Absence of lymphatic vessels in the developing human sclera

- The fetal human sclera contains CD31 + blood vessels as early as week 13.
- The fetal sclera is alymphatic, with singular LYVE1 + cells surrounding blood vessels.
- Lymphangiogenesis in the conjunctiva were detected as early as week 17.
- Lymphangiogenesis in the conjunctiva is associated with a decrease of LYVE1 + cells.
- LYVE1 + cells seem to be involved in conjunctival lymphangiogenesis.

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.