Dependence of corneal stem/progenitor cells on ocular surface innervation

Abstract

Purpose: Neurotrophic keratopathy (NK) is a corneal degeneration associated with corneal nerve dysfunction. It can cause corneal epithelial defects, stromal thinning, and perforation. However, it is not clear if and to which extent epithelial stem cells are affected in NK. The purpose of this study was to identify the relationship between corneolimbal epithelial progenitor/stem cells and sensory nerves using a denervated mouse model of NK.

Methods: NK was induced in mice by electrocoagulation of the ophthalmic branch of the trigeminal nerve. The absence of corneal nerves was confirmed with β-III tubulin immunostaining and blink reflex test after 7 days. ATP-binding cassette subfamily G member 2 (ABCG2), p63, and hairy enhancer of split 1 (Hes1) were chosen as corneolimbal stem/progenitor cell markers and assessed in denervated mice versus controls by immunofluorescent microscopy and real-time PCR. In addition, corneolimbal stem/progenitor cells were detected as side population cells using flow cytometry, and colony-forming efficiency assay was performed to assess their function.

Results: ABCG2, p63, and Hes1 immunostaining were significantly decreased in denervated eyes after 7 days. Similarly, the expression levels of ABCG2, p63, K15, Hes1, and N-cadherin transcripts were also significantly decreased in denervated eyes. Stem/progenitor cells measured as side population from NK mice were decreased by approximately 75% compared with normals. In addition, the authors found a significant (P = 0.038) reduction in colony-forming efficiency of stem/progenitor cells harvested from denervated eyes.

Conclusions: Corneolimbal stem/progenitor cells are significantly reduced after depletion of sensory nerves. The data suggest a critical role of innervation in maintaining stem cells and/or the stem cell niche.

Figure 1.

NK induced in a mouse model by means of TSE. Representative biomicroscopy of a normal cornea (A), and of a denervated cornea (B) after 7 days. (B) Beta III tubulin immunostaining of normal and denervated corneas. (C) Normal cornea, note regular subbasal and epithelial nerve plexus. (D) Denervated cornea, note massive disruption of the subbasal nerve plexus.

Figure 2.

Decreased expression of stem cell markers after denervation. Representative immunostained micrographs of cross section of normal (A, C, E) and denervated (B, D, F) corneas showing expression of stem cell markers. ABCG2 (B), p63 (D), and Hes1 (F) expression were decreased in denervated eyes after 7days compared with those in normal eyes (A, C, E).

Figure 3.

Diminished mRNA expression levels of stem cell markers after denervation. (A) ABCG2, Hes1, Keratin15, and N-cadherin were significantly decreased in denervated corneas. An approximately twofold reduction of p63 was also observed, although this was not statistically significant. (B) Denervated corneas showed decreased expression of p75 and TrkA, and increased expression of NGF compared with normal corneas. The dotted line represents expression of mRNA in normal corneas. Bars represent mean ± SEM. *P < 0.01.

Figure 4.

Reduced SP cells in denervated eyes. (A) Representative flow cytometric dot plots showing analyses of the frequencies of corneal stem cells by means of SP cells. Note the disappearance of the low Hoechst 33342 blue-red fluorescence population, which represents stem cells, after verapamil addition. (B) Mean SP cells in normal and denervated corneas from three different experiments. SP cell frequencies were 1.99 ± 0.03% and 0.42 ± 0.08% of total epithelial cells in normal and denervated corneas, respectively. Bars represent mean ± SEM. *P < 0.001.

Figure 5.

Decreased stem cell function measured by CFE assay after denervation. Representative micrographs of CFE colonies (A) as seen under phase contrast microcopy, and after rhodamine staining at magnification ×200 (dotted circles), and (B) in culture wells from normal and denervated corneal epithelial cells. (C) Quantification of CFE. CFE of normal corneal epithelial cells were 5.73 ± 1.10%, whereas CFE of denervated corneal epithelial cells were 3.0 ± 0.4%. Bars represent mean ± SEM. *P < 0.05.