How Does Aqueous Humor Cross the Inner Wall of Schlemm's Canal?

描述（由申请人提供）：青光眼特征性眼内压（IOP）升高通常是由于通过常规流出途径的房水流出阻力增加所致。不幸的是，我们对流出道组织如何产生流出阻力和控制IOP的理解存在差距。流出道中的假定阻力屏障是Schlemm管的内壁内皮及其下方的小管结缔组织，但尚不清楚这些组织如何调节流出以产生流出阻力。本项目的目标是确定水状体如何穿过施累姆氏管的内壁。在体内，内壁暴露于苛刻的机械力，使细胞变形并使它们与相邻细胞和基底层的连接应变。这些力来自于穿过内壁的房水流动的基底至顶端方向。我们假设，机械力动态地塑造内壁形态，导致短暂的开放和关闭的跨内皮通道的流动通过内壁。这种流动有两种潜在的途径：（i）涉及细胞间连接和细胞旁空间（所谓的“B孔”）扩张的细胞旁途径和（ii）涉及在细胞内穿过单个细胞的微米尺寸的“I孔”的跨细胞途径。这两种途径可能与“巨空泡”有关-当一个或多个相邻细胞从基膜分离时，内壁细胞形成降落伞样的外囊。实验测试我们的假设进行根据两个特定的目的：1）确定跨舒累姆氏管内皮细胞的跨内皮流动的途径和巨大的空泡，孔，和细胞间连接在这个运输过程中的作用。2)确定增加机械力（通过增加灌注压）如何影响水力传导率和通过巨泡、孔和细胞间连接的运输动力学。我们的实验设计的核心是一种新的方法，动态可视化Schlemm管内皮（SCE）细胞单层和跨内皮流动的途径，在基底-心尖定向灌注。这种方法使用一个专门的体外灌注系统的显微镜物镜的工作距离内的SCE单层定位，同时精确地控制transendothelial灌注压，使用计算机化系统。最重要的是，该系统允许在灌注期间对内皮进行延时光学切片，以同时观察巨空泡、孔和细胞间连接（使用荧光活细胞染色成像）和跨内皮流动通路（使用灌注液中不同颜色的荧光示踪剂纳米颗粒成像）的动态变化。青光眼是失明的主要原因，通常与眼内压升高有关，眼内压升高是由眼房水流出阻力增加引起的。本研究探讨了外流阻力产生的基本机制，以了解青光眼眼压升高的根本原因。最终，这项研究将有助于开发更成功的治疗策略，通过靶向外流阻力的来源来降低青光眼患者的IOP。

DESCRIPTION (provided by applicant): Elevated intraocular pressure (IOP) characteristic of glaucoma typically results from increased resistance of aqueous humor outflow through the conventional outflow pathway. Unfortunately, there is a gap in our understanding of how outflow pathway tissues function to generate outflow resistance and control IOP. The putative resistive barrier in the outflow pathway is the inner wall endothelium of Schlemm's canal and its underlying juxtacanalicular connective tissue, but it is unclear how these tissues regulate outflow to generate outflow resistance. The goal of this project is to determine how aqueous humor crosses the inner wall of Schlemm's canal. In vivo, the inner wall is exposed to demanding mechanical forces that deform the cells and strain their connections to neighboring cells and to the basal lamina. These forces result from the basal-to-apical direction of aqueous humor flow across the inner wall. We hypothesize that mechanical forces dynamically shape inner wall morphology, leading to transient opening and closing of transendothelial pathways for flow across the inner wall. There are two potential pathways for such flow: (i) the paracellular pathway involving inter-cellular junctions and dilations of the paracellular space (the so-called "B-pores") and (ii) the transcellular pathway involving micron-sized "I-pores" that pass intra-cellularly through individual cells. Both pathways may be associated with "giant vacuoles" - parachute-like outpouchings of inner wall cells formed when one or more contiguous cells separate from the basal lamina. Experiments testing our hypothesis proceed according to two Specific Aims: 1) Determine the pathway of transendothelial flow across Schlemm's canal endothelium and the role of giant vacuoles, pores, and intercellular junctions in this transport process. 2) Determine how increased mechanical force (by increasing perfusion pressure) affects hydraulic conductivity and transport dynamics through giant vacuoles, pores, and intercellular junctions. The centerpiece of our experimental design is a novel approach to dynamically visualize Schlemm's canal endothelial (SCE) cell monolayers and the pathways for transendothelial flow during basal-to-apical directed perfusion. This approach uses a specialized in vitro perfusion system to position SCE monolayers within the working distance of a microscope objective, while precisely controlling the transendothelial perfusion pressure using a computerized system. Most importantly, this system allows for time-lapse optical sectioning of the endothelium during perfusion to simultaneously observe dynamic changes in giant vacuoles, pores and intercellular junctions (imaged using a fluorescent vital cell stain) and transendothelial flow pathways (imaged using a different color of fluorescent tracer nano-particles in the perfusion fluid). Glaucoma is a leading cause of blindness that is typically associated with elevated intraocular pressure caused by increased resistance of aqueous humor outflow from the eye. This research investigates the fundamental mechanism of outflow resistance generation to understand the underlying cause of elevated IOP in glaucoma. Ultimately, this research will contribute to the development of more successful therapeutic strategies to reduce IOP in glaucoma patients by targeting the source of outflow resistance.