Quantifying retinal nerve fiber layer thickness histologically: A novel approach to sectioning of the retina

PURPOSE. To present a technique of ocular sectioning that enables continuous histologic measurements of peripapillary retinal nerve fiber layer (RNFL) thickness in a concentric ring around the optic disc, corresponding to similar regions measured by in vivo imaging techniques. METHODS. Two pig eyes and two normal human eyes were processed using the "umbrella" technique, in which peripapillary concentric ring sections were obtained, at increasing diameters, all centered on the optic disc. Each histologic ring section contains a continuous circumferential 360degrees retinal slice, oriented approximately perpendicular to the retinal surface. Every histologic slice contains each axon of the retina, sectioned perpendicular to each axon's long axis and at an equal set distance from the disc margin. RESULTS. Ring sections from pig and human eyes are presented and correlated to known RNFL anatomy. For the two human eyes, peripapillary RNFL thickness was quantified and plotted, resulting in the expected double-hump pattern. CONCLUSIONS. The umbrella technique provides, on a single histologic section, all necessary information for quantifying the entire RNFL layer of that eye, in a standardized fashion. This technique can simplify the process of analyzing the RNFL thickness histologically, assist in obtaining a normative database of RNFL thickness in humans, and be implemented as a histologic end point in animal studies evaluating new treatment modalities for glaucoma.