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(Investigative Ophthalmology and Visual Science. 2004;45:4423-4432.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0816
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Aquaporin-Dependent Water Permeation at the Mouse Ocular Surface: In Vivo Microfluorimetric Measurements in Cornea and Conjunctiva

Marc H. Levin and A. S. Verkman

From the Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California San Francisco, San Francisco, California.

PURPOSE. Fluorescence methods were developed to quantify membrane and tissue water permeabilities at the ocular surface and to compare water transport in wild-type mice versus transgenic mice lacking each of the water channels, aquaporin (AQP)-1, -3, and -5, normally expressed in cornea or conjunctiva.

METHODS. Membrane water permeabilities (Pfmem) of calcein-stained surface epithelial cells were measured from the kinetics of fluorescence quenching in response to rapid (<0.2 seconds) changes in extraocular fluid osmolarity. Tissue water permeabilities (Pftiss) across intact cornea and conjunctiva—the relevant parameters describing water movement into the hyperosmolar tear film in vivo—were determined by a dye-dilution method from the fluorescence of Texas red-dextran in an anisosmolar solution in a microchamber at the ocular surface.

RESULTS. Osmotic equilibration occurred with an exponential time constant ({tau}) of 1.3 ± 0.2 seconds (Pfmem = 0.045 cm/s) in calcein-loaded corneal epithelial cells of wild-type mice, slowing 2.1 ± 0.4-fold in AQP5-deficient mice; {tau} was 2.4 ± 0.1 seconds in conjunctiva (Pfmem = 0.025 cm/s), slowing 3.6 ± 0.7-fold in AQP3-deficient mice. In dye-dilution experiments, Pftiss of cornea was 0.0017 cm/s and decreased by greater than fivefold in AQP5-deficient mice. Pftiss in AQP5-null mice was restored to 0.0015 cm/s after removal of the epithelium. Pftiss of conjunctiva was 0.0011 cm/s and was not sensitive to AQP3 deletion.

CONCLUSIONS. These results define for the first time the water-transporting properties of the two principal ocular surface barriers in vivo. The permeability data were incorporated into a mathematical model of tear film osmolarity, providing insights into the pathophysiology of dry eye disorders.




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