In Situ Fluorescence Measurement of Tear Film [Na+], [K+], [Cl−], and pH in Mice Shows Marked Hypertonicity in Aquaporin-5 Deficiency

Abstract

Tear film composition depends on water and ion transport across ocular surface epithelia and on fluid secretion by lacrimal glands. The purpose of this study was to establish in situ fluorescence methods to measure tear film ionic concentrations and pH in mice and to determine whether tear film composition is sensitive to deficiency of the major ocular surface aquaporin water channels. Tear film ionic concentrations and pH were measured in anesthetized mice by ratio imaging fluorescence microscopy after topical application of ion/pH-sensing, dual-wavelength fluorescent indicators. [Na(+)], [K(+)], and [Cl(-)] were measured with membrane-impermeant indicators developed by our laboratory, and pH was measured with bis(carboxyethyl)-carboxyfluorescein fluorescence-conjugated dextran. Measurements were performed on wild-type mice and on knockout mice lacking aquaporins AQP1, AQP3, and AQP5. In wild-type mice, tear film [Na(+)] was 139 +/- 8 mM, [K(+)] was 48 +/- 1 mM, [Cl(-)] was 127 +/- 4 mM, and pH was 7.59 +/- 0.2 (SE; n = 5-8). pH did not differ significantly in the AQP knockout mice. [Na(+)] was increased by approximately twofold in AQP5 null mice (230 +/- 20 mM) and was greatly reduced after exposure of the ocular surface to a humidified atmosphere. [K(+)] was mildly reduced in AQP1 null mice. These results establish an in situ optical methodology to measure tear film [Na(+)], [K(+)], [Cl(-)], and pH in living mice, without the need for fluid sampling. Tear film hypertonicity in AQP5 deficiency is likely caused by reduced transcorneal water secretion in response to evaporative water loss.