An in vitro Perfusion/Microscopy System to Visualize Aqueous Humor Transport Dynamics Across Schlemm's Canal Endothelium

Abstract

Purpose:: The bulk of aqueous humor outflow resistance is generated near the inner wall endothelium of Schlemm’s canal, but it is unknown how aqueous humor crosses the inner wall and how the inner wall contributes to the generation of outflow resistance. To investigate the mechanism of aqueous humor transport across the inner wall, we designed and characterized an in vitro perfusion system mounted on a fluorescence microscope to simultaneously visualize endothelial morphology and transendothelial flow patterns during basal-to-apical directed perfusion.

Methods:: The perfusion/microscopy system was designed to position a living monolayer of Schlemm’s canal endothelial cells cultured on a filter insert within the working distance of an inverted microscope objective. A computerized perfusion system was built to measure transendothelial hydraulic conductivity while maintaining a constant basal-to-apical directed perfusion pressure (0.5-5.0 mmHg) to mimic the hydrodynamic stress acting on the inner wall in vivo. Time-lapse images of endothelial morphology and transendothelial flow patterns were acquired using fluorescent CellTracker^® stain and a different color of fluorescent tracer FluoSpheres^® (200 nm; 10⁸ #/mL) contained in the perfusion fluid (Dulbecco’s PBS). Characterization studies were performed to confirm perfusion control and cell/tracer visualization using human umbilical vein endothelial cells (HUVECs) or human Schlemm’s canal endothelial cells (SCEs) seeded on the bottom surface of collagen-coated PTFE filter membrane inserts (Transwell^®-COL).

Results:: The perfusion system was able to quickly maintain (<4 min) and accurately control perfusion pressure at a constant value between 0.5 to 5.0 mmHg. Fluorescently stained HUVEC and SCE monolayers cultured on filter inserts (10⁵ cells/cm²) were successfully visualized within the perfusion system. Fluorescent time-lapse sequences of sub-confluent HUVEC monolayers perfused with fluorescent tracer at 1 mmHg revealed tracer passage through open spaces between cells.

Conclusions:: The in vitro perfusion system is compatible with time-lapse fluorescence microscopy and allows dynamic visualization of endothelial monolayers and transendothelial flow patterns during pressure-controlled basal-to-apical directed perfusion. This perfusion/microscopy system offers a novel approach to investigate the mechanism of aqueous humor transport across the inner wall and the role of giant vacuoles, pores and intercellular junctions in this transport process.Acknowledgements: We thank Dr. Dan Stamer for providing SCE cells.

Keywords: outflow: trabecular meshwork • intraocular pressure • microscopy: light/fluorescence/immunohistochemistry