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J Trauma. 1999 Feb;46(2):280-5.

The effect of hypoxia/reoxygenation on the cellular function of intestinal epithelial cells.

Author information

1
Department of Surgery, University of Medicine and Dentistry of New Jersey Medical School, Newark 07103, USA.

Abstract

BACKGROUND:

Previously, using in vivo models, we have demonstrated that ischemia/reperfusion can increase intestinal mucosal permeability, promote bacterial translocation, and induce gut cytokine production. Because of the cellular heterogeneity of the gut, however, studies investigating the direct effects of hypoxia/reoxygenation on intestinal epithelial cells are confounded in in vivo model systems. Consequently, this study examines oxidant-mediated enterocyte injury using an in vitro intestinal enterocyte hypoxia/reoxygenation model system.

METHODS:

Two intestinal epithelial cell lines, IEC-6 and Caco-2, were seeded onto 3-microm filters in a Transwell bicameral system and grown until tight junction integrity was established. Cells were subjected to hypoxia in a sealed chamber with 95% nitrogen and 5% carbon dioxide and incubated at 37 degrees C for 60 or 90 minutes. Reoxygenation was initiated by replacing the media and putting the cells in an environment of room air plus 5% carbon dioxide. Permeability and bacterial translocation were assayed by measuring the phenol red concentration and culturing the bacteria that crossed the cell monolayer and reached the basal chamber of the bicameral system. Monolayer tight junction integrity was monitored by serial measurements of transepithelial electrical resistance (TEER), and cell viability was assessed by trypan blue dye.

RESULTS:

IEC-6 cell monolayers subjected to 60 or 90 minutes of hypoxia showed significantly higher permeability to phenol red, with 54+/-5% and 57+/-5% of the dye crossing the monolayers, respectively, compared with normoxic control (38+/-2%; p < 0.01). Caco-2 cell monolayers also had increased permeability to phenol red, with 24+/-6% and 20+/-4% of the phenol red crossing the monolayer after 60 or 90 minutes of hypoxia, respectively, compared with 8+/-3% in the normoxic controls (p < 0.01). At 3 hours after challenge with Escherichia coli, the monolayers subjected to 60 or 90 minutes of hypoxia had significantly increased bacterial translocation (IEC-6 cells, p < 0.05; Caco-2 cells, p < 0.01) compared with controls. The increased permeability of the hypoxic Caco-2 and IEC-6 monolayers was associated with a decrease in TEER beginning as early as 1 hour after reoxygenation (p < 0.01). Cell viability, however, was not decreased.

CONCLUSION:

These results indicate that hypoxia/reoxygenation can directly impair cellular function as manifested by increased monolayer permeability to phenol red, increased E. coli bacterial translocation, and a decrease in TEER values.

[Indexed for MEDLINE]

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