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Ryan M. Pedrigi, Ritika Gupta, Sietse T. Braakman, W. Daniel Stamer, C. Ross Ethier, Darryl R. Overby; Pore Formation is Impaired in Glaucomatous SC Cells Perfused In Vitro. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3278.
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© ARVO (1962-2015); The Authors (2016-present)
Schlemm’s canal endothelial (SC) cells form pores, likely to facilitate aqueous humor outflow into the canal lumen. In primary open-angle glaucoma, SC pore density is reduced compared to non-glaucomatous eyes (Johnson et al., IOVS, 2002), which may contribute to ocular hypertension. We hypothesize that impaired pore formation is an intrinsic property of glaucomatous SC cells that is retained in culture.
SC cells were isolated from 3 normal and 3 glaucomatous human donor eyes, seeded at confluence (4.5x104 cells/cm2) onto track-etch filters and cultured for 2 days. To test our hypothesis, SC cell layers were perfused in the basal-to-apical direction (same direction that aqueous humor crosses the inner wall in vivo) at 6 mmHg for 30 minutes. Cell layers were then immersed in fixative while continuing perfusion with medium for an additional 30 minutes. For controls, cell layers were either not perfused (immersion fixed at 0 mmHg) or perfusion-fixed in the (opposite) apical-to-basal direction at 6 mmHg. All pore-like structures were imaged in 12 randomly selected regions (5500 µm2 each) per cell layer using scanning electron microscopy. Four masked observers classified each pore-like structure as either a true pore or artifact. We then quantified the total area and density (pores/mm2) of all true pores in each cell layer. ANOVA was used for statistical analysis.
Pores were identified as micron-sized elliptical openings with smooth edges that passed through or between individual cells, consistent with pores observed along the SC inner wall in situ. Glaucomatous SC cell layers had an 84% lower total pore area (averaged over all cell lines) of 5.2 ± 1.3 (N = 20 cell layers) versus 32.5 ± 4.4 (N = 14) µm2 in normal cell layers (mean ± SE, p < 0.0001). Similarly, pore density was 63% lower in glaucomatous versus normal cell layers, with 74.5 ± 12.8 versus 201.9 ± 25.8 pores per mm2 (p < 0.0001). Pore area and density were not statistically different between control cell layers that were not perfused and those perfused in the (opposite) apical-to-basal direction.
These data demonstrate that glaucomatous SC cells have impaired pore-forming ability in vitro. This study establishes the first cell culture model that detects a specific pathology associated with ocular hypertension in glaucoma.
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