title: Noninvasive in vivo fluorescence measurement of airway-surface liquid depth, salt concentration, and pH.
abstract: The concentration of salt in the thin layer of fluid at the surface of large airways, the airway-surface liquid (ASL), is believed to be of central importance in airway physiology and in the pathophysiology of cystic fibrosis. Invasive sampling methods have yielded a wide range of ASL [NaCl] from 40 to 180 mM. We have developed novel fluorescent probes and microscopy methods to measure ASL thickness, salt concentration, and pH quantitatively in cell-culture models and in the trachea in vivo. By rapid z-scanning confocal microscopy, ASL thickness was 21 +/- 4 microm in well-differentiated cultures of bovine tracheal epithelial cells grown on porous supports at an air-liquid interface. By ratio imaging fluorescence microscopy using sodium, chloride, and pH-sensitive fluorescent indicators, ASL [Na+] was 97 +/- 5 mM, [Cl-] was 118 +/- 3 mM, and pH was 6.94 +/- 0.03. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 45 +/- 5 microm, [Na+] was 115 +/- 4 mM, [Cl-] was 140 +/- 5 mM, and pH was 6.95 +/- 0.05. Similar ASL tonicity and pH were found in cystic fibrosis (CFTR-null) mice. In freshly harvested human bronchi, ASL thickness was 55 +/- 5 microm, [Na+] was 103 +/- 3 mM, [Cl-] was 92 +/- 4 mM, and pH was 6.78 +/- 0.2. These results establish by a noninvasive approach the key properties of the ASL and provide direct evidence that the ASL is approximately isotonic and not saltier in cystic fibrosis.
authors.affiliation: Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
authors.forename: S
authors.initials: S
authors.lastname: Jayaraman
authors.forename: Y
authors.initials: Y
authors.lastname: Song
authors.forename: L
authors.initials: L
authors.lastname: Vetrivel
authors.forename: L
authors.initials: L
authors.lastname: Shankar
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D001999:Bronchoscopy; D002417:Cattle; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000503:physiopathology; D005336:Fiber Optic Technology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005466:Fluorocarbons; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007552:Isotonic Solutions; D051379:Mice; D018613:Microscopy, Confocal / Q000295:instrumentation; D023421:Models, Animal; D009993:Osmium Tetroxide; D010477:Perfusion / Q000295:instrumentation; D020545:Respiratory Mucosa / Q000737:chemistry* / Q000473:pathology; D014132:Trachea / Q000737:chemistry* / Q000473:pathology
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D005466:Fluorocarbons; D007552:Isotonic Solutions; C072954:fluorocarbon 72; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D009993:Osmium Tetroxide