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Ryan M. Pedrigi, C. R. Ethier, W. D. Stamer, Darryl R. Overby; Pores Form in Cultured Schlemm’s Canal Endothelial Cells During Transendothelial Perfusion. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6265.
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© ARVO (1962-2015); The Authors (2016-present)
Aqueous humor transport across Schlemm’s canal (SC) endothelium likely involves flow through micron-sized transendothelial pores, but the existence of pores is debated and their role in outflow regulation is not well understood. This project investigates whether pores form in cultured SC cells during basal-to-apical directed perfusion (the direction that flow crosses SC endothelium in vivo).
SC cells were isolated from post-mortem human eyes, cultured, and seeded (4.5x104 cells/cm2) on track-etch filters for 2 days. Cell layers were perfused in the basal-to-apical direction for 30 minutes at 2 or 6 mmHg pressure drop (N=3 each), followed by perfusion with a constant volume of fixative (120µL). Unperfused cell layers were immersion-fixed at 0 mmHg (N=4). Samples were processed for scanning electron microscopy, and 12 randomly selected regions (5500 µm2 each) were examined per sample using masked observers to identify pores.
Pores appeared as elliptical micron-sized openings with smooth edges that passed either through or between individual cells (Figure). Pore diameter increased with pressure: 1.3±1.0, 1.7±1.1, 2.3±1.7 µm for 0 (N=6 pores), 2 (25), and 6 (25) mmHg, respectively (p=0.045; mean±SD). Total pore area (per 66,000µm2 cell area) also increased with pressure: 2.1±3.5, 17.2±7.5, 37.2±20.1 µm2 for 0, 2, and 6 mmHg (p=0.003).
Transcellular and paracellular pores form due to the pressure difference across cultured SC cells during basal-to-apical directed perfusion, mimicking pores observed in the inner wall in situ. This suggests that at least some fraction of inner wall pores are non-artifactual, and it introduces a novel system to study pore formation to better understand how inner wall pores influence outflow resistance regulation.
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