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

感觉神经保护角膜免受损伤，对维持角膜上皮的完整性是必不可少的。缺乏角膜神经支配的患者发生进行性角膜变性和干细胞功能受损，导致不可逆转的视力丧失，称为神经营养性角膜病变(NK)。然而，角膜干细胞和轴突之间的潜在细胞信号通路仍然未知，传统的治疗方法无法恢复角膜上皮细胞的再生能力。因此，NK仍然是世界范围内导致角膜失明的主要原因。目前已有两种有前景的实验方法：局部应用神经生长因子(NGF)和感觉神经纤维移植到角膜(角膜神经化)，目前尚不清楚这些方法是否能够长期恢复角膜的神经和上皮功能，以及是否存在潜在的协同治疗效果。目的：本项目旨在研究局部应用NGF、角膜神经化和联合治疗(NGF+角膜神经化)在临床前NK长期模型中的治疗效果，并确定轴突和角膜干细胞之间的信号通路。方法：体内：使用最近建立的转基因Thy1GFP+大鼠模型，三个治疗组将被随机分成三组：A)局部应用NGF；B)角膜神经化；C)联合治疗(NGF+角膜神经化)。以失神经对照组和常规神经支配的假去神经组为参照。将在六个月内定期评估NK的四个主要方面：1.角膜再神经支配和敏感度(角膜再神经支配的密度和方式、再神经支配神经元的数量、角膜四象限敏感度和流泪)。上皮完整性和愈合(自发性上皮损伤的量化和标准化上皮损伤后角膜愈合的进展)细胞凋亡和增殖的定量(TUNEL法、原位细胞死亡检测试剂盒和Ki-67染色)。体外角膜干细胞功能(角膜干细胞的数量和增殖能力)：利用建立的体外共培养模型(角膜上皮细胞+背根节神经元)，将确定两种细胞类型在受损和完整的角膜上皮中的表达谱。在受损的角膜中，将筛选相应的配体和受体，以识别与再生相关的信号。预期的结果：该项目将展示NGF替代、角膜神经化或两者结合是否能够在临床前的长期NK模型中恢复角膜神经支配和上皮完整性。此外，角膜上皮干细胞和角膜轴突之间的调节信号通路将被确定为NK的病理生理关键方面。

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