Corneal epithelial and neuronal interactions: Role in wound healing

doi:10.1016/j.exer.2014.05.006

Experimental Eye Research

Volume 125, August 2014, Pages 53-61

https://doi.org/10.1016/j.exer.2014.05.006 Get rights and content

Highlights

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We present the study of corneal epithelial and trigeminal neuron interactions.
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TG neurons show increased neurite outgrowth in epithelial co-cultures.
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TG neurons show an increase in the expression of SP mRNA in the presence of CEM.
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Evidence for EMT-like transition in primary mouse corneal epithelial cells.
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Corneal epithelial cells show a decrease in BMP7 expression in the presence of CNM.

Abstract

Impaired corneal innervation and sensitivity are the main causes of corneal neurotrophic keratopathy which simultaneously also leads to poor epithelial wound healing. Restoration of the diminished communication between the corneal epithelium and trigeminal nerve is indispensable for the proper functioning of the epithelium. The present study aims to investigate corneal epithelial and trigeminal neuron interactions to shed light on corneal wound healing during neurotrophic keratopathy. Mouse trigeminal neurons and corneal epithelial cells were cultured according to standard methods. To study the effect of corneal epithelial cells on trigeminal neurons as well as the effect of trigeminal neurons on corneal epithelial cells during wound healing, conditioned media from the cultures of pure trigeminal neurons (CNM) and corneal epithelial cells (CEM) were collected freshly and applied on the other cell type. Neurite outgrowth assay and RT-PCR analysis using primers specific for substance P (SP), Map1a, Map1b were performed on trigeminal neurons in the presence of CEM. We observed an increase in the neurite outgrowth in the presence of CEM and also in co-culture with corneal epithelial cells. Increase in the expression of SP mRNA and a decrease in the expression of Map1b mRNA was observed in the presence of CEM. We also observed the presence of epithelial-to-mesenchymal transition (EMT)-like phenomenon during wound healing using a scratch assay in primary corneal epithelial cultures. This system was further employed to study the effect of CNM on corneal epithelial cells in the context of wound healing to find the effect of trigeminal neurons on epithelial cells. RT-PCR analysis of Pax6 expression in corneal epithelial cell cultures with scratch served as a positive control. Further, we also show the expression of bone morphogenetic protein 7 (BMP7) mRNA in corneal epithelial cells which is decreased gradually along with Pax6 mRNA when cultured together in the presence of CNM. The expression and down regulation of BMP7 in the presence of CNM was further confirmed at the protein level by western blotting. From this study it seems that the epithelial and neuronal interactions in the cornea may contribute to the corneal innervation as well as recovery of corneal epithelial cells during injury. Appraising the differences in the expression of various signalling molecules during EMT of epithelial cells in the presence of SP and BMP7 gives an insight into the detailed dissection of the involved signalling pathways to develop future therapeutics.

Introduction

The cornea, the outermost layer of the eye, is a transparent, clear, convex avascular structure that is one of the most highly innervated tissues in the body. Its outer epithelial layer is the region where nerve endings are localized. The ophthalmic branch of the trigeminal ganglion (TG) sensory nerve fibres entering the corneal stroma through the limbal area is responsible for the innervation of the corneal epithelium (de Leeuw and Chan, 1989). Before penetrating Bowman's layer in the cornea, the trigeminal sensory nerve fibres lose their myelin sheath and densely innervate the apical wing cell level of the epithelium (Müller et al., 2003). These corneal nerves include peptidergic, sympathetic and parasympathetic nerve fibres and, by secreting neurotransmitters such as calcitonin gene-related peptide, neuropeptide Y, catecholamines, and acetylcholine (Shimizu, 1982, Tervo et al., 1982), they modulate the proliferation and differentiation of the epithelium and also play an important role in corneal epithelial wound healing.

Diminished TG nerve function causes epithelial alterations and adversely affects the wound healing process (Beuerman and Schimmelpfennig, 1980). Through the release of neurotrophic factors, TG sensory nerves influence signal transduction cascades involved in epithelial wound healing and homeostasis (Garcia-Hirschfeld et al., 1994, Kim et al., 2009). Altered corneal epithelial barrier function and delayed wound healing have been observed following disruption of TG (Gallar et al., 1990, Goins, 2005), leading to decreased vision, which can be treated by administration of substance P (SP) and insulin-like growth factor-1 (Nagano et al., 2003). These findings highlight the influence of sensory nerves on corneal epithelial functional restoration and suggest that the trophic effects of sensory nerves on corneal epithelium are mediated partly by the neuropeptides secreted from the nerve terminals (Gallar et al., 1990).

Impairment of trigeminal nerve function and insufficient supply of neural factors are the principal causes of corneal neurotrophic keratopathy, a condition that may consequently also lead to dry eye because of the disruption of neural input to the cornea (Nishida and Yanai, 2009). Clinical application of autologous serum eye drops as well as other neurotransmitters and peptides (Matsumoto et al., 2004) promotes corneal reinnervation and accelerates wound healing (Freire et al., 2012, Müller et al., 2003, Rao et al., 2010). Similarly, secreted neurotrophic factors from cultured corneal epithelial cells can stimulate the outgrowth of neurites from TG neurons and prolong their survival (Chan and Haschke, 1981, Chan and Haschke, 1982). Development of a tissue culture model of the innervated ocular surface for studying functional relationships of various cornea–trigeminal interactions (Forbes et al., 1987) has further helped to show the trophic effect of TG neurons on corneal epithelial cells (Baker et al., 1993, Garcia-Hirschfeld et al., 1994). These reports demonstrate that, during treatment for corneal epithelial disorders, it is necessary to maintain the cooperative interplay between the corneal epithelial and neural cell microenvironments: the resultant activation of neural cells in turn provides sufficient neural signals to support the corneal epithelial cells, and vice versa (Nishida and Yanai, 2009).

In the process of corneal wound healing, transforming growth factor-β and upregulation of Slug (Aomatsu et al., 2011, Aomatsu et al., 2012, Chandler et al., 2007) are known to induce epithelial-to-mesenchymal transition (EMT)-like cellular morphology, migration and cadherin switching in corneal epithelial cells. Since the cornea is highly innervated with nerve endings, studies explaining the role of neuronal involvement in the induction of EMT help to increase our understanding of epithelial–neuronal interactions during wound healing.

The aim of the present study was to analyse epithelial–neuronal interactions and their involvement in controlling EMT during wound healing using murine corneal epithelial and TG neuron primary cell cultures. To assess the influence of TG neurons on epithelial cells and vice versa, we collected the conditioned media from corneal epithelial and TG neuron cultures, applied them to the other cell type and studied the expression of different molecules in the presence of conditioned medium.

Section snippets

Materials

Serum-free culture media for the culture of mouse primary neurons (NeuroCult SM1 and SM2 Neuronal Culture Kit) was purchased from Stemcell Technologies (Grenoble, France). Keratinocyte-serum free medium (KSFM) for mouse corneal epithelial cell culture was purchased from Life Technologies (Darmstadt, Germany). Neurite outgrowth staining kit was obtained from Molecular Probes (Darmstadt, Germany). Antibodies used in this study were: anti-BMP7 (ab56023, Abcam, Cambridge, UK), anti-Substance P

TG neurons in culture

After seeding the dissociated TG neurons in culture dishes, gradual development of profuse axonal arborizations from the cultured TG neurons was observed until 3 days as shown in Fig. 1A–D. Neuron specific anti-β-III tubulin and anti-NeuN antibodies as well as glial cell- specific GFAP antibody were used to confirm the presence of neurons in TG culture (Fig. 1E–J).

Neuron–epithelial interactions and effect of CEM on TG neurons in culture

To study the effect of epithelial cells on neurons, we cultured neurons in freshly collected CEM and measured cell viability and

Discussion

Establishing reliable and reproducible culture conditions is an essential step towards studying the role of corneal epithelial and neuronal interactions. In order to confirm the accuracy of our TG neuron and corneal epithelial culture conditions, we first studied the effect of epithelial cells on the neurite outgrowth of TG neurons as a functional parameter in culture. It had previously been shown that the presence of corneal epithelial cells alone can induce neurite outgrowth from dissociated

Declaration of interest

The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Author's contributions

BSK designed the study, performed the cell cultures, biochemical studies and their analysis and drafted the manuscript; TS participated in the design of the study and carried out the biochemical studies and their analysis; AW, RG and OS participated in the design and coordination of the study. All authors read and approved the final manuscript.

Acknowledgements

The authors thank C. Leyh for her technical skill and expertise and M. Lietz for timely supply of experimental mice throughout the project. We also thank Prof. Dr. S. Baltrusch for supporting our molecular biology experiments, M. Reichard for stimulating scientific discussions, and D. Beattie for editorial assistance in preparing the manuscript for publication. This work was financially supported by the FORUN programme of the University of Rostock Medical Faculty (889011) (grant to BS

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Corneal epithelial and neuronal interactions: Role in wound healing

Highlights

Abstract

Introduction

Section snippets

Materials

TG neurons in culture

Neuron–epithelial interactions and effect of CEM on TG neurons in culture

Discussion

Declaration of interest

Author's contributions

Acknowledgements

Exp. Neurol.

Exp. Eye Res.

Exp. Eye Res.

Mol. Cell. Neurosci.

Exp. Eye Res.

Dev. Cell.

Ocul. Surf.

Mol. Cell. Neurosci.

Ophthalmology

Exp. Eye Res.

Cell. Immunol.

Lab. Invest.

TGF-β induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways in a human corneal epithelial cell line

Invest. Ophthalmol. Vis. Sci.

Slug is upregulated during wound healing and regulates cellular phenotypes in corneal epithelial cells

Invest. Ophthalmol. Vis. Sci.

Substance P-induced cadherin expression and its signal transduction in a cloned human corneal epithelial cell line

J. Cell. Physiol.

Trigeminal ganglion neurons affect corneal epithelial phenotype. Influence on type VII collagen expression in vitro

Invest. Ophthalmol. Vis. Sci.

Microtubule-associated protein 1B controls directionality of growth cone migration and axonal branching in regeneration of adult dorsal root ganglia neurons

J. Neurosci.

Action of a trophic factor(s) from rabbit corneal epithelial culture on dissociated trigeminal neurons

J. Neurosci.

Corneal nerve regeneration. Correlation between morphology and restoration of sensitivity

Invest. Ophthalmol. Vis. Sci.

Co-culture of rat trigeminal ganglion neurons and corneal epithelium

Curr. Eye Res.