A Workflow for Three-Dimensional Reconstruction and Quantification of the Monkey Optic Nerve Head Vascular Network

A comprehensive characterization of the three-dimensional (3D) vascular network of the optic nerve head (ONH) is critical to understanding eye physiology and pathology. Current in vivo imaging technologies, however, do not have simultaneous high spatial resolution and imaging depth to resolve the small vessels deep within the ONH. We describe a workflow for the 3D reconstruction and quantitative morphological analysis of the ONH vasculature. The vessels of a normal monkey ONH were perfusion labeled. Serial cryosections of the ONH were imaged using fluorescence microscopy (FM) and instant polarized light microscopy (IPOL) to visualize the labeled vessels and label-free collagen, respectively. The IPOL images were registered and used to form a stack of FM images from which the vessels were segmented and skeletonized to reconstruct the 3D vascular network. The network consisted of 12,966 vessel segments, 7989 branching points, and 1100 terminal points at the boundaries. For each vessel segment, we measured its length, tortuosity, inclination (theta), and polar orientation (phi). The length followed a lognormal distribution, whereas the distribution of the tortuosity followed an exponential decay. The vessels were mainly oriented toward the coronal plane (theta = 90 deg). For orientation, there were nearly as many vessels aligned circumferentially (phi = 90 deg) and radially (phi = 0 deg). Our results demonstrate the workflow for 3D eye-specific reconstruction and quantification of the monkey ONH vascular network. This is a critical first step to analyze the blood flow and oxygenation within the ONH, which will help understand the role of vascular dysfunction in glaucoma.