Spontaneous lymphatic vessel formation and regression in the murine cornea

Abstract

Purpose: Lymphatic dysfunctions are associated with many diseases, ranging from cancer metastasis to transplant rejection, for which there is little effective treatment. To date, there is no natural model with which to study lymphatic regression. This study was conducted to investigate whether murine cornea, an extensively exploited tissue for vascular studies, derives its lymphatic-free status from a natural regression mechanism. The differential behaviors between the lymphatic and blood vessels under normal development and inflammation conditions are also compared.

Methods: Normal mouse eyeballs or whole-mount corneas encompassing the entire course of corneal development and maturation and adult inflamed corneas were used for immunofluorescent microscopic studies.

Results: The data demonstrated, for the first time, that mouse cornea was endowed with a significant number of lymphatic vessels that underwent spontaneous formation and regression during a critical period after birth, which was not observed for blood vessels. Because lymphatic growth can be reactivated in the adult cornea after inflammatory stimulation, the cornea thereby becomes the first tissue ever identified to have a full range of lymphatic plasticity.

Conclusions: These novel findings not only provide a new concept in defining the cornea and its related diseases, they also reveal a completely natural model with which to study both lymphatic regression and formation. It is hoped that further studies will divulge novel and potent pro- or anti-lymphatic factors to treat lymphatic disorders inside and outside the eye.

Figure 1.

LYVE-1 expression in embryonic corneas (A–F). Representative cross-sectional micrographs demonstrate LYVE-1⁺ signals detected in the mouse corneas at E12.5 (B) and thereafter (C–E), as indicated by the arrowheads. (F) Higher magnification view of the boxed area in (D) showing LYVE-1 expression in the central cornea. L, lens; Cor, cornea; EL, eyelid; E, embryonic. Red: LYVE-1; blue: DAPI (nuclei staining). Scale bars, 100 μm.

Figure 2.

Spontaneous lymphatic vessel formation followed by regression in postnatal corneas (A–F). (A–E) Representative whole-mount micrographs demonstrating the gradual appearance and disappearance of lymphatic vessels in the corneas from E18.5 to the adult age. Red: LYVE-1. Scale bars, 200 μm. White circles: demarcation between the cornea and the conjunctiva. (F) Summarized data from repetitive experiments illustrating the trend of lymphatic changes over the time period. Results are expressed as the mean ± SEM. Statistical analysis was performed by one-way ANOVA (P < 0.0001). E, embryonic; P, postnatal; LV, lymphatic vessels.

Figure 3.

Comparison between early- and late-stage lymphatic vessels. (A–C) Representative whole-mount micrographs showing that the lymphatic vessels of the early stages had larger diameters, were longer, and had more branches as indicated by the arrows. (D–F) Summarized data from repetitive experiments. Results are expressed as the mean ± SEM. Statistical analysis was performed by one-way ANOVA. (D) P < 0.05. (E, F) P < 0.0001. Red: LYVE-1. Scale bars, 100 μm. P, postnatal.

Figure 4.

(A–C) Lymphatic origin of LYVE-1⁺ vessels in developing cornea. Representative whole-mount micrographs showing LYVE-1⁺ vessels in P6 corneas also expressed the Prox-1, another lymphatic specific marker. Green: LYVE-1; red: Prox-1. Scale bars, 50 μm.

Figure 5.

Differential behavior of lymphatic and blood vessels in the cornea. (A–C) Representative whole-mount micrographs demonstrating that though lymphatic vessels underwent dramatic changes from natural formation to regression, the blood vessels constantly remained at the peripheral or limbal areas. (D) However, at the adult age after inflammatory stimulation, both vessel types were reactivated and introduced into the cornea in parallel. Green: CD31; red: LYVE-1. Scale bars, 200 μm. Arrows: limbal peripheral vessels. P, postnatal.

Figure 6.

Schematic illustration summarizing the full range of lymphatic plasticity in the cornea under both physiologic (top half) and pathologic (bottom half) situations. Yellow: common pathways for lymphatic formation (phases A and C). Green: common pathways for lymphatic regression (phases B and D).