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

描述(由申请人提供)：青光眼的高眼压(IOP)特征通常是由于房水流出的阻力通过传统的流出途径增加所致。不幸的是，我们对流出道组织如何产生流出阻力和控制眼压的理解存在差距。流出通道中的阻性屏障是Schlemm管的内壁内皮及其下方的结缔组织，但这些组织如何调节流出以产生流出阻力尚不清楚。这个项目的目标是确定房水如何穿过Schlemm管的内壁。在活体中，内壁暴露在苛刻的机械力下，使细胞变形，并使它们与邻近细胞和基底板的连接变得紧张。这些力来自房水流过内壁的从底部到顶端的方向。我们假设，机械力动态地塑造内壁形态，导致穿过内壁的跨内皮细胞通道的瞬时开放和关闭。这种流动有两条可能的途径：(I)涉及细胞间连接和细胞旁空间扩张的细胞旁途径(所谓的“B孔”)和(Ii)涉及微米大小的“I孔”的跨细胞途径，这些孔在细胞内穿过单个细胞。这两条通路都可能与“巨型空泡”有关--当一个或多个相邻的细胞从基膜分离时，内壁细胞形成降落伞状突起。实验验证我们的假设是基于两个特定的目的：1)确定穿过Schlemm管内皮细胞的跨内皮流的途径，以及巨大的空泡、毛孔和细胞间连接在这一运输过程中的作用。2)确定增加的机械力(通过增加灌流压力)如何通过巨大的空泡、气孔和细胞间连接影响水力传导性和运输动力学。我们实验设计的核心是一种新的方法来动态显示Schlemm管内皮细胞单层和从基底到根尖定向灌流过程中的跨内皮细胞流动的途径。这种方法使用专门的体外灌流系统在显微镜物镜的工作距离内定位SCE单层，同时使用计算机系统精确控制跨内皮灌流压力。最重要的是，该系统允许在灌流过程中对内皮进行延时光学切片，以同时观察巨大的空泡、毛孔和细胞间连接(使用荧光生命细胞染色成像)和跨内皮血流路径(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。青光眼是导致失明的主要原因，通常与眼部房水流出阻力增加引起的眼压升高有关。本研究探讨流出阻力产生的基本机制，以了解青光眼高眼压的根本原因。最终，这项研究将有助于开发更成功的治疗策略，通过瞄准流出阻力的来源来降低青光眼患者的眼压。

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.