Novel discovery of a lymphatic bridge connecting Schlemm's canal to limbal and conjunctival lymphatic pathway

Abstract

Purpose: Schlemm's canal (SC) is a critical structure regulating aqueous humor (AH) drainage and intraocular pressure (IOP). It is known that in the conventional outflow pathway, AH flows from SC to episcleral veins. We recently reported a high-resolution three-dimensional (3D) imaging technology for intact eyeballs, SC and ocular surface. Using this advanced technology, we herein report the discovery of a new structure, termed lymphatic bridge, that directly connects SC to the limbal and conjunctival lymphatic pathway. Further investigation on this novel outflow pathway may provide new mechanisms and therapeutic approaches for glaucoma.

Methods: As reported previously, intact eyeballs were harvested from Prox-1-GFP (green fluorescent protein) mice and processed by a tissue clearing technique with CLARITY. Samples were immunolabeled with specific antibodies for CD31 (pan-endothelial marker) and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) and imaged by light-sheet fluorescent microscopy. The limbal areas were examined to locate connecting channels between SC and limbal and conjunctival lymphatic vessels. Moreover, in vivo anterior chamber dye injection was performed with Texas Red dextran for functional analysis on AH outflow.

Results: A novel lymphatic bridge structure that expressed both Prox-1 and LYVE-1 was discovered between the SC and limbal lymphatic vessels connected with conjunctival lymphatic pathway. Results from the anterior chamber dye injection also confirmed AH drainage into the conjunctival lymphatic outflow pathway.

Conclusions: This study provides the first evidence on the direct connection between SC and conjunctival lymphatic pathway. This new pathway is distinctive from the traditional episcleral vein pathway and merits further investigation.

Keywords: Aqueous humor drainage pathway; Conjunctiva; Limbus; Lymphatic vessel; Schlemm's canal.

Figure 1.. Representative images showing the lymphatic bridge between the Schlemm’s canal and limbal lymphatic vessels.

A. The anterior segment of an adult Prox-1-GFP mouse was imaged along the Z-axis demonstrated in the upper left panel. Lower left panel, Prox-1⁺ vascular structures (green) were detected at the Schlemm’s canal and the ocular surface. White asterisk: nictating membrane. Right panel, the enlarged boxed area showing the location of the lymphatic bridge (LB, red arrow and outlines) between the Schlemm’s canal (SC, yellow outlines) and limbal lymphatic vessels (LL, blue outlines), which were continuously connected with conjunctival lymphatics (CL, purple outlines). B. Side (left) and top (right) view from 3D imaging showing the lymphatic bridge (LB, red arrows and outlines) between the Schlemm’s canal (SC) and limbal lymphatic vessels (LL). Scale bars: 100 μm (A), 50 μm (B).

Figure 2.. Representative images showing two distinctive outflow pathways from the Schlemm’s canal.

Images were taken at the limbal area of an adult Prox-1-GFP mouse eyeball labeled with CD31. A. Top view of 3D imaging of the limbal area. The stacked image shows various limbal vascular structures detected including Schlemm’s canal (SC), limbal lymphatics (LL), and conjunctival lymphatics (CL). The white and yellow boxed area indicate where the lymphatic bridge and collector channel were detected, respectively. B. Extended sectional views from the boxed areas in panel A. Upper panels: the lymphatic bridge pathway where the lymphatic bridge (LB, white arrow) connected the Schlemm’s canal (SC) with the Prox-1^positive CD31^positive limbal lymphatics (LL). Lower panels: the collector channel (CC, yellow arrow) pathway where the Schlemm’s canal (SC) was connected to the Prox-1^negative CD31^positive blood vessel (BV) belonging to the episcleral vein pathway. Prox-1: green; CD31, red. Scale bars: 200 μm (A), 20 μm (B).

Figure 3.. Representative images showing the lymphatic bridge was present during development.

Images were taken at the limbal area of a P14 Prox-1-GFP mouse eyeball labeled with CD31 and presented in the extended sectional view. The lymphatic bridge (red arrows and red outlines) was detected between the Schlemm’s canal (SC, yellow outlines) and the limbal lymphatics (LL, blue outlines). Prox-1: green; CD31, red. Scale bars: 30 μm.

Figure 4.. Representative images showing the lymphatic bridge expressed both Prox-1 and LYVE-1.

Images were taken from an adult Prox-1-GFP mouse eyeball labeled by LYVE-1. The Prox-1^positive LYVE-1^positive lymphatic bridge (LB, red arrows and red outlines) was detected between the Prox-1^positive LYVE-1^negative Schlemm’s canal (SC, yellow outlines) and Prox-1^positive LYVE-1^positive limbal lymphatics (LL, blue outlines). Similar to the limbal lymphatics, the lymphatic bridge expressed both Prox-1 and LYVE-1. Prox-1: green; LYVE-1, red. Scale bars: 20 μm.

Figure 5.. Representative images showing the drainage of fluorescent dye into the conjunctival lymphatic vessels after anterior chamber injection.

Intravital images showing that the Texas Red fluorescent dye (red) injected into the anterior chamber (AC) was drained into Prox-1⁺ conjunctival lymphatic vessels (green) post-operation (PO, upper panels) and signals at 10 minutes after the injection (middle panels), as indicated by the white arrows. Bottom panels: enlarged images from the boxed areas. Scale bars: 200 μm (PO, 10 minutes), 100 μm (boxed areas).

Figure 6.. Schematic diagram showing the difference between the conjunctival lymphatic outflow pathway and the episcleral vein outflow pathway.

Left, the episcleral vein outflow pathway where aqueous humor flows from Schlemm’s canal to collector channels and intrascleral plexus/aqueous vein and drains into the episcleral veins, which are blood vessels. Right, the conjunctival lymphatic outflow pathway where aqueous humor drains from Schlemm’s canal to lymphatic bridge and limbal and conjunctival lymphatic vessels. In the episcleral vein pathway, only the initial segment around the Schlemm’s canal expresses Prox-1, while the entire conjunctival outflow pathway is Prox-1 positive. Structures of the conjunctival outflow pathway are typical lymphatic vessels that express both Prox-1 and LYVE-1.