Corneal Reinnervation for Neurotrophic Keratopathy – Evaluation of Topical and Microsurgical Treatment Approaches in a Standardized, Preclinical, In-vivo Long-term Model of Neurotrophic Keratopathy in Thy1-GFP+ Rats

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Sensory innervation protects the cornea from injury and is essential for maintaining its epithelial integrity. Patients with absent corneal innervation develop progressive corneal degeneration and impaired stem cell function leading to irreversible vision loss, known as neurotrophic keratopathy (NK). Yet, the underlying cellular signaling pathways between corneal stem cells and axons remain unknown and conventional therapeutic approaches are unable to restore the regenerative capacity of the corneal epithelium. Thus, NK remains a worldwide leading cause of corneal blindness. Recently, two promising experimental approaches have been introduced: topical nerve growth factor (NGF) application and a microsurgical transfer of sensory nerve fibers to the cornea (corneal neurotization) It remains unclear whether these approaches are able to restore the corneal innervation and epithelial function in the long term and whether potential synergistic treatment effects arise.Objective: This project aims at investigating the treatment effect of topical NGF, corneal neurotization and a combination treatment (NGF + corneal neurotization) in a preclinical NK long-term model and identifying signaling pathways between axons and corneal stem cells.Methods: In-vivo: Using a recently established transgenic Thy1GFP+ rat model, the cornea will be denervated and three treatment groups will be randomized: A) topical NGF application B) corneal neurotization C) combination therapy (NGF + corneal neurotization). A denervated control group and a regularly innervated sham-denervation group will serve as a reference. Four main aspects of NK will be assessed regularly over six months: 1.) Corneal reinnervation and sensitivity (Density and pattern of corneal reinnervation, number of reinnervating neurons corneal 4-quadrant-sensitivity and lacrimation) 2.) Epithelial integrity and healing (quantification of spontaneous epithelial defects and progress of corneal healing following standardized epithelial lesion) 3.) Quantification of epithelial apoptosis and proliferation (TUNEL-method, in situ cell death detection kit and KI-67-staining) 4.) Corneal stem cell function (number and proliferative capacity of corneal stem cells) In-vitro: Using an established in-vitro co-culture model (corneal epithelial cells + DRG-neurons), the expression profile of both cell types in lesioned and intact corneal epithelium will be defined. Corresponding ligands and receptors undergoing up regulation in lesioned cornea will be screened for both cell types to identify regeneration associated signaling.Expected results: This project will demonstrate whether NGF-substitution, corneal neurotization or a combination of both approaches are capable to restore the corneal innervation and epithelial integrity in a preclinical long-term model of NK. Moreover, regulatory signaling pathways between corneal epithelial stem cells and corneal axons as a pathophysiological key aspect of NK will be identified