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Lymphatic Markers in the Adult Human Choroid
Falk Schrödl1, Alexandra Kaser-Eichberger2, Andrea Trost2
1University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, Paracelsus Medical University, SALK, Salzburg, Austria 2Department of Anatomy, Paracelsus Medical University, Salzburg, Austria.
Insights
This study found no evidence of classical lymphatic vessels in the adult human choroid using multiple lymphatic markers. The posterior human uvea appears to be alymphatic, challenging previous contradictory reports.
Area of Science:
- Ocular immunology
- Vascular biology
- Anatomy
Background:
- Lymphatic vessels in the posterior human uvea have been inconsistently reported.
- Accurate characterization of ocular lymphatics is crucial for understanding immune surveillance and disease pathogenesis.
Purpose of the Study:
- To systematically investigate the presence of lymphatic vessels in the human choroid.
- To clarify contradictory reports regarding lymphatics in the posterior human uvea using established immunohistochemical guidelines.
Main Methods:
- Human choroids were analyzed using flat mount serial cryosectioning and immunohistochemistry for multiple lymphatic markers (LYVE-1, PDPN, PROX1, FOXC2, VEGFR3, CCL21).
- Confocal microscopy was used to examine marker colocalization, alongside α-smooth muscle actin and DAPI staining.
- Messenger RNA analysis of lymphatic markers was performed on choroid and skin tissues.
Main Results:
- While single cells in the choroid expressed some lymphatic markers (CCL21, LYVE-1, PDPN), no classical lymphatic vessels were detected.
- PROX1-positive nuclei were absent in the choroid, and LYVE-1, PDPN, or CCL21-positive vessel-like structures (excluding blood vessels) were not observed.
- VEGFR3 showed varied immunoreactivity in choroidal blood vessels, including choriocapillaris, but not in other structures.
Conclusions:
- Classical lymphatic vessels are not present in the healthy adult human choroid based on the markers and methods used.
- The human choroid should be considered alymphatic in its healthy adult state.
- Further research may be needed to fully elucidate lymphatic drainage in the eye.
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.
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