High-Resolution, Three-Dimensional Reconstruction of the Outflow Tract Demonstrates Segmental Differences in Cleared Eyes

TL;DR

This study used high-resolution 3D imaging to analyze the outflow structures in pig eyes, revealing regional differences in outflow pathways and vessel sizes across the limbus.

Contribution

It demonstrated a novel application of ribbon-scanning confocal microscopy combined with optical clearing to visualize and analyze the entire outflow tract in three dimensions.

Findings

Largest collector channels are in the superonasal and inferonasal quadrants.

Proximal collector channels are larger and more elliptical nasally.

The scleral vascular plexus is larger in the inferior limbus.

Abstract

Purpose: The rate of conventional aqueous humor outflow is the highest nasally. We hypothesized that this is reflected in regionally different outflow structures and analyzed the entire limbus by high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM). Methods: We perfused pig eyes by anterior chamber cannulation with eight lectin-fluorophore conjugates, followed by optical clearance with benzyl alcohol benzyl benzoate (BABB). RSCM and an advanced analysis software (Imaris) were used to reconstruct a three-dimensional, whole-specimen rendering of the perilimbal outflow structures. We performed morphometric analyses of the outflow tract from the level of the trabecular meshwork (TM) to the scleral vascular plexus (SVP). Results: Except for pigmented structures, BABB cleared the entire eye. Rhodamine-conjugated Glycine max agglutinin (soybean, SBA) labeled the outflow tract evenly and retained fluorescence for months. RSCM produced terabyte-sized files allowing for in silico dissection of outflow tract vessel at a high resolution and in 3D. Networks of interconnected lumina were traced from the TM to downstream drainage structures. The collector channel (CC) volumes were ten times smaller than the receiving SVP vessels, the largest of which were in the inferior limbus. Proximal CC diameters were up to four times the size of distal diameters and more elliptical at their proximal ends. The largest CCs were found in the superonasal and inferonasal quadrants where the highest outflow occurs. Conclusions: RSCM of cleared eyes enabled high-resolution, volumetric analysis of the outflow tract. The proximal structures had greater diameters nasally while the SVP was larger in the inferior limbus.