Identification and Mapping of a Posterior Ocular Lymphatic Outflow (POLO) Pathway Through Choroidal Lymphatics

Purpose: To identify, map, and characterize a posterior ocular lymphatic outflow (POLO) pathway from the suprachoroidal space and assess its drainage dynamics and cervical nodal uptake using time-resolved imaging. Methods: Albumin-based tracers (galbumin and CF770/BSA) were delivered into the suprachoroidal space of adult mice under general anesthesia. In vivo imaging included magnetic resonance imaging (MRI) and near-infrared fluorescence imaging using confocal scanning laser ophthalmoscopy (cSLO) of the ocular surface and orbital soft tissues. Ex vivo assessments included in situ head and neck fluorescence imaging 20 minutes after tracer injection and hyperspectral microscopy of ocular and orbital sections. Lymphatic identity was assessed by immunofluorescence staining (podoplanin and vascular endothelial growth factor receptor-3 [VEGFR-3]) in Prox1-tdTomato reporter mice. Results: Following suprachoroidal tracer injection, in vivo MRI and cSLO fluorescence imaging showed preferential drainage toward the nasal orbit. Hyperspectral microscopy showed higher tracer signal in the ipsilateral sclera and orbit than contralateral tissues, and in situ fluorescence imaging detected near-infrared tracer in the ipsilateral cervical lymph node at 20 minutes. Confocal microscopy of the choroid showed co-localization of podoplanin with VEGFR-3 and Prox1 and demonstrated tracer within podoplanin-positive choroidal lymphatic vessels, consistent with a posterior drainage route from the suprachoroidal space to cervical lymph nodes. Conclusions: This study identifies a previously unrecognized POLO pathway and expands ocular drainage physiology beyond the anterior segment to include a posterior lymphatic route through choroidal lymphatic vessels. Translational Relevance: Identification of a POLO pathway reveals a measurable route for fluid clearance, intraocular pressure regulation, and drug delivery, offering new strategies to preserve vision in retinal and posterior segment diseases characterized by fluid accumulation, impaired clearance, or inflammation.