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

项目摘要 青光眼是全球不可逆性失明的主要原因，是一种不同类型的眼科疾病。 以视神经损伤和视力受损为特征的疾病。大多数青光眼患者是老年人 由于年龄相关，视神经疾病，而有早发性青光眼的形式：原发性先天性青光眼 青光眼和发育性青光眼。前房水引流的抵抗力 眼球进入体循环的节段会导致眼压(IOP)升高， 眼压是青光眼的主要且唯一可改变的危险因素。尽管目前的治疗方案旨在 为了降低眼压，迫切需要开发新的、更有效的治疗方法。房水 作为传统的流出途径，引流是通过小梁网(TM)介导的，这是 来源于神经脊(NC)谱系，进入一种特殊的淋巴管，称为Schlemm管 (SC)。最近的证据表明，SC具有特化和独特的血管内皮细胞(ECs)，并且 Angiopoietin/Tie2等信号通路在SC的形成和维持中起重要作用 脉管系统。我们小组的长期目标是阐明监管SC的基本机制 维护/功能，并了解这些机制的中断如何导致受损的流出 病理情况下眼压升高。为了在小鼠身上成像全圆SC并评估TM硬度，我们的团队 最近开发了一种环形扫描可见光OCT(VIS-OCT)系统。FOXC2是福克斯的成员之一 (叉头盒)转录因子家族，在血管发育和疾病中起关键作用。失活 人类FOXC2基因突变主要与淋巴水肿有关，淋巴水肿的特征是阻塞 四肢淋巴引流，最近的证据表明FOXC2变异体具有假定的作用 治疗原发性先天性青光眼的改良剂。然而，它在TM和SC细胞中的作用尚未 接受全面调查。我们的初步实验结果通过VIS-OCT成像，免疫组织化学， 单细胞RNA测序表明NC-FOXC2-/-突变与TM形成缺陷有关， SC形态异常，或SC缺失，PROX1、VEGFR3和TEK表达减少 EC-FOXC2-/-突变导致SC形成受损。我们的中央 假说是新的诊断VIS-OCT成像技术阐明了FOXC2功能在 NC来源的TM细胞和SC ECs在SC形成/维持和TM刚性方面都是如此。为了检验这一假设， 我们有两个具体目标。首先，我们将开发一种新的平衡检测相对于OCT来确定解剖学上的 FOXC2调控网络在SC形成/维持中的影响。第二，我们将发展新的 基于高速散斑后VIS-OCT相位成像的TM硬度评估方法 还原以确定FOXC2调节网络在TM刚性和ECM中的功能影响 作文。

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.