Diabetic retinopathy, the leading cause of blindness among working-age Americans, is characterized by increased retinal microvascular permeability which results in the formation of lipid exudates, macular edema and blindness. Recent data suggest that water and solutes cross the blood-retinal barrier through separate and differentially regulated transport pathways. To date, the only systems available to study changes in permeability are based on transit measurement of non-specific ion permeability or the accumulation of labeled solutes. To better investigate the basis of these pathways, we developed a prototype instrument to determine transport rates of water and solute flux across cell monolayers in realtime and under a variety of physiologic conditions. Water flux is measured by tracking an air bubble hydraulically coupled to a cell culture chamber. Solute flux is measured by monitoring flux of a fluorescent-labeled protein. Computer-controlled filter wheels, combined with gating and multiplexing techniques are used to monitor up to four solutes in two or more separate cell chambers simultaneously. Cells are cultured in plastic transwetl fitters and are then placed in the cell chamber. Opticat fibers and tubing are used to couple the cell chambers to the main instrument. The chambers can be placed either on a bench top or in a cell incubator for climate control. Our optohydraulic instrument enables investigators to easily monitor and compare water and solute transport properties of cell monolayers in response to various vasoactive substances. Furthermore, the ability to measure both of these parameters in the same preparation allows analysis of previously unobtainable data for study of cellular permeability. We anticipate that this instrument will significantly advance the study of vascular permeability and facilitate developments in pharmacotherapy. PROPOSED COMMERCIAL APPLICATION: This is a novel instrument that has a high potential for commercialization. The control of vascular permeability is a research area that is being aggressivly studied by scientists in academia and the private sector. No other instruments of its type exist.
