Abstract
Abstract: :
Purpose: To develop biophysical methods to measure the properties (ionic composition, pH and volume) of the tear film layer in situ in living mice, as well as water and ionic permeability of the corneal epithelium. Direct assessment of tear film physiology will facilitate definition of the pathophysiology of tear film deficiency. Dry eye results from a diverse set of etiologies where diminished tear volume represents a common endpoint. Elevated ion concentrations and osmolality have been implicated in the subsequent inflammatory changes that compromise epithelial integrity. Tear film thickness may also be a reliable measure of dry eye severity over time and during therapy. Methods: To measure ionic composition and pH, ratioable small–molecule fluorescent indicators were dissolved in mouse tear film for quantitative ratio imaging microscopy. Techniques are also under development to measure with submicron z–resolution tear film thickness. Given challenges posed by the mouse eye’s curved geometry, the need to maintain physiologic tear film volume, eye movements associated with respiration, and sub–layer heterogeneity, three methods are being evaluated for their suitability: scanning fluorescence confocal microscopy, scanning resistance measurements using patch–clamp technology, and scanning fluorescence measurements using fiber–optic probes designed for near–field microscopy. Also, fluorescence techniques have been adapted to study ocular surface fluid and ion flux across the corneal epithelium. Microliter–sized perfusion chambers were constructed to make quantitative measurements of corneal fluorescence during perfusion of the external corneal surface and in response to rapid solute exchange. A calcein fluorescence quenching method was used in an initial study to measure the osmotic water permeability of corneal epithelial cells. Results: In anesthesized CD1 wild–type mice, tear [Na+] was 123 ± 5 mM, [Cl–] was 127 ± 4 mM, and pH was 7.63 ± 0.06 (SE, n=9–12 mice). Corneal epithelial swelling studies showed that osmotic permeability was reduced two–fold in transgenic mice deficient in the membrane water channel, aquaporin–5. Cytoplasmic calcein fluorescence changed reversibly in response to cell swelling induced by rapid changes in perfusate osmolality, with a half–equilibration time of ∼1.5 second in the wild–type and ∼3 seconds in the knock–out. Conclusions: Quantitative fluorescence methods for measuring water and ion content and transport in tear film/cornea in living mouse eye were developed and validated. The first in situ measurements of tear film composition were made.
Keywords: cornea: tears/tear film/dry eye • cornea: epithelium • ion transporters