In Vivo Architecture and Functional Dynamics of Conjunctival Lymphatic Vessels: A Three-Plexus Model Revealed by Modified Trypan Blue Lymphangiography

The conjunctival lymphatic network is critical for ocular surface immunity, aqueous humor drainage after glauco-ma surgery, and subconjunctival drug delivery, yet its in vivo architecture in humans remains incompletely char-acterized. This study aimed to visualize and describe the in vivo architecture and functional dynamics of conjunc-tival lymphatic vessels using a modified trypan blue injection technique. Ninety eyes of 78 patients undergoing rou-tine cataract surgery were enrolled. A modified intratissue injection technique using a 34 gauge needle, a silicone tube adapter, and 0.08 % trypan blue was applied at standardized locations (limbus, 4 mm, 7 mm, and fornix), with real time video recording under the operating microscope in all cases. A total of 249 investigations were performed, with successful lymphatic visualization in 143 (57.4 %). Three distinct lymphatic plexuses were identified: superfi-cial, intermediate, and deep. The superficial plexus showed a valve like “pearl beads” morphology with blind ending segments; the intermediate plexus consisted of larger collectors draining toward the fornix; and the deep plexus—previously undescribed—comprised flat, interconnected vessels without visible valves, filled exclu-sively via inter plexus lymphatic bridges. Limbal lymphatic capillaries originated only from the intermediate and deep plexuses. Drainage followed a consistent nasal to temporal and posterior to fornix pattern, and blood ele-ments were observed replacing trypan blue within lymphatic vessels during and after surgical manipulation. This study provides the first in vivo description of a three plexus conjunctival lymphatic architecture in humans and identifies inter plexus bridges as a novel anatomical feature. The modified trypan blue technique proved effective for mapping both structure and dynamics, even when subconjunctival dye depots formed, confirming its utility for in vivo lymphatic visualization.