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

描述（由申请人提供）：青光眼特征的眼压升高（IOP）通常是由于房水通过常规流出通道流出时阻力增加所致。不幸的是，我们对流出通道组织如何产生流出阻力和控制IOP的理解存在空白。流出通道中假定的阻力屏障是Schlemm管的内壁内皮及其下的导管旁结缔组织，但尚不清楚这些组织如何调节流出以产生流出阻力。这个项目的目标是确定房水如何穿过施勒姆管的内壁。在体内，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板的连接断裂。这些力是由房水流过内壁的基底到根尖方向产生的。我们假设机械力动态地塑造内壁形态，导致流过内壁的跨内皮通道的短暂打开和关闭。这种流动有两种可能的途径：(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）涉及通过单个细胞的细胞内微米大小的“i孔”的细胞外途径。这两种途径都可能与“巨液泡”有关——当一个或多个相邻的细胞从基底层分离时形成的内壁细胞的伞状突起。为了验证我们的假设，实验主要有两个目的：1)确定穿越施莱姆氏管内皮的内皮细胞流动途径，以及大液泡、气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（通过增加灌注压力）如何影响通过大液泡、孔隙和细胞间连接的水力传导和运输动力学。我们的实验设计的核心是一种新的方法来动态可视化施勒姆管内皮（SCE）单层细胞和基底到根尖定向灌注过程中跨内皮血流的途径。该方法使用专门的体外灌注系统在显微镜物镜的工作距离内定位SCE单层，同时使用计算机化系统精确控制跨内皮灌注压力。最重要的是，该系统允许在灌注过程中对内皮进行延时光学切片，同时观察大液泡、毛孔和细胞间连接（使用荧光重要细胞染色成像）和跨内皮流动途径（使用灌注液中不同颜色的荧光示踪纳米颗粒成像）的动态变化。青光眼是致盲的主要原因，通常与眼内房水流出阻力增加引起的眼压升高有关。本研究探讨了流出阻力产生的基本机制，以了解青光眼IOP升高的潜在原因。最终，这项研究将有助于开发更成功的治疗策略，通过针对流出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.