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Darryl R Overby, Sietse Braakman, Alice Spenlehauer, Justino R Rodrigues, Carter Teal, A Thomas Read, W Daniel Stamer, C Ross Ethier; Stretch-Dependent Pore Formation in Glaucomatous Schlemm’s Canal Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3773.
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Micron-sized pores provide a pathway for aqueous humor drainage across Schlemm’s canal (SC) endothelium. SC pore density is reduced in glaucoma, and glaucomatous SC cells have impaired pore-forming ability compared to normal SC cells exposed to the same transendothelial pressure drop (Overby et al., PNAS 2014). In this study, we compare pore density between normal and glaucomatous SC cells exposed to the same levels of mechanical stretch. Imposing stretch (vs. imposing transendothelial pressure drop) minimizes the influence of cellular stiffness that correlates with pore density in pressure-controlled experiments.
Human SC cells were isolated from 1 glaucomatous (SC57g) and 1 non-glaucomatous donor (SC67) and characterized by established protocols (Perkumas and Stamer, EER 2012). SC cells were seeded at 24x103 cells/cm2 on elastic PDMS membranes coated with biotinylated gelatin. Once confluent, cells were exposed to 0%, 10% or 20% equibiaxial stretch (N=2-4 samples each), incubated with FITC-avidin tracer for 3 minutes and then fixed at 6 minutes post-stretch. We identified pores based on accumulation of tracer where it crossed the SC monolayer and bound to the PDMS membrane, following Dubrovskyi et al. (Lab Invest 2013) and Braakman et al. (ARVO 2014). We focused on transcellular ‘I’ pores, identified as circular tracer spots underneath individual cells away from cell borders. Tracer patterns were imaged at 20x by epifluorescence microscopy covering 3 mm2 (15-20 non-overlapping images) per sample. One masked observer (STB) identified and counted I-pores. Pore density was analyzed using Poisson statistics.
At 0% stretch, I-pore density was similar between SC67 (41±17 pores/mm2; mean±SD) and SC57g (47±19 pores/mm2). Pore density increased with stretch in SC67 (p<10-5) and SC57g (p<0.03; Figure). However, the linear slope of pore density versus stretch was significantly lower in SC57g versus SC67 (p<0.003; ANCOVA).
Transcellular pore formation was reduced in glaucomatous relative to normal SC cells when controlling for mechanical stretch. This suggests a deficiency in the biomolecular machinery responsible for I-pore formation in glaucomatous SC cells that is maintained in culture. Ongoing studies will compare tracer-based pore detection against scanning electron microscopy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Tracer-based measurements of I-pore density as a function of stretch in normal (SC67) and glaucomatous (SC57g) SC cells.
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