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

The conjunctival lymphatic network is critical for ocular surface immunity, aqueous humor drainage after glaucoma surgery, and subconjunctival drug delivery, yet its in vivo architecture in humans remains incompletely characterized. This study aimed to visualize and describe the in vivo architecture and functional dynamics of conjunctival lymphatic vessels using a modified trypan blue injection technique. Ninety eyes of 78 patients (66 unilateral, 12 bilateral contributions) undergoing routine 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: superficial, 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 exclusively 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 elements 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.