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Sietse T Braakman, W Daniel Stamer, Darryl R Overby; A fluorescent permeability assay for Schlemm’s canal endothelial cells in response to stretch. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5983.
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All conventional outflow must cross the inner wall endothelium of Schlemm’s canal (SCE), and the increased outflow resistance in glaucoma is located near to SCE. Understanding the factors that regulate SCE permeability may thereby facilitate development of novel glaucoma therapies. SCE cells experience extraordinary biomechanical stretch as a consequence of basal-to-apical directed outflow and giant vacuole formation. We hypothesize that cellular stretch increases SCE permeability.
Permeability was measured based on the intensity of a fluorescent tracer (FITC-avidin) as it crosses the SCE cell monolayer and binds to an elastic (PDMS) membrane coated with biotinylated gelatin, following Dubrovskyi et al. (Lab Invest, 2013). The elastic membrane was mounted in a cell stretching device (Lee et al., AJP, 1996). SCE cells isolated from a 44-y/o donor were seeded in cloning rings onto the membrane. Once confluent, cells were stretched to 0%, 21% or 44% areal increase, exposed to FITC-avidin for 3 mins and fixed at 5 mins post-stretch. A plate reader was used to measure membrane fluorescence. Permeability measurements were compared against porosity measurements from a previous study of stretch-induced pore formation in the same cell line (Braakman et al., ISER, 2012).
Membrane fluorescence increased by 66% when cells were stretched from 0% to 44% (p=0.0009). To calculate relative permeability, fluorescence values were normalized between 0 (fluorescence of cells on non-biotinylated membranes) and 1 (fluorescence of biotinylated membranes without cells). Relative SCE permeability also increased with stretch (p=0.001) and mimicked the stretch-induced porosity increase (p=0.03) observed in the prior study, see figure.
The permeability of cultured SCE monolayers increases within minutes of stretch, suggesting that biomechanical deformation regulates SCE barrier function to possibly influence outflow resistance. The fluorescent permeability assay may provide a surrogate measure of SCE porosity that requires time-intensive pore counting and may provide a screening platform for drugs that target SCE permeability.
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