In Vivo Three-Dimensional Analysis of Conjunctival Epithelial Microcysts in Glaucoma

Abstract

Objective: To analyze the three-dimensional (3D) features of conjunctival epithelial microcysts (CEM) in eyes affected with primary open angle glaucoma (POAG). Methods: This was a case series study. Nine patients that underwent successful trabeculectomy and four eyes with medically controlled POAG were enrolled. Patients were examined with confocal laser-scanning microscope (Heidelberg Retina Tomograph/Rostock Cornea Module). Sequential images 300 × 300 μm (384 × 384 pixels) derived from automatic scans were acquired throughout the upper bulbar conjunctiva, 2 mm from the limbus. Image acquisition was performed in z-scan automatic volume mode and a series of 40 images to a maximum depth of 40 μm were captured. The 3-D volume tissue reconstruction with a maximal size of 300 × 300 × 40 μm and voxel size of 0.78 × 0.78 × 0.95 μm was performed with the AMIRA volume-rendering software package, to provide a 3-D characterization of conjunctival epithelial microcysts (CEM). Results: In the enface view, CEM appeared as empty, optically clear, round or oval shaped sub-epithelial structures. In eyes that underwent trabeculectomy CEM showed greater density and larger area compared to medically controlled glaucomatous eyes. The 3-D spatial reconstruction showed microcysts as oval-shaped, optically clear, differently sized structures, often surrounded by a well-defined and mildly thick wall. All microcysts were embedded in the extra cellular spaces and located 10 μm below the epithelial surface. Conclusions: Conjunctival epithelial microcysts were proposed as hallmark of trans-scleral aqueous humor outflow in eyes with glaucoma. They can be effectively imaged with a 3-D reconstruction system, which permits to better clarify their microscopic anatomy and patho-physiological significance.