Steger B, Palme C, Romano V, Ahmad S, Seifarth C, Williams B, Zheng Y, Parekh M, Kaye SB. En-face morphometric analysis of the human limbal lymphatic vasculature. Investigative Ophthalmology & Visual Science. 2019 Jul 22;60(9):953-.
https://iovs.arvojournals.org/article.aspx?articleid=2745911
Abstract
Purpose
Confocal microscopy (CM) can be used to visualize clinically invisible corneal lymphatic neovessels, the primary mediator of corneal allograft rejection. No descriptive parameters have been validated to morphologically differentiate blood and lymphatic vessels in vivo. We aimed to identify morphometric characteristics distinguishing limbal blood and lymphatic vasculature in a laboratory investigation.
Methods
6 human corneoscleral rings preserved in cornea cold (Eurobio, France) medium at 4°C were examined using CM, 6 human corneoscleral rings preserved in organ culture medium at 31°C were en-face sectioned (200µm thickness) and examined using immunofluorescence (IF) staining for CD-31 and D2-40. The architecture of identified blood and lymphatic vascular complexes was compared using a semi-automated program (Matlab, The Mathworks, Natick, MA). Analyzed parameters included vessel depth, diameter, segment length, intersegmental angle, tortuosity index, centripetal corneal extension.
Results
Two limbal corneal vascular networks of distinct location and architecture were identified using both techniques. The marginal corneal blood vessel arcade (MCA) was located more centrally (Figure 1), and more superficially (24±9 vs 43±13 µm, p=0.004) than the peripherally adjacent lymphatic plexus (Figure 2). Vessel diameters were significantly higher for lymphatic compared to blood vessels (36±38 vs 13±8 µm, p=0.01). Significant differences were found for vessel segment length using both IF (54±16 vs 65±25 µm, p=0.04) and CM (52±13 vs 64±21 µm, p=0.01). Intravascular red blood cells were identified in all blood vessels using both techniques, lymphocytes were rarely identified in lymphatic vessels. No significant differences were found for intersegmental angle and tortuosity index.
Figure 1. Immunofluorescence micrograph showing the limbal lymphatic (red) and haematic (green) vascular arcade.
Figure 2. CM images showing the limbal lymphatic (2A, focus depth 34-40 µm) and haematic MCA (2B, focus depth 20-26 µm). The termination of Bowman’s layer is seen in proximity to the MCA’s terminal loops (2B).
Conclusions
The limbal lymphatic vascular arcade lies peripheral to and deeper than the MCA. Both vascular networks can be visualized by CM on corneoscleral tissue. CM potentially differentiates blood and lymphatic vessels in vivo based on the assessment of depth, location, vascular segment diameter and length, and intravascular cells.
