title: A novel method for imaging sites of paracellular passage of macromolecules in epithelial sheets.
abstract: Understanding the dynamics of intestinal barrier function is key to elucidating oral delivery routes of therapeutics as well as to understanding various diseases that involve the mucosal immune system. Passage of macromolecules across barrier-forming epithelia is classically analyzed by means of various tracer flux measurements. This approach averages over contributions from many cells and lacks labeling of passage-sites. Thus, abundance and nature of involved cells have remained unidentified. We present a novel method that allowed for optical analysis of passage of various macromolecules on large-scale and single-cell level. To achieve tracking of passage loci in epithelia at submicrometer resolution we used biotinylated and fluorescent macromolecules that bind to basolateral membranes pre-labeled with cell-adherent avidin. We applied this method to epithelial cell lines and isolated mucosae in order to 3-dimensionally determine barrier leak properties over time. Tracer passage was found in all epithelia examined. However, it was infrequent, strikingly inhomogeneous, depended on culture duration and tightness of the monolayer. Stimulating passage with barrier-perturbing agents increased the number of leaks exposition time-dependently in cell lines and explanted mucosae. After stepwise opening of the paracellular passage pathway, integrated tracer-signal measured by our assay strictly correlated to simultaneously performed standard fluxes. Thus, our assay allows for the study of transepithelial macromolecule passage in various physiological and pathological conditions.
authors.affiliation: Institute of Anatomy II, Jena University Hospital, 07743 Jena, Germany. Electronic address: Jan.Richter@med.uni-jena.de.
authors.forename: Jan F
authors.initials: JF
authors.lastname: Richter
authors.affiliation: Institute of Physiology II, Jena University Hospital, 07743 Jena, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Schmauder
authors.affiliation: Institute of Clinical Physiology, Charité - Universitäts medizin Berlin, 12203 Berlin, Germany.
authors.forename: Susanne M
authors.initials: SM
authors.lastname: Krug
authors.affiliation: Institute of Anatomy II, Jena University Hospital, 07743 Jena, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gebert
authors.affiliation: Dept. of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Charité - Universitäts medizin Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schumann
mesh_terms: D000818:Animals; D001360:Avidin / Q000378:metabolism; D001681:Biological Assay; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D003106:Colon / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000378:metabolism*; D046911:Macromolecular Substances / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D010539:Permeability; D017208:Rats, Wistar
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D001360:Avidin; D001710:Biotin
keywords: Confocal imaging; Epithelial barrier function; Inflammatory bowel disease; Intestinal permeability; Leak pathway; Macromolecule passage; Tight junction