title: The synergistic effects of hypoxia/reoxygenation or tissue acidosis and bacteria on intestinal epithelial cell apoptosis.
abstract: BACKGROUND
  Clinical data indicate that gut perfusion deficits must be rectified within 24 hours after traumatic injury to decrease organ failure and death. Ischemia/reperfusion injury to the gut causes enterocyte apoptosis (Apo), which may contribute to intestinal barrier failure. The temporal response of enterocyte Apo to acidosis and hypoxia/reoxygenation (H/R) in vitro is unknown. The purpose of this study was to examine the effect of various time points of acidosis or H/R on enterocyte apoptosis and monolayer integrity in an in vitro model.
  
  
  METHODS
  Caco-2 cell monolayers were made acidic (Dulbecco's modified Eagle's medium, pH 6.9) by hydrochloric acid or exposed to 95% nitrogen/5% carbon dioxide (hypoxia) and then 21% oxygen (reoxygenation). Escherichia coli C-25 were added to the apical media in subsets. Apo and necrosis were quantified by flow cytometry. Permeability was determined by fluorescein isothiocyanate-dextran. Transepithelial electrical resistance (TEER) indexed monolayer.
  
  
  RESULTS
  Extracellular acidosis and C-25 significantly increased apoptosis of Caco-2 cells at 18 hours (extracellular acidosis [EC] + C-25, 14.5 +/- 3.0; control, 3.8 +/- 0.8; p < 0.001 by analysis of variance). Similarly, the H/R + C-25 group showed a significant increase in apoptosis at 12 hours (H/R + C-25 vs. control, 22.86 +/- 2.12 vs. 3.74 +/- 0.7; p < 0.001 by analysis of variance). The permeability difference was not significant for EC + C-25 versus control at 18 hours (0.68 +/- 0.25 vs. 0.43 +/- 0.0.0.36, respectively; p > 0.05). The H/R + C-25 group had a profound increase in permeability over control at 12 hours (10.8 +/- 0.5 vs. 2.1 +/- 0.3, respectively; p < 0.001). The TEER was significantly lowered for EC versus control at 18 hours (458 +/- 1.5 vs. 468 +/- 8.2) and at 0, 6, and 18 hours for EC + C-25 (409 +/- 28.1, 443 +/- 16.8, and 438 +/- 8.9 vs. 455 +/- 6.5, 467 +/- 6.5, and 469 +/- 8.2, respectively). There was no significant change in the H/R and H/R + C-25 groups.
  
  
  CONCLUSIONS
  Synergism of H/R or tissue acidosis and bacteria caused increased Apo, TEER, and permeability in vitro.
authors.affiliation: Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. ldiebel@med.wayne.edu
authors.forename: Alfred E
authors.initials: AE
authors.lastname: Baylor
authors.forename: Lawrence N
authors.initials: LN
authors.lastname: Diebel
authors.forename: David M
authors.initials: DM
authors.lastname: Liberati
authors.forename: Scott A
authors.initials: SA
authors.lastname: Dulchavsky
authors.forename: William J
authors.initials: WJ
authors.lastname: Brown
authors.forename: Clement A
authors.initials: CA
authors.lastname: Diglio
mesh_terms: D000138:Acidosis / Q000503:physiopathology*; D017209:Apoptosis / Q000502:physiology*; D018938:Caco-2 Cells; D015687:Cell Hypoxia / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000502:physiology; D017097:Electric Impedance; D020895:Enterocytes / Q000502:physiology*; D004926:Escherichia coli / Q000502:physiology*; D006801:Humans; D006931:Hyperbaric Oxygenation; D066298:In Vitro Techniques; D015427:Reperfusion Injury / Q000503:physiopathology*; D013997:Time Factors