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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的人类接触呋喃丹（CF；一种杀虫剂）和氯（Cl2；一种漂白剂和战剂）是一个主要的危险因素。超过 300 万人暴露于 CF/Cl2，眼部发病率较高。知识缺乏机制数据“CF/Cl2 暴露如何导致角膜损伤和视力丧失”？ mTORC1 介导功能失调的自噬体形成和溶酶体生物发生的新假设是接触这些威胁化学物质 (CF/Cl2) 导致角膜损伤的主要操作机制。自噬和溶酶体生物发生在角膜稳态和透明度维持中发挥关键作用。我们的假设基于一项人类-患者研究，该研究确定了自噬缺陷是缓慢和缓慢的原因对小鼠和供体进行的圆锥角膜患者进行性角膜变薄和视力丧失的初步研究。人类角膜强烈支持我们的新假设该项目的主要目标是检验我们的假设。采用高度严格的方法，使用 2 种威胁化学品（CF 和 Cl2）和 2 种动物（C57BL/6J 小鼠）和新西兰白兔）以及假设机制在使用供体人角膜的人体中的适用性派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 CF 暴露引起的晚期/慢性角膜毒性的临床症状和强调机制眼睛使用 3 个子目标：(1a) 记录临床体征以及角膜表型和密度的变化 CF +/- 活体兔和小鼠暴露眼中的上皮细胞、基质角膜细胞和内皮细胞间隔两周使用裂隙灯、HRT3-RCM 共焦、镜面反射和 Spectralis 光学相干断层扫描显微镜系统直至 6 个月，(1b) 通过分析自噬体和溶酶体确定机制 CF +/- 暴露角膜中的特征基因（ATG、LC3、SQSTM1/p62、LAMP1、mTORC1、TFEB 和 vATPase）在 1、2、4 和 6 个月收集的 2 个物种的样本，以及 (1c) 验证假设机制在人类中的适用性使用尸体角膜定义了临床症状并强调了晚期/慢性角膜的机制。 Cl2 接触眼睛引起的毒性，使用 3 个子目标：(2a) 记录临床体征和眼部变化 Cl2 +/- 活体眼睛中角膜上皮、基质角膜细胞和内皮细胞的表型和密度兔子和小鼠体内实验，(2b) 通过研究自噬体和在 2 个物种的 Cl2 +/- 角膜中，aim-1b 中所述的溶酶体特征，以及 (2c) 验证机制的适用性在人类中使用对照供体人类角膜，采用实验方法和技术 Aim-1a-c. 通过拟议的研究，我们期望确定 CF 和之后症状的开始时间、程度和持续时间。活体动物中的 Cl2 暴露、mTORC1 在 CF/Cl2 暴露的角膜中介导的自噬事件的作用以及与 CF/Cl2 介导的角膜毒性相关的特征自噬体和溶酶体基因。研究的完成预计将填补许多知识空白并显着推进眼科 counterACT 领域的发展。