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

房水如何穿过施累姆氏管内壁？

• 批准号: 7296961
• 负责人: DARRYL R OVERBY
• 金额: $ 20.44万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7296961
• 关键词: Adherent Culture; Affect; Apical; Aqueous Humor; Basal lamina; Blindness; Cell Wall; Cells; Characteristics; Color; Connective Tissue; Contracts; Development; Endothelial Cells; Endothelium; Experimental Designs; Eye; Generations; Glaucoma; Goals; Image; Imagery; In Vitro; Individual; Intercellular Junctions; Life; Liquid substance; Measures; Mechanics; Microscope; Morphology; Open-Angle Glaucoma; Optics; Parachuting; Pathogenesis; Pathway interactions; Patients; Perfusion; Physiologic Intraocular Pressure; Positioning Attribute; Process; Publishing; Research; Resistance; Role; Shapes; Source; Staining method; Stains; Structure of sinus venosus of sclera; System; Testing; Therapeutic; Time; Tissues; Tracer; Transport Process; Vacuole; Work; aqueous humor flow; computerized; digital; improved; in vivo; mechanical drive; monolayer; nanoparticle; novel strategies; pressure; prevent; research study; response; size

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.

描述（由申请人提供）：青光眼特征性的眼内压（IOP）升高通常是由于房水通过传统流出路径流出的阻力增加所致。不幸的是，我们对流出通路组织如何产生流出阻力和控制眼压的理解存在差距。流出通路中假定的阻力屏障是施累姆氏管的内壁内皮及其下方的管旁结缔组织，但尚不清楚这些组织如何调节流出以产生流出阻力。该项目的目标是确定房水如何穿过施累姆氏管内壁。在体内，内壁受到苛刻的机械力的作用，使细胞变形并拉紧它们与邻近细胞和基底层的连接。这些力是由房水流过内壁的基底到心尖方向产生的。我们假设机械力动态地塑造内壁形态，导致跨内皮通路的短暂打开和关闭以穿过内壁。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扩张（所谓的“B孔”）的细胞旁途径和（ii）涉及在细胞内穿过单个细胞的微米大小的“I孔”的跨细胞途径。这两种途径都可能与“巨型液泡”有关，即当一个或多个连续细胞与基底层分离时形成的内壁细胞的降落伞状外突。测试我们的假设的实验根据两个具体目标进行：1）确定跨施累姆氏管内皮的跨内皮流动的路径以及巨大液泡、孔和细胞间连接在该运输过程中的作用。 2) 确定增加的机械力（通过增加灌注压力）如何影响水力传导性和通过巨大液泡、孔隙和细胞间连接的运输动力学。我们实验设计的核心是一种动态可视化施累姆氏管内皮 (SCE) 细胞单层和基底到心尖定向灌注过程中跨内皮血流通路的新颖方法。该方法使用专门的体外灌注系统将 SCE 单层定位在显微镜物镜的工作距离内，同时使用计算机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，以同时观察巨型液泡、孔和细胞间连接（使用荧光活细胞染色成像）和跨内皮血流通路（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。青光眼是导致失明的主要原因，通常与房水流出眼睛的阻力增加引起的眼内压升高有关。本研究调查了流出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，通过针对流出阻力的来源来降低青光眼患者的眼压。