Novel ocular imaging and molecular analysis of anterior eye segment for glaucoma

项目摘要 青光眼是世界范围内不可逆性失明的主要原因，是一组异质性的眼部疾病， 以视神经损伤和视力受损为特征的疾病。青光眼患者大多数是老年人 由于年龄相关，视神经疾病，而有早发性青光眼：原发性先天性 青光眼和发育性青光眼。房水从前部引流的阻力 眼段进入体循环导致眼内压（IOP）升高， IOP是青光眼的主要和唯一可改变的风险因素。尽管目前的治疗方案旨在 为了降低IOP，迫切需要开发新的、更有效的治疗方法。房水 作为常规流出途径的引流是通过小梁网（TM）介导的， 起源于神经嵴（NC）谱系，成为一个专门的，类似神经管的血管，称为Schlemm管 （SC）。最近的证据表明，SC具有专门和独特的血管内皮细胞（EC）， Angiopoietin/Tie2等信号通路对SC的形成和维持至关重要 脉管系统我们小组的长期目标是阐明调节SC的基本机制 维持/功能，并了解这些机制的破坏如何导致流出受损， 病理环境中的IOP升高。为了对小鼠的全环SC进行成像并评估TM刚度，我们的小组 最近开发了一种圆扫描可见光OCT（vis-OCT）系统。FOXC2是FOX的成员。 （叉头盒）转录因子家族，并且在血管发育和疾病中具有关键作用。灭活 人FOXC 2的突变主要与水肿相关，其特征是阻塞性水肿。 最近的证据表明，FOXC 2变异体具有假定的作用， 原发性先天性青光眼的发展。然而，它在TM和SC细胞中的作用还没有得到证实。 进行全面调查。我们通过vis-OCT成像，免疫组织化学， 和单细胞RNA测序表明NC-Foxc2-/-突变与缺陷性TM形成相关， SC形态异常，或SC缺失，PROX1、VEGFR3和TEK表达减少 在SC EC中，和升高的IOP，并且EC-Foxc2-/-突变导致SC形成受损。我们的中央 假设是新诊断性vis-OCT成像技术阐明了Foxc2功能在 NC衍生的TM细胞和SC EC在SC形成/维持和TM刚度中的作用。为了验证这个假设， 我们有两个具体目标。首先，我们将开发一种新的平衡检测vis-OCT来确定解剖结构， Foxc2调控网络对SC形成/维持的影响。第二，我们将开发新的 基于vis-OCT相位成像的高速时域散斑后TM刚度评估方法 减少以定义Foxc2调节网络在TM刚度和ECM中的功能影响 组合物的

Project Summary Glaucoma is the leading cause of irreversible blindness worldwide and is a heterogeneous group of ocular diseases characterized by optic nerve damage and impaired vision. The majority of glaucoma patients are elderly due to the age-related, optic nerve disease, whereas there are early-onset forms of glaucoma: primary congenital glaucoma and developmental glaucoma. The resistance to drainage of aqueous humor from the anterior segment of the eye into the systemic circulation leads to the elevation of intraocular pressure (IOP), and elevated IOP is the primary and only modifiable risk factor for glaucoma. Despite the current treatment options aimed at lowering IOP, there are urgent needs to develop new, more effective therapeutic approaches. Aqueous humor drainage as the conventional outflow pathway is mediated through the trabecular meshwork (TM), which is derived from the neural crest (NC) lineage, into a specialized, lymphatic-like vessel known as Schlemm's canal (SC). Recent evidence indicates that SC has specialized and unique vascular endothelial cells (ECs) and that the signaling pathways such as Angiopoietin/Tie2 are essential for the formation and maintenance of SC vasculature. The long-term goal of our group is to elucidate the fundamental mechanisms that regulate SC maintenance/function and to understand how disruption of these mechanisms leads to impaired outflow causing increased IOP in pathological settings. To image the full-circle SC and evaluate TM stiffness in mice, our group has recently developed a circular-scanning visible-light OCT (vis-OCT) system. FOXC2 is a member of the FOX (Forkhead box) transcription factor family and has critical roles in vascular development and disease. Inactivating mutations in human FOXC2 are dominantly associated with lymphedema, which is characterized by obstructed lymph drainage in the limbs, and recent evidence has shown that FOXC2 variants possess a role as putative modifiers for the development of primary congenital glaucoma. However, its role in TM and SC cells has yet to be fully investigated. The results from our preliminary experiments via vis-OCT imaging, immunohistochemistry, and single-cell RNA sequencing indicate that the NC-Foxc2-/- mutation is associated with defective TM formation, abnormal SC morphology, or the absence of the SC, a reduction in expression of PROX1, VEGFR3, and TEK in SC ECs, and elevated IOP, and that the EC-Foxc2-/- mutation results in impaired SC formation. Our central hypothesis is that novel diagnostic vis-OCT imaging techniques elucidate the significance of Foxc2 function in both NC-derived TM cells and SC ECs in SC formation/maintenance and TM stiffness. To test this hypothesis, we have two Specific Aims. First, we will develop a new balanced-detection vis-OCT to determine the anatomical influence of the Foxc2 regulatory network in SC formation/maintenance. Second, we will develop new methodologies to evaluate TM stiffness based on vis-OCT phase imaging after high-speed temporal speckle reduction to define the functional influence of the Foxc2 regulatory network in TM stiffness and ECM compositions.