Late/chronic corneal injury following threat chemical exposure

威胁化学品暴露后的晚期/慢性角膜损伤

• 批准号: 10676950
• 负责人: Suneel Gupta
• 金额: $ 45.6万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676950
• 关键词: Acute; Agriculture; Anatomy; Animals; Autophagocytosis; Autophagosome; Biogenesis; Blindness; Cadaver; Carbamates; Chemical Exposure; Chemicals; Chlorine; Chronic; Clinical; Cornea; Corneal Injury; Data; Defect; Diagnostic Imaging; Disinfectants; Endothelial Cells; Euthanasia; Event; Exposure to; Eye; Fluorescein; Genes; Goals; Health; Histologic; Homeostasis; Human; In Vitro; Keratoconus; Knowledge; Link; Maintenance; Mediating; Methods; Microscope; Microscopy; Modeling; Molecular; Molecular Target; Morbidity - disease rate; Mus; New Zealand; Optical Coherence Tomography; Organ Culture Techniques; Oryctolagus cuniculus; Patients; Periodicals; Persons; Pesticides; Phenotype; Pilot Projects; Play; Primary Cell Cultures; Public Health; Publications; Publishing; Records; Reporting; Rodent Model; Role; Symptoms; System; Techniques; Testing; Thinness; Time; Toxic effect; Transmission Electron Microscopy; United States National Institutes of Health; chemical threat; clinical diagnostics; corneal epithelium; density; experience; genetic signature; in vivo; microscopic imaging; multimodality; novel; novel strategies; ophthalmic examination; prevent; programs; tomography; tonometry; tool

Abstract Exposure of Carbofuran (CF; a pesticide) and Chlorine (Cl2; a bleaching and warfare agent) to humans is a major public health issue. Over 3 million people exposed to CF/Cl2 show high ocular morbidity. A major gap in knowledge is lack of mechanistic data “how CF/Cl2 exposure causes corneal injury and vision loss”? We test a novel hypothesis that mTORC1-mediated dysfunctional autophagosome formation and lysosomal biogenesis are a dominant operating mechanism for corneal damage from exposure of these threat chemicals (CF/Cl2). Autophagy and lysosomal biogenesis play a key role in corneal homeostasis and transparency maintenance. Our hypothesis is based on a human-patient study identifying defective autophagy the cause of slow and progressive corneal thinning and vision loss in keratoconus patients. Pilot studies performed with mice and donor human corneas strongly supports our novel hypothesis. The main goal of this project is to test our hypothesis employing highly rigorous approach using 2 threat chemicals (CF and Cl2) and 2 animal species (C57BL/6J mice and New Zealand White rabbits) and applicability of postulated mechanism in human using donor human cornea derived organ culture and primary cell culture models using two entirely independent but integrated specific aims. Aim-1 defines clinical signs and underline mechanism of late/chronic corneal toxicity caused by CF exposure to the eye using 3 sub-aims: (1a) records clinical signs and changes in the phenotype and density of corneal epithelial, stromal keratocytes, and endothelial cells in CF +/- exposed eyes of live rabbits and mice in vivo every two weeks interval with slit-lamp, HRT3-RCM confocal, Specular, and Spectralis optical coherence tomography microscopy system until 6 months, (1b) defines the mechanism by analyzing autophagosomal and lysosomal signature genes (ATGs, LC3, SQSTM1/p62, LAMP1, mTORC1, TFEB, & vATPase) in CF +/- exposed corneas of 2 species collected at 1, 2, 4, and 6 months, and (1c) verifies applicability of postulated mechanism in human using cadaver corneas. Aim-2 defines clinical symptoms and underline mechanism of late/chronic corneal toxicity caused by Cl2 exposure to the eye using 3 sub-aims: (2a) records clinical signs and changes in the phenotype and density of corneal epithelial, stromal keratocytes, and endothelial cells in Cl2 +/- eyes of live rabbits and mice in vivo, (2b) elucidates Cl2-induced corneal damage by studying autophagosomal and lysosomal signatures stated in aim-1b in Cl2 +/- corneas of 2 species, and (2c) verifies applicability of mechanism in humans using control donor human corneas employing an experimental approach and techniques stated in Aim-1a-c. With proposed studies, we expect to define start time, extent, and duration of symptoms after CF and Cl2 exposure in live animals, role of mTORC1 mediated autophagic events in CF/Cl2 exposed corneas and signature autophagosomal and lysosomal genes linked to CF/Cl2 mediated corneal toxicity. Successful completion of studies is expected to fill many knowledge gaps and advance ocular counterACT field significantly.

摘要 克百威(一种杀虫剂)和氯(一种漂白和战剂)对人类的暴露是主要的 公共卫生问题。超过300万人暴露在CF/Cl2中，表现出很高的眼部发病率。在以下方面存在重大差距 缺乏“CF/Cl2暴露如何导致角膜损伤和视力丧失”的机制数据？我们测试了一个 MTORC1介导的功能障碍自噬小体形成和溶酶体生物发生的新假说 是接触这些威胁化学物质(CF/Cl2)造成角膜损伤的主要操作机制。 自噬和溶酶体的生物发生在维持角膜动态平衡和透明度方面起着关键作用。 我们的假设是基于一项人类-患者研究，该研究确定了有缺陷的自噬是迟缓和 圆锥角膜患者的进行性角膜变薄和视力丧失。对小鼠和供体进行的初步研究 人类的角膜强烈支持我们的新假设。这个项目的主要目标是检验我们的假设。 使用2种具有威胁性的化学物质(CF和Cl2)和2种动物(C57BL/6J小鼠)采用高度严格的方法 和新西兰大白兔)，以及使用供体人角膜的假设机制在人类中的适用性 衍生器官培养和原代细胞培养模型使用两个完全独立但综合的特定目标。 AIM-1定义了慢性和迟发性角膜毒性的临床体征和发病机制 使用3个子目标的眼睛：(1)记录临床体征以及角膜表型和密度的变化 活体兔和小鼠CF+/-暴露眼内皮细胞、基质角质细胞和内皮细胞 间隔两周使用裂隙灯、HRT3-RCM共聚焦、镜面和光谱光学相干断层扫描 显微镜系统直到6个月，(1b)通过分析自噬小体和溶酶体来确定其机制 暴露于CF+/-的角膜中的标志性基因(ATGs、Lc3、SQSTM1/p62、LAMP1、mTORC1、TFEB和vATPase) 在1、2、4和6个月收集的2种物种，以及(1c)验证了假设的机制在人类中的适用性 使用的是身体角膜。AIM-2定义迟发性/慢性角膜病变的临床症状和发病机制 使用3个子目标：(2)记录临床体征和眼部的变化 活体Cl2+/-眼角膜上皮细胞、基质角质细胞和内皮细胞的表型和密度 兔和小鼠体内，(2b)通过研究自噬和自噬来阐明Cl2引起的角膜损伤 在2个物种的Cl2+/-角膜中，Aim-1b中所述的溶酶体特征，以及(2c)验证了机制的适用性 在使用对照供体人类角膜的人类中，采用了 AIM-1a-c.在拟议的研究中，我们希望确定CF和CF后症状的开始时间、程度和持续时间 活体动物的Cl2暴露，mTORC1介导的自噬事件在CF/Cl2暴露的角膜和 与Cf/Cl2介导的角膜毒性相关的特征自噬和溶酶体基因。成功 研究的完成有望填补许多知识空白，显着推进眼部中和领域。