title: Epithelial barrier function in vivo is sustained despite gaps in epithelial layers.
abstract: OBJECTIVE
  Epithelial cells of the small intestine migrate to the tip of the villus at which they are shed. It is not understood how the intestinal barrier is maintained during this high cell turnover. The aim of this study was to use high-resolution in vivo light microscopy to investigate the mechanism of epithelial shedding and the site of the permeability barrier during cell shedding.
  
  
  METHODS
  A laparotomy was performed on anesthetized mice, and a segment of small intestine was opened. The exposed epithelial surface of the intestine was imaged by multiphoton microscopy. Nuclei, cytosol, and cell membranes were imaged using the dyes Hoescht 33258, BCECF, a transgenically expressed fluorescent protein, and the membrane dye DiI. The fluorescent caspase substrate PhiPhiLux was used to detect apoptosis.
  
  
  RESULTS
  In the epithelial monolayer, gaps were observed that lacked nuclei or cytosol but appeared to be filled with an impermeable substance. Studies with membrane impermeant fluorophores (Lucifer Yellow and Alexa-dextran) showed that the impermeable substance completely fills the void left by the absent cell. Only a fraction of gaps have either ZO-1 staining or cytoplasmic extensions from neighboring cells at the basal pole. Time-lapse studies reveal that cell shedding results in genesis of a gap and that shedding usually occurs prior to detectable cellular activation of caspase 3 or nuclear condensation.
  
  
  CONCLUSIONS
  Results suggest that epithelial barrier function is sustained at the apical pole of the epithelial layer, despite discontinuities in the cellular layer.
authors.affiliation: Department of Medicine, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Alastair J M
authors.initials: AJ
authors.lastname: Watson
authors.forename: Shaoyou
authors.initials: S
authors.lastname: Chu
authors.forename: Leah
authors.initials: L
authors.lastname: Sieck
authors.forename: Oleg
authors.initials: O
authors.lastname: Gerasimenko
authors.forename: Tim
authors.initials: T
authors.lastname: Bullen
authors.forename: Fiona
authors.initials: F
authors.lastname: Campbell
authors.forename: Michael
authors.initials: M
authors.lastname: McKenna
authors.forename: Tracy
authors.initials: T
authors.lastname: Rose
authors.forename: Marshall H
authors.initials: MH
authors.lastname: Montrose
mesh_terms: D000818:Animals; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000166:cytology* / Q000502:physiology* / Q000601:surgery; D051379:Mice; D008871:Microvilli / Q000502:physiology / Q000648:ultrastructure; D023421:Models, Animal