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

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. 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. 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. 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.