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Item A Novel Technique Identifies Valve-Like Pathways Entering and Exiting Schlemm's Canal in Macaca nemestrina Primates With Similarities to Human Pathways(Frontiers Media, 2022-07-04) Martin, Elizabeth A.; Johnstone, Murray A.; Ophthalmology, School of MedicinePurpose: The aim of the study was 1) to describe a novel combination of techniques that permit immunohistochemistry imaging of Schlemm's canal inlet (SIV) and outlet (SOV) valve-like structures, 2) to identify tissue-level SIV adhesive relationships linking the trabecular meshwork (TM) to hinged collagen leaflets at the Schlemm's canal (SC) external wall, and 3) to determine whether the SIV lumen wall's adhesive vascular markers are similar to those of the SC inner wall endothelium. Materials and Methods: Anterior segments of 16 M. nemestrina primates underwent immunohistochemistry (IHC) labeling. We perfused fluorescent microspheres into 12 of the eyes. Limbal tissues were divided into quadrants, viscoelastic introduced into SC, tissues fixed, immunohistochemistry performed, radial segments cut, tissues clarified, and confocal microscopy performed. Finally, we generated ImageJ 3D projections encompassing the TM, SC, and distal pathways. Results: IHC imaging identified 3D relationships between SIV, collector channel ostia, collector channels (CC), SOV, and intrascleral channels. Imaging depth increased 176.9%, following clarification (p < 0.0001). Imaging demonstrated CD31, collagen type 1 and 4 in the walls of the SIV lumen and more distal pathways. In eight eyes, 384 segments were examined, 447 SIV identified, and 15.4% contained microspheres. Conclusion: Our technique's imaging depth permitted the identification of SIV linkage between the TM and SOV. We found comparable cell-cell adhesion molecules (CD31) and basement membrane components in the SC inner wall and SIV lumen walls. Recent OCT studies have suggested that SIV tensional relationships may control CC entrance dimensions that regulate distal resistance. Cellular adhesive properties sustain SIV tensional relationships. These SIV cell-cell and cell-basement membrane properties warrant further study because abnormalities could be a factor in the IOP elevation of glaucoma.Item Trabecular Meshwork Movement Controls Distal Valves and Chambers: New Glaucoma Medical and Surgical Targets(MDPI, 2023-10-18) Johnstone, Murray; Xin, Chen; Martin, Elizabeth; Wang, Ruikang; Ophthalmology, School of MedicineHerein, we provide evidence that human regulation of aqueous outflow is by a pump-conduit system similar to that of the lymphatics. Direct observation documents pulsatile aqueous flow into Schlemm's canal and from the canal into collector channels, intrascleral channels, aqueous veins, and episcleral veins. Pulsatile flow in vessels requires a driving force, a chamber with mobile walls and valves. We demonstrate that the trabecular meshwork acts as a deformable, mobile wall of a chamber: Schlemm's canal. A tight linkage between the driving force of intraocular pressure and meshwork deformation causes tissue responses in milliseconds. The link provides a sensory-motor baroreceptor-like function, providing maintenance of a homeostatic setpoint. The ocular pulse causes meshwork motion oscillations around the setpoint. We document valves entering and exiting the canal using real-time direct observation with a microscope and multiple additional modalities. Our laboratory-based high-resolution SD-OCT platform quantifies valve lumen opening and closing within milliseconds synchronously with meshwork motion; meshwork tissue stiffens, and movement slows in glaucoma tissue. Our novel PhS-OCT system measures nanometer-level motion synchronous with the ocular pulse in human subjects. Movement decreases in glaucoma patients. Our model is robust because it anchors laboratory studies to direct observation of physical reality in humans with glaucoma.Item Valve-Like Outflow System Behavior With Motion Slowing in Glaucoma Eyes: Findings Using a Minimally Invasive Glaucoma Surgery–MIGS-Like Platform and Optical Coherence Tomography Imaging(Frontiers Media, 2022-04-29) Johnstone, Murray; Xin, Chen; Acott, Ted; Vranka, Janice; Wen, Joanne; Martin, Elizabeth; Wang, Ruikang K.; Ophthalmology, School of MedicinePurpose: This study aimed to investigate anatomic relationships and biomechanics of pressure-dependent trabecular meshwork and distal valve-like structure deformation in normal and glaucoma eyes using high-resolution optical coherence tomography (HR-OCT). Methods: We controlled Schlemm's canal (SC) pressure during imaging with HR-OCT in segments of three normal (NL) and five glaucomatous (GL) ex vivo eyes. The dissected limbal wedges were studied from 15 locations (5 NL and 10 GL). A minimally invasive glaucoma surgery (MIGS)-like cannula was inserted into the SC lumen, whereas the other end was attached to a switch between two reservoirs, one at 0, the other at 30 mm Hg. A steady-state pressure of 30 mm Hg was maintained to dilate SC and collector channels (CC) during 3D volume imaging. The resulting 3D lumen surface relationships were correlated with internal structural features using an image mask that excluded tissues surrounding SC and CC. While imaging with HR-OCT, real-time motion responses in SC and CC areas were captured by switching pressure from 0 to 30 or 30 to 0 mm Hg. NL vs. GL motion differences were compared. Results: Lumen surface and internal relationships were successfully imaged. We identified SC inlet and outlet valve-like structures. In NL and GL, the mean SC areas measured at the steady-state of 0 and 30 mm Hg were each significantly different (p < 0.0001). Synchronous changes in SC and CC lumen areas occurred in <200 ms. Measured SC area differences at the steady-state 0 and 30 mmHg, respectively, were larger in NL than GL eyes (p < 0.0001). The SC motion curves rose significantly more slowly in GL than NL (p < 0.001). Pressure waves traveled from the cannula end along the SC lumen to CC and deep intrascleral channels. Conclusion: HR-OCT provided simultaneous measurements of outflow pathway lumen surfaces, internal structures, and biomechanics of real-time pressure-dependent dimension changes. We identified SC inlet and outlet valve-like structures. GL tissues underwent less motion and responded more slowly than NL, consistent with increased tissue stiffness. A MIGS-like shunt to SC permitted pulse waves to travel distally along SC lumen and into CC.