Quantitation of Microcomputed Tomography-Imaged Ocular Microvasculature

Abstract

To quantitatively assess microvascular dimensions in the eyes of neonatal wild-type and VEGF(120)-tg mice, using a novel combination of techniques which permit three-dimensional (3D) image reconstruction. A novel combination of techniques was developed for the accurate 3D imaging of the microvasculature and demonstrated on the hyaloid vasculature of the neonatal mouse eye. Vascular corrosion casting is used to create a stable replica of the vascular network and X-ray microcomputed tomography (muCT) to obtain the 3D images. In-house computer-aided image analysis techniques were then used to perform a quantitative morphological analysis of the images. With the use of these methods, differences in the numbers of vessel segments, their diameter, and volume of vessels in the vitreous compartment were quantitated in wild-type neonatal mice or littermates over-expressing a labile (nonheparin binding) isoform of vascular endothelial growth factor (VEGF(120)) from the developing lens. This methodology was instructive in demonstrating that hyaloid vascular networks in VEGFA(120) over-expressing mice have a 10-fold increase in blind-ended, a six-fold increase in connected vessel segments, in addition to a sixfold increase (0.0314 versus 0.0051 mm(3)) in total vitreous vessel volume compared with wild type. These parameters are not readily quantified via histological, ultrastructural, or stereological analysis. The combination of techniques described here provides the first 3D quantitative characterization of vasculature in an organ system; i.e., the neonatal murine intra-ocular vasculature in both wild-type mice and a transgenic model of lens-specific over-expression of VEGF.