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, 59 (6), 2371-2380

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


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

Susannah Waxman et al. Invest Ophthalmol Vis Sci.


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 advanced analysis software (Imaris) were used to reconstruct a three-dimensional (3D), 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 vessels at a high resolution and in 3D. Networks of interconnected lumens were traced from the TM to downstream drainage structures. The collector channel (CC) volumes were 10 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.

Conclusion: RSCM of cleared eyes enabled high-resolution, volumetric analysis of the outflow tract. The proximal structures had greater diameters nasally, whereas the SVP was larger in the inferior limbus.


Figure 1
Figure 1
Macroscopic view of a BABB-cleared eye. Anterior segments of perfusion fixed eyes were cleared with BABB. Left: anterior segment before and after clearing protocol. Right: an eye flat-mounted for demonstration purposes shows a high transparency except for parts that are heavily pigmented.
Figure 2
Figure 2
Volumetric limbal reconstruction. SBA-labeled samples were visualized as maximum intensity projections (MIPs) of the superior (A) and inferior (B) limbus as seen from the anterior. In this right eye, nasal limbus is shown at the right and temporal at the left (A, bottom right). MIPs A2 and B2 show fine elements of the aqueous humor outflow tract distal to the TM as seen from the anterior chamber angle, viewed through the cornea and facing the z-plane. A3 and B3 show surface reconstruction and labeling of morphology with TM in yellow, CC in red, and SVP in blue. A4 and B4 show merged with corresponding surfaces set to 50% transparency. Vertical, solid white lines demarcate boundaries within which representative sagittal subregions (A, B 2.1, 3.1, 4.1) are segmented and positioned with the Z plane facing inward. ES, episclera.
Figure 3
Figure 3
Collector morphology and connectivity from the TM to the SVP. Top: a MIP of the inferior scan (green) with 3D reconstructions of representative collector channel luminal (red). A1C1 show the detail of delineated areas of the top panel at the depth of each channel's connection to the TM. A2C2 show a 2.43-μm-thick slice of this site of connection (solid arrows). A3C3 show the same for site of the lumen's connection to the SVP (solid arrows) as vasculature begins to run parallel to the surface of the episclera. Hollow arrow marks a hinge-like flap by collector channel opening. A4C4 show sagittal sections of regions of interest in the top panel, with an uninterrupted connections between TM and SVP. The depth of regions delineated in A1C1 in white and orange are shown.
Figure 4
Figure 4
Outflow vessels and location. (A) The average CC and SVP volumes in the nasal and temporal regions consisting of the two nasal and temporal quadrants, respectively. Average volumes of SVP were approximately 10 times larger than those of CC. (B) Frontal view of a right eye. Volume measurements from 20 full-thickness surface segments are plotted corresponding to their anatomical locations. Larger CC and SVP volumes were found mainly in the inferior limbus.
Figure 5
Figure 5
Cross-sectional area of CC sections. (A) CC openings at the level of the trabecular meshwork (proximal CC) had a 1.7-fold larger section area nasally than temporally (P = 0.035). Both nasally and temporally, the proximal portion of CCs was larger than their distal portion. (B) Frontal view of a right eye. The greatest section areas were found in the superonasal and inferonasal quadrant. (C) Proximal nasal and temporal CCs were more oval compared with their distal ends as expressed by the ratio of the maximal to the minimal cross-sectional width. (D) In the frontal view of this right eye, cross-sectional areas were more elliptical in the superonasal and inferonasal quadrant.

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