Route of Fluid Transport Across Corneal Endothelium From the Observation of Movement of Microscopic Particles

Abstract

Abstract: : Purpose: In contrast with the local osmosis theory, the electro–osmotic transport hypothesis predicts the existence of relatively sizable fluid motions in the anterior chamber at or near the apical openings of the corneal endothelial cells tight junctions, as the fluid is funneled through them. The purpose of this study was to test whether such motions can be demonstrated. Methods: Cultured bovine corneal endothelial cells (CBCECs) were seeded on Anopore 0.2 µm pore membranes coated with fibronectin. Cells grew to confluence in DMEM with 10% FBS. Cell layers on their supports were placed in a chamber (Warner) with separate perfusions for the apical and basolateral sides. The chamber was mounted on a temperature–controlled (37 C) stage of an Olympus IX71 fluorescence microscope with a CARV confocal attachment and Cascade 512B CCD camera. Fluorescent polystyrene spheres (∼0.1 µm diameter) were placed in the apical compartment, and their motions in a plane immediately close to the apical membrane were serially recorded in frames taken every 3 s for 200 s. Sequential images were processed with IPlab software to detect particle tracks. Results: Particles exhibited two types of behavior: (1) random (Brownian) motions with average velocity of ∼ 1 um/s; (2) much faster translations (jumps) when particles crossed certain areas of the preparation. The jump velocities could be 6–10 times faster than random motions. Conclusions: From these initial experiments, CBCEC appears to transport water inhomogeneously, with some regions being more active than others. This is consistent with electro–osmotic transport. Presumably there can be areas with comparatively low resistance through which electrical current and fluid movement will be maximized. These findings are also in line with prior evidence (Hirsch and colleagues, 1994) indicating that corneal endothelial tight junctions are not morphologically uniform; thus, they may have areas with lower electrical resistance and higher hydraulic permeability. In contrast, localized fluid motions movements are difficult to explain if fluid transport is transcellular and driven by local osmosis.

Keywords: cornea: endothelium • pump/barrier function • microscopy: confocal/tunneling