Differential distribution of blood and lymphatic vessels in the murine cornea

Abstract

Purpose: Because of its unique characteristics, the cornea has been widely used for blood and lymphatic vessel research. However, whether limbal or corneal vessels are evenly distributed under normal or inflamed conditions has never been studied. The purpose of this study was to investigate this question and to examine whether and how the distribution patterns change during corneal inflammatory lymphangiogenesis (LG) and hemangiogenesis (HG).

Methods: Corneal inflammatory LG and HG were induced in two most commonly used mouse strains, BALB/c and C57BL/6 (6-8 weeks of age), by a standardized two-suture placement model. Oriented flat-mount corneas together with the limbal tissues were used for immunofluorescence microscope studies. Blood and lymphatic vessels under normal and inflamed conditions were analyzed and quantified to compare their distributions.

Results: The data demonstrate, for the first time, greater distribution of both blood and lymphatic vessels in the nasal side in normal murine limbal areas. This nasal-dominant pattern was maintained during corneal inflammatory LG, whereas it was lost for HG.

Conclusions: Blood and lymphatic vessels are not evenly distributed in normal limbal areas. Furthermore, corneal LG and HG respond differently to inflammatory stimuli. These new findings will shed some light on corneal physiology and pathogenesis and on the development of experimental models and therapeutic strategies for corneal diseases.

Figure 1.

Schematic picture illustrating the suture placement method used to compare the nasal and temporal distributions of vessels. Two sutures were placed at the 3 o'clock and 9 o'clock positions of the cornea, respectively. Outer circle: limbus. Inner circle: demarcation of the trephine. Dashed line: demarcation between the nasal and the temporal sides.

Figure 2.

Comparison of blood and lymphatic vascular distribution in the nasal versus temporal side of the normal BALB/c limbi. (A) Representative immunofluorescence micrographs demonstrating that both blood and lymphatic vessels were more prominent in the nasal side. Green: CD31. Red: LYVE-1. Yellow: merged. BV, blood vessels; LV, lymphatic vessels. Original magnification, ×100. (B) Summary of results. Significant differences were observed for both blood and lymphatic vessels. *P < 0.05. Error bars represent SEM (n = 10).

Figure 3.

Comparison of HG and LG in the nasal versus temporal sides of the inflamed BALB/c corneas. (A) Representative immunofluorescence micrographs demonstrating more lymphatic vessels in the nasal side. Green: CD31. Red: LYVE-1. Yellow: merged. HG, hemangiogenesis; LG, lymphangiogenesis. Original magnification, ×100. (B) Summary of the results. Although no significant difference was observed for corneal inflammatory HG (P = 0.83), LG was more prominent in the nasal side. *P < 0.05. Error bars represent SEM (n = 10).

Figure 4.

Comparison of blood and lymphatic vascular distribution in the nasal versus temporal sides of the normal C57BL/6 limbi. (A) Representative immunofluorescence micrographs demonstrating that both blood and lymphatic vessels were more prominent in the nasal side. Green: CD31. Red: LYVE-1. Yellow: merged. BV, blood vessels; LV, lymphatic vessels. Original magnification, ×100. (B) Summary of the results. Significant differences were observed for both blood and lymphatic vessels. *P < 0.05. Error bars represent SEM (n = 10).

Figure 5.

Comparison of HG and LG in the nasal versus temporal sides of the inflamed C57BL/6 corneas. (A) Representative immunofluorescence micrographs demonstrating more lymphatic vessels in the nasal side. Green: CD31. Red: LYVE-1. Yellow: merged. HG, hemangiogenesis; LG, lymphangiogenesis. Original magnification, ×100. (B) Summary of the results. Although no significant difference was observed for corneal inflammatory HG, corneal LG was more prominent in the nasal side. *P < 0.05. Error bars represent SEM (n = 10).