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. [unreadable] [unreadable] [unreadable]