitors on the appropriate surface and then treated with PGE2 (10−6 mol/L) on the serosal side of the tissue (t = 30 min). For each in vitro experiment, a total of 6 pigs were used (n = 6). For each pig, multiple mucosal samples were harvested and mounted in Ussing chambers, with each tissue exposed to a distinct treatment. A total of 12 pigs were used for in vivo recovery experiments. For each experiment, general anesthesia was induced in 2 pigs with xylazine (1.5 mg/kg, intramuscularly) and ketamine (11 mg/kg, intramuscularly). Pigs then were intubated and maintained on isoflurane throughout the duration of the experiment. Pigs were placed on heating pads and lactated Ringer’s solution was administered intravenously at a maintenance rate of 15 mL/kg/h. Body temperature, heart rate, and respiration were measured throughout the experiment. Intestinal injury was induced by cross-clamping 10-cm segments of ileum, and clamping local vasculature for 1 hour, after which the clamps were removed, and the intestine was allowed to reperfuse for 2 hours. Pigs remained anesthetized for the entire experiment. Just before reperfusion, furosemide (1 mg/kg, intravenously) was administered to one of each of the pairs of pigs while the other pig received an equal volume of saline and served as a control. At the end of the 2-hour recovery period the pigs were euthanized. Ileal tissues were harvested and mounted on Ussing chambers for determination of TER and mucosal-to-serosal flux of 3H-mannitol. To assess transmucosal Na+ and Cl− fluxes, 22Na or 36Cl were added to the mucosal or serosal solutions of tissues paired according to their conductance (conductance within 25% of each other). After a 15-minute equilibration period and before addition of treatments, standards were taken from the bathing reservoirs. Thirty minutes after the addition of treatments, 3 successive 60-minute flux periods (from 30 to 210 minutes of the experiments) were performed by taking samples from the bathing reservoirs opposite the side of isotope addition. Samples were counted for 22Na and 36Cl in a liquid scintillation counter, and unidirectional fluxes were calculated as previously described.7Argenzio R.A. Lecce J. Powell D.W. Prostanoids inhibit intestinal NaCl absorption in experimental porcine cryptosporidiosis.Gastroenterology. 1993; 104: 440-471PubMed Google Scholar Similar methods were used to quantify mucosal-to-serosal fluxes of 3H-labeled mannitol. Tissues were taken at 0, 60, and 240 minutes for routine histologic evaluation. Tissues were sectioned (5 μm) and stained with H&E. For each tissue, 3 sections were evaluated. Four well-oriented villi were identified in each section, and the villus height and the height of the epithelial-covered portion of each villus were measured. The percentage of the villous surface area that remained denuded was calculated from the total surface area of the villus and the surface area of the villus covered by epithelium, as previously described.7Argenzio R.A. Lecce J. Powell D.W. Prostanoids inhibit intestinal NaCl absorption in experimental porcine cryptosporidiosis.Gastroenterology. 1993; 104: 440-471PubMed Google Scholar Ileal scrapings from control and ischemia-injured mucosa were snap-frozen and stored at −70°C before sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE). Tissue aliquots were thawed at 4°C and added to 3 mL of chilled lysis buffer, including protease inhibitors at 4°C, as previously described. This mixture was homogenized on ice and then centrifuged at 4°C, and the supernatant was saved. Protein analysis of extract aliquots was performed (DC Protein Assay; Bio-Rad, Hercules, CA). Tissue extracts (amounts equalized by protein concentration) were mixed with an equal volume of 2× SDS-PAGE sample buffer and boiled for 4 minutes. Lysates were loaded on a 10% SDS-polyacrylamide gel, and electrophoresis was performed according to standard protocols. Proteins were transferred to a nitrocellulose membrane (Hybond ECL; Amersham Life Science, Birmingham, UK) by using an electroblotting minitransfer apparatus. Membranes were blocked at room temperature for 60 minutes in Tris-buffered saline plus 0.05% Tween 20 and 5% dry powdered milk. Membranes were washed and incubated with primary antibody (rabbit ClC-2 polyclonal antibody; Alpha Diagnostic International, San Antonio, TX; or rabbit cystic fibrosis transmembrane conductance regulator [CFTR] polyclonal antibody; Research Diagnostics Inc, Flanders, NJ). After additional washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibody, and developed for visualization of protein by the addition of enhanced chemiluminescence reagent (Amersham, Piscataway, NJ) as previously described.24Little D. Dean R.A. Young K.M. McKane S.A. Martin L.D. Jones S.L. Blikslager A.T. PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum.Am J Physiol. 2003; 284: G46-G56Google Scholar Densitometry analysis was performed by using appropriate software (IP gel; Scanalytics, Fairfax, VA). Tissue extracts were prepared according to the Western blotting protocol described previously.24Little D. Dean R.A. Young K.M. McKane S.A. Martin L.D. Jones S.L. Blikslager A.T. PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum.Am J Physiol. 2003; 284: G46-G56Google Scholar Extracted proteins (500 μg) were solubilized in RIPA buffer with protease inhibitors and incubated with rabbit ClC-2 polyclonal antibody (5 μg/mL) for 1 hour at 37°C. The antibody-protein complexes were adsorbed from solution with protein A/G-Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). Protein samples were washed 3× in ice-cold RIPA buffer and centrifuged at 25,000 × g for 1 minute at 4°C. Protein pellets were solubilized in 2× SDS-PAGE sample buffer and electrophoresis was performed according to standard procedures described previously.24Little D. Dean R.A. Young K.M. McKane S.A. Martin L.D. Jones S.L. Blikslager A.T. PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum.Am J Physiol. 2003; 284: G46-G56Google Scholar Nitrocellulose membranes were incubated with anti-ClC-2 (50 μg/mL). For co-immunoprecipitation experiments, immunoprecipitated ClC-2 or rabbit pre-immune serum were subjected to SDS-PAGE and probed using an occludin antibody (2 μg/mL; Santa Cruz Biotechnology). Immunofluorescence labeling was performed on ileal tissues that were embedded in optimal cutting temperature medium, frozen, sectioned at 5-μm thickness, and fixed in cold acetone. Tissue sections were blocked with 2% bovine serum albumin before incubation with rabbit anti-ClC-2 polyclonal antibody (40 μg/mL) in BLOTTO (Bovine Lacto Transfer Technique Optimizer) for 2 hours at 4°C. Sections were washed with BLOTTO and incubated for 45 minutes with Cy3-conjugated anti-rabbit secondary antibody (1:300; Zymed Laboratories Inc., San Francisco, CA) in the dark. Sections were washed in phosphate-buffered saline, mounted, cover slipped, and viewed with an immunofluorescence microscope. Ileal tissues were fixed in 4% formaldehyde and processed for transmission electron microscopy using standard techniques.28Dykstra M.J. A manual of applied techniques for biological electron microscopy. Plenum Press, New York1993Crossref Google Scholar In brief, tissue samples were dehydrated in a graded ethanol series. Tissues were placed in fresh LR white resin (Electron Microscopy Sciences, Fort Washington, PA) in gelatin capsules and polymerized at 55°C-60°C overnight. Ultrathin sections (80–90 nm) were mounted on stainless steel grids (Electron Microscopy Sciences, Fort Washington, PA). For immunolocalization, grid sections were blocked in 5% normal goat serum (Biogenix, San Ramon, CA) before incubation with rabbit anti-ClC-2 polyclonal antibody (200 μg/mL) for 1 hour. Sections were washed and incubated with gold-conjugated anti-rabbit immunoglobulin G secondary antibody (Sigma, St. Louis, MO; 10-nm particle size) in normal goat serum for 1 hour. Tissue sections were rinsed with phosphate-buffered saline, dried, and stained with 1% methanolic uranyl acetate and viewed on an electron microscope (Phillips/FEICO Model 208s Transmission Electron Microscope; Hillsboro, OR). Indomethacin, 16,16 dimethyl PGE2, 4,4′-diaminostilbene-2,2′-disulfonic acid (DNDS), cadmium chloride (CdCl2), zinc chloride (ZnCl2), bumetanide, PD-098,059, and 5-nitro 2-(3-phenylpropylamino) benzoic acid were purchased from Sigma Chemical. N-(4-methylphenylsulfonyl)-N′-(4-trifluoromethylphenyl)urea (DASU-02) can be obtained from B. D. Schultz (Kansas State University, Manhattan, KS). Data were reported as means ± SE based on the experimental number (n). All data were analyzed by using an analysis of variance (ANOVA) for repeated measures, except when the peak response was analyzed by using a standard one-way ANOVA (Sigmastat; Jandel Scientific, San Rafael, CA). A Tukey’s test was used to determine differences between treatments after ANOVA. Forty-five minutes of intestinal ischemia resulted in diminished TER values compared with nonischemic control tissue (27 ± 1.1 Ω · cm2 in ischemic tissue vs. 53 ± 2.5 Ω · cm2 in control tissue), which is indicative of impaired barrier function. Application of 10−6 mol/L PGE2 to the serosal side of ischemia-injured mucosa resulted in rapid and significant (P < 0.01) restoration of TER (▵TER = 26 Ω · cm2, Figure 1A), achieving control levels within 30 minutes of PGE2 application. In line with previous observations,23Blikslager A.T. Roberts M.C. Argenzio R.A. Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion.Am J Physiol. 1999; 276: G28-G36PubMed Google Scholar, 24Little D. Dean R.A. Young K.M. McKane S.A. Martin L.D. Jones S.L. Blikslager A.T. PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum.Am J Physiol. 2003; 284: G46-G56Google Scholar PGE2-induced increases in TER were preceded by sharp and significant (P < 0.01) increases in short circuit current (▵Isc = 22 μA/cm2, Figure 1B, C), an indicator of electrogenic Cl− secretion. Histologic (Figure 2) and morphometric (Table 1) evaluation of ischemia-injured mucosa revealed a significant decrease in villus height (160 ± 20 μm in control vs. 100 ± 30 μm in ischemic tissue) and a significant denuding of ischemic mucosa (30% ± 2.7% denuded villus surface area). Denuded villous tips were near-fully restituted within 60 minutes from the time they were mounted in Ussing chambers (Table 1). Furthermore, restitution was not enhanced by administration of PGE2, and treatment with indomethacin alone did not retard restitution. Given these results, we concluded that the significant increases in TER in the presence of PGE2 were attributable to changes in paracellular rather than transcellular resistance, as has been shown in previous studies.23Blikslager A.T. Roberts M.C. Argenzio R.A. Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion.Am J Physiol. 1999; 276: G28-G36PubMed Google Scholar, 24Little D. Dean R.A. Young K.M. McKane S.A. Martin L.D. Jones S.L. Blikslager A.T. PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum.Am J Physiol. 2003; 284: G46-G56Google Scholar, 25Gookin J.L. Galanko J.A. Blikslager A.T. Argenzio R.A. Prostaglandin-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum.Am J Physiol. 2003; 285: G967-G979Google ScholarTable 1Morphometric Assessment of Epithelial Restitution in Ischemia-Injured Porcine Ileal MucosaTreatmentRecovery time (min)Epithelial surface area denuded (%)Villus height (mm)Nonischemic control000.16 ± 0.02aP < 0.05 vs. ischemia alone at 0 minutes.Ischemic030.2 ± 4.70.10 ± 0.01Ischemic/Indo603.1 ± 2.6aP < 0.05 vs. ischemia alone at 0 minutes.0.16 ± 0.01aP < 0.05 vs. ischemia alone at 0 minutes.Ischemic/PGE2602.8 ± 1.6aP < 0.05 vs. ischemia alone at 0 minutes.0.10 ± 0.02Ischemic/Indo/PGE2604.4 ± 1.6aP < 0.05 vs. ischemia alone at 0 minutes.0.14 ± 0.02aP < 0.05 vs. ischemia alone at 0 minutes.NOTE. Values represent means ± SE for % villus surface area denuded and villus height; n = 6. Tissues were subjected to 45 minutes ischemia in vivo. Tissues were harvested at 0 and 60 minutes after ischemia, fixed in 10% buffered formalin, and processed for histologic examination according to standard protocols. Indomethacin (Indo) was administered to select tissues at 5 × 10−6 mol/L, and PGE2 was given at 1 × 10−6 mol/L.a P < 0.05 vs. ischemia alone at 0 minutes. Open table in a new tab NOTE. Values represent means ± SE for % villus surface area denuded and villus height; n = 6. Tissues were subjected to 45 minutes ischemia in vivo. Tissues were harvested at 0 and 60 minutes after ischemia, fixed in 10% buffered formalin, and processed for histologic examination according to standard protocols. Indomethacin (Indo) was administered to select tissues at 5 × 10−6 mol/L, and PGE2 was given at 1 × 10−6 mol/L. Because basolateral Cl− uptake is mediated predominantly by the Na+-K+-2 Cl− cotransporter and represents a critical component of Cl− secretion, we treated tissues with the specific Na+-K+-2 Cl− transport inhibitor bumetanide, and measured its effect on Isc and TER. Application of bumetanide (10−4 mol/L) to the serosal side of is}, number={3}, journal={GASTROENTEROLOGY}, author={Moeser, AJ and Haskell, MM and Shifflett, DE and Little, D and Schultz, BD and Blikslager, AT}, year={2004}, month={Sep}, pages={802–815} }