Topography of Lymphatic Markers in Human Iris and Ciliary Body

Reports of lymphatics in the anterior human uvea are contradictory. This might be caused due to a certain topography, which has not been considered yet. Therefore, here we systematically analyze iris and adjacent ciliary body with immunohistochemistry by combining various lymphatic markers.

Human iris and ciliary body were obtained from cornea donors and prepared for cryosectioning. Cross sections of tissue blocks at 12/3/6/9 o'clock position and at corresponding intersections (1:30/4:30/7:30/10:30) were processed for immunohistochemistry of LYVE-1, PDPN, PROX1, FOXC2, VEGFR3, and CCL21, and when necessary, these lymphatic markers were combined with CD31, α-smooth muscle-actin, CD68, and 4',6-diamidino-2 phenylindole dihydrochloride (DAPI). Double, triple, and quadruple marker combinations were documented using confocal microscopy.

Numerous podoplanin+ cells were mainly located at the anterior border of the iris while LYVE-1+ cells were distributed throughout the nonpigmented part. Both cell populations were PROX1/FOXC2/CCL21/VEGFR3-. Blood vessels, iris smooth muscles, and individual cells were VEGFR3+. While PDPN+ cells were rarely detected posteriorly of the iris root, many LYVE-1+ cells were present within the ciliary body muscle and villi. Within the muscle, occasionally PDPN+ vessel-like structures were detectable, but these were never colocalized with LYVE-1. Similar vessel-like structures were VEGFR3+/PROX1-/CCL21-, but CD31+. Further, ciliary muscle fibers and ciliary epithelium were immunoreactive for VEGFR3/CCL21, but were LYVE-1/PDPN-. A certain topography of structures at the various uvea-positions investigated was not obvious. The majority of LYVE-1+ cells displayed immunoreactivity for CD68.

Lymphatic vessels colocalizing for at least two lymphatic markers were not detectable. Therefore, if present, putative lymphatic channels of the anterior uvea might display a different marker panel than generally presumed.