Absence of lymphatic vessels in the developing human sclera.

The adult sclera is free of lymphatic vessels, but contains a net of blood vessels. Whether and when this selectively lymphangiogenic privilege is achieved during embryologic development is not known yet. Therefore, we investigated the developing human sclera for blood- and lymphatic vessels in 34 abortions/stillborns (12-38 weeks of gestation). The probes were subdivided into three groups (group 1: 12-18 weeks of gestation, n = 10; group 2: 19-23 weeks of gestation, n = 13; group 3: 24-38 weeks of gestation, n = 11), and prepared for paraffin sections followed by immunohistochemistry against CD31 to detect blood vessels, and against lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1)/podoplanin to detect lymphatic vessels. We could show, that in the human episclera distinct CD31 + blood vessels are present as early as week of gestation 13. Their amount increased during pregnancy, whereas stromal CD31 + blood vessels were elevated in early pregnancy and regressed with ongoing pregnancy. In the lamina fusca CD31 + blood vessels were absent at any time point investigated. Single LYVE1 + cells were identified primarily in the episclera; their amount decreased significantly with increasing gestational ages (group 1 compared to group 3: p < 0.01). However, LYVE1+/podoplanin + lymphatic vessels were not detectable in the sclera at any gestational ages analyzed. In contrast to the conjunctiva where LYVE1+/podoplanin + lymphatic vessels were detectable as early as week 17, the amount of LYVE1 + cells in the sclera was highest in early pregnancy (group 1), with a significant decrease during continuing pregnancy (p < 0.001). These findings are the first evidence for a fetal lymphangiogenic privilege of the sclera and show, that the fetal human sclera contains CD31 + blood vessels, but is primarily alymphatic. Our findings suggest a strong expression of selectively antilymphangiogenic factors, making the developing sclera a potential model to discern antilymphangiogenic mechanisms.

Enrichment of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-positive macrophages around blood vessels in the normal human sclera.

PURPOSE: To investigate whether the normal adult human sclera contains lymphatic vessels and to study their relation to immune cells and blood vessel anatomy. METHODS: Scleral tissue probes from 35 adult human donor bulbi were analyzed by immunohistochemistry and confocal microscopy for blood vessels (CD31+), lymphatic vessels (lymphatic vessel endothelial hyaluronan receptor 1 [LYVE1]+, podoplanin+), and macrophages (CD68+) at 12 locations (anterior, equatorial, and posterior at 3, 6, 9, and 12 o'clock positions of the eye) in all three scleral layers (episclera, stroma, and lamina fusca). Approval for scientific examination was obtained. RESULTS: CD31+ blood vessels were detectable in the human sclera, where the percentage area covered by CD31+ blood vessels was highest in the anterior episclera, followed by equatorial and posterior episclera, and was lowest in the scleral stroma (regardless of location). LYVE1+ podoplanin+ lymphatic vessels were not detectable in any location investigated, although there was a high number of LYVE1+ CD68+ macrophages. These macrophages were concentrated around blood vessels. In contrast, in the episclera, the number of detected LYVE1+ CD68+ macrophages was comparable in all locations; within the stroma, their number increased toward the posterior part of the eye. CONCLUSIONS: The adult sclera contains blood vessels but lacks, as revealed by immunohistochemistry and confocal microscopy, true lymphatic vessels. LYVE1+ CD68+ macrophages are located adjacent to the longitudinal axis of blood vessels. The function of these cells needs further investigation, but could be a next step toward a better understanding of pathological disorders such as inflammation, tumor, trauma, or glaucoma.