Purpose: Reports of lymphatics in the posterior human uvea are contradictory. We systematically analyzed the choroid by combining various lymphatic markers, following recently established guidelines for the immunohistochemical detection of ocular lymphatics.

Methods: Human choroids were prepared for flat mount serial cryosectioning. Sections were processed for immunohistochemistry of the lymphatic markers LYVE-1, PDPN, PROX1, FOXC2, VEGFR3, CCL21, and combined with α-smooth muscle-actin and 4′, 6-diamidino-2-phenylendole (DAPI). Single, double, and triple marker combinations were documented using confocal microscopy. Messenger RNA analysis for CCL21, FOXC2, LYVE-1, PDPN, PROX, and VEGFR3 was performed in choroid and skin.

Results: In the choroid, CCL21 immunoreactivity was detected in choroidal blood vessels, intrinsic choroidal neurons, and numerous small cells of the choroidal stroma. These small cells were not colocalized with PROX1 and PDPN, while a subpopulation of cells showed immunoreactivity for CCL21 and LYVE-1, and very occasionally PDPN-only+ cells were detected. Nuclei positive for PROX1 were never detected in the choroid, and vessel-like structures immunoreactive for LYVE-1, PDPN, or CCL21 (other than blood vessels) were never observed. Immunoreactivity of VEGFR3 was absent in the majority of choroidal blood vessels, but present in choriocapillaris, while other structures positive for VEGFR3 were not detected. Nonvascular smooth muscle cells were lacking VEGFR3-immunoreactivity. Messenger RNA analysis detected all lymphatic markers investigated and confirmed immunohistochemical results.

Conclusions: By combining several lymphatic markers, single cells expressed these markers, but classical lymphatic vessels were not detected in the human choroid. Therefore, the healthy adult human choroid must be considered alymphatic, at least with the markers applied here.