Late/chronic corneal injury following threat chemical exposure

摘要 呋喃丹（CF;一种杀虫剂）和氯（Cl 2;一种漂白剂和战争剂）对人类的暴露是一个主要的威胁。 公共卫生问题。超过300万人暴露于CF/Cl 2，显示出高眼部发病率。一个主要的差距， 知识是缺乏“CF/Cl 2暴露如何导致角膜损伤和视力丧失”的机制数据？我们测试一个 mTORC 1介导的自噬体形成和溶酶体生物发生功能障碍的新假说 是暴露于这些威胁化学品（CF/Cl 2）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的稳态和透明度维持中起关键作用。 我们的假设是基于一项人类患者的研究，该研究确定了有缺陷的自噬是导致缓慢和 圆锥角膜患者的进行性角膜变薄和视力下降。对小鼠和供体进行的初步研究 人类角膜有力地支持了我们的新假设。这个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Cl 2）和2种动物物种（C57 BL/6 J小鼠 和新西兰白色兔）和使用供体人角膜的假设机制在人中的适用性 衍生的器官培养和原代细胞培养模型，使用两个完全独立但整合的特定目标。 目的-1定义了CF暴露于以下物质引起的晚期/慢性角膜毒性的临床体征和强调机制： 使用3个子目标的眼睛：（1a）记录临床体征和角膜上皮细胞表型和密度的变化， 在活体兔和小鼠的CF +/-暴露眼中， 间隔两周，使用裂隙灯、HRT 3-RCM共聚焦、Specular和Spectralis光学相干断层扫描 显微镜系统，直到6个月，（1b）通过分析自噬体和溶酶体来定义机制 CF +/-暴露角膜中的特征基因（ATG、LC 3、SQSTM 1/p62、LAMP 1、mTORC 1、TFEB和vATPase） 在1个月、2个月、4个月和6个月时采集的2个种属，以及（1c）验证假设机制在人类中的适用性 使用尸体角膜。目的-2定义晚期/慢性角膜炎的临床症状和强调机制 使用3个子目标评估Cl 2暴露于眼睛引起的毒性：（2a）记录临床体征和 Cl ~（2+）/~-活体眼角膜上皮细胞、基质细胞和内皮细胞的表型和密度 兔和小鼠体内，（2b）通过研究自噬体阐明Cl 2诱导的角膜损伤， 目的-1b中所述的2个物种的Cl 2 +/-角膜中的溶酶体特征，以及（2c）验证了机制的适用性 在人类中使用对照供体人角膜，采用在 Aim-1a-c.通过拟议的研究，我们希望确定CF后症状的开始时间、程度和持续时间， 活体动物中的Cl 2暴露，mTORC 1介导的自噬事件在CF/Cl 2暴露角膜中的作用， 与CF/Cl 2介导的角膜毒性相关的标记性自噬体和溶酶体基因。成功 研究的完成有望填补许多知识空白，并显着推进眼部对抗ACT领域。

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.