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R. Pedrigi, D. R. Overby; Giant Vacuoles Formed in vitro Are Morphologically Similar to Those in vivo. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5840.
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© ARVO (1962-2015); The Authors (2016-present)
Giant vacuoles likely facilitate aqueous humor outflow across Schlemm’s canal (SC) endothelium, but we have little understanding of how these structures form or relate to IOP regulation. Previous work has shown that "giant vacuole like" structures (GVLs) form in vitro during basal-to-apical directed perfusion of SC cells. In this study, we determined whether the three-dimensional morphology of GVLs were similar to giant vacuoles observed in vivo.
Human SC cells were seeded at confluence (5x104 cells/cm2) on track-etch filter inserts and cultured for 2-3 days under standard conditions. SC cell monolayers were perfused in the basal-to-apical direction (mimicking the flow direction across the inner wall in vivo) for 30 minutes at controlled pressure drops of 2 or 6 mmHg, using a computerized perfusion system. Cells were then perfusion-fixed and processed by phalloidin staining to visualize F-actin architecture via confocal microscopy. Z-series confocal stacks were acquired through the entire thickness of the most prominent GVLs and 3D reconstructions of GVL morphology were performed using Velocity software.
The 3D morphology of GVLs was similar to giant vacuoles observed in vivo, based upon the typical "signet ring" appearance, ellipsoidal geometry, and perinuclear localization. GVL height depended upon pressure drop and was significantly larger at 6 versus 2 mmHg (21 ± 8 vs. 13 ± 3 µm; p < 0.0001; n = 26 and 22, respectively). GVL heights were larger than giant vacuoles reported in vivo (typically ≤ 7 µm), which is possibly due to SC cells tending to be larger in culture or because the pressure drop across SC cells in vivo is less than 2 mmHg.
GVLs are pressure-dependent and appear morphologically similar to giant vacuoles observed in vivo. This establishes the first in vitro perfusion model of giant vacuole formation in SC cell monolayers, offering novel opportunities to investigate the mechanics of giant vacuoles and their role in IOP regulation.Acknowledgements: We thank Prof. Dan Stamer (U. of Arizona) for providing SC cells.Support: Supported by the Whitaker International Scholars Program (RMP), American Health Assistance Foundation (DRO), and NEI (DRO).
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