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

神经营养性角膜病的角膜神经再生 â 在 Thy1-GFP 大鼠神经营养性角膜病的标准化临床前体内长期模型中评估局部和显微手术治疗方法

• 批准号: 430619860
• 负责人: Dr. Simeon Däschler
• 金额: --
• 依托单位: Klinik für Plastisch-Rekonstruktive, Ästhetische Chirurgie und Präventive Medizin
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/430619860?language=en
• 关键词: Corneal; Reinnervation; Neurotrophic; Keratopathy; Evaluation

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

感觉神经支配可以保护角膜免受损伤，对于维持其上皮完整性至关重要。角膜神经支配缺失的患者会出现进行性角膜变性和干细胞功能受损，导致不可逆的视力丧失，称为神经营养性角膜病（NK）。然而，角膜干细胞和轴突之间的潜在细胞信号传导途径仍然未知，传统的治疗方法无法恢复角膜上皮的再生能力。因此，NK 仍然是全球角膜失明的主要原因。最近，引入了两种有前途的实验方法：局部神经生长因子（NGF）应用和感觉神经纤维显微外科转移到角膜（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神经支配和上皮功能以及是否产生潜在的协同治疗效果。 目的：本项目旨在研究局部NGF、角膜的治疗效果 在临床前 NK 长期模型中进行神经化和联合治疗（NGF + 角膜神经化），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，将角膜去神经化，并随机分配三个治疗组：A) 局部 NGF 应用 B) 角膜神经化 C) 联合治疗 （NGF + 角膜神经化）。去神经控制组和定期神经支配的假去神经组将作为参考。将在六个月内定期评估 NK 的四个主要方面： 1.) 角膜神经支配和敏感性（角膜神经支配的密度和模式、角膜神经支配神经元的数量、角膜四象限敏感性和流泪） 2.) 上皮完整性和愈合（标准化上皮病变后自发上皮缺陷的量化和角膜愈合进展） 3.) 上皮细胞凋亡和增殖（TUNEL 法、原位细胞死亡检测试剂盒和 KI-67 染色） 4.) 角膜干细胞功能（角膜干细胞的数量和增殖能力） 体外：使用已建立的体外共培养模型（角膜上皮细胞 + DRG 神经元），病变和完整角膜上皮中两种细胞类型的表达谱将 定义的。将针对两种细胞类型筛选在病变角膜中经历上调的相应配体和受体，以识别再生相关信号传导。预期结果：该项目将证明 NGF 替代、角膜神经化或两种方法的组合是否能够在 NK 临床前长期模型中恢复角膜神经支配和上皮完整性。此外，作为 NK 病理生理学关键方面的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

项目成果

Effect of moist heat reprocessing of N95 respirators on SARS-CoV-2 inactivation and respirator function

• DOI: 10.1503/cmaj.201203
• 发表时间: 2020-10-13
• 期刊: CANADIAN MEDICAL ASSOCIATION JOURNAL
• 影响因子: 14.6
• 作者: Daeschler, Simeon C.;Manson, Niclas;Borschel, Gregory H.
• 通讯作者: Borschel, Gregory H.