Nerves and neovessels inhibit each other in the cornea

Abstract

Purpose: To evaluate the regulatory cross-talk of the vascular and neural networks in the cornea.

Methods: b-FGF micropellets (80 ng) were implanted in the temporal side of the cornea of healthy C57Bl/6 mice. On day 7, blood vessels (hemangiogenesis) and nerves were observed by immunofluorescence staining of corneal flat mounts. The next group of mice underwent either trigeminal stereotactic electrolysis (TSE), or sham operation, to ablate the ophthalmic branch of the trigeminal nerve. Blood vessel growth was detected by immunohistochemistry for PECAM-1 (CD31) following surgery. In another set of mice following TSE or sham operation, corneas were harvested for ELISA (VEGFR3 and pigment epithelium-derived factor [PEDF]) and for quantitative RT-PCR (VEGFR3, PEDF, and CD45). PEDF, VEGFR3, beta-3 tubulin, CD45, CD11b, and F4/80 expression in the cornea were evaluated using immunostaining.

Results: No nerves were detected in the areas subject to corneal neovascularization, whereas they persisted in the areas that were neovessel-free. Conversely, 7 days after denervation, significant angiogenesis was detected in the cornea, and this was associated with a significant decrease in VEGFR3 (57.5% reduction, P = 0.001) and PEDF protein expression (64% reduction, P < 0.001). Immunostaining also showed reduced expression of VEGFR3 in the corneal epithelial layer. Finally, an inflammatory cell infiltrate, including macrophages, was observed.

Conclusion: Our data suggest that sensory nerves and neovessels inhibit each other in the cornea. When vessel growth is stimulated, nerves disappear and, conversely, denervation induces angiogenesis. This phenomenon, here described in the eye, may have far-reaching implications in understanding angiogenesis.

Figure 1.

Growth of neovessels is associated with corneal nerve degeneration observed in both the epithelium and stroma. “P” = intracorneal pellet. Note that vessels are growing toward the pellet, whereas no vessels are visible opposite to the pellet. Beta-3 tubulin (red staining) reveals corneal nerves in the epithelium and stroma on the opposite side of the pellet (arrows). In contrast, adjacent to the pellet no nerves are detected, whereas PECAM staining (green) confirms the presence of neovessels (indicated by arrowheads).

Figure 2.

Corneal nerves and vessels inhibit each other in the cornea. (A) Corneal nerves are undetectable 48 hours following trigeminal stereotactic electrolysis (beta-3 tubulin staining), right: denervated eye; left: normal eye. (B) Neovascularization occurs 7 days after denervation in the cornea (right), in contrast to the normal cornea (left). (C) Immunostaining for PECAM showing growth of neovessels following denervation (right).

Figure 3.

Reduction of normally present angiostatic molecules following denervation of the cornea (A, mRNA expression; B, protein expression). PEDF and VEGFR3 are both significantly reduced 7 days after TSE. Bars indicate standard error.

Figure 4.

Differential expression of PEDF, beta-3 tubulin, and CD45 in the corneal epithelium (A), stroma (B), and corneal nerves (C). (A) The normal corneal epithelium diffusely expresses PEDF (red). (B) Epithelial bone-marrow derived CD45⁺ cells (in green, arrows) do not double stain for PEDF (red). (C) Epithelial nerves, stained by anti-beta-3 tubulin (green), are not stained by anti-PEDF antibody. Stromal nerves co-stain for anti-PEDF antibody and anti–beta-3 tubulin in some areas (a, inset I), but not others (b).

Figure 5.

Confocal micrographs in X-Y plane showing that VEGFR3 expression is present in the normal corneal epithelium (A), and reduced following denervation (B). Z-plane stacking of all epithelial images in X-Y plane shows reduced expression in the denervated corneal epithelium (C).

Figure 6.

Characterization of inflammatory cell infiltrate following corneal denervation. Both CD11b (A, green) and F4/80 (B, green) expression is significantly increased in denervated corneas.