Department of Bioengineering, Imperial College London, London SW7 2AZ, UK.
Exp Eye Res. 2011 Jan;92(1):57-66. doi: 10.1016/j.exer.2010.11.003. Epub 2010 Nov 12.
Aqueous humour transport across the inner wall endothelium of Schlemm's canal likely involves flow through giant vacuoles and pores, but the mechanics of how these structures form and how they influence the regulation of intraocular pressure (IOP) are not well understood. In this study, we developed an in vitro model of giant vacuole formation in human Schlemm's canal endothelial cells (HSCECs) perfused in the basal-to-apical direction (i.e., the direction that flow crosses the inner wall in vivo) under controlled pressure drops (2 or 6 mmHg). The system was mounted on a confocal microscope for time-lapse en face imaging, and cells were stained with calcein, a fluorescent vital dye. At the onset of perfusion, elliptical void regions appeared within an otherwise uniformly stained cytoplasm, and 3-dimensional reconstructions revealed that these voids were dome-like outpouchings of the cell to form giant vacuole-like structures or GVLs that reproduced the classic "signet ring" appearance of true giant vacuoles. Increasing pressure drop from 2 to 6 mmHg increased GVL height (14 ± 4 vs. 21 ± 7 μm, p < 0.0001) and endothelial hydraulic conductivity (1.15 ± 0.04 vs. 2.11 ± 0.49 μl min⁻¹ mmHg⁻¹ cm⁻²; p < 0.001), but there was significant variability in the GVL response to pressure between cell lines isolated from different donors. During perfusion, GVLs were observed "migrating" and agglomerating about the cell layer and often collapsed despite maintaining the same pressure drop. GVL formation was also observed in human umbilical vein and porcine aortic endothelial cells, suggesting that giant vacuole formation is not a unique property of Schlemm's canal cells. However, in these other cell types, GVLs were rarely observed "migrating" or contracting during perfusion, suggesting that Schlemm's canal endothelial cells may be better adapted to withstand basal-to-apical directed pressure gradients. In conclusion, we have established an in vitro model system to study giant vacuole dynamics, and we have demonstrated that this system reproduces key aspects of giant vacuole morphology and behaviour. This model offers promising opportunities to investigate the role of endothelial cell biomechanics in the regulation of intraocular pressure in normal and glaucomatous eyes.
房水通过施莱姆管内壁内皮细胞的运输可能涉及通过巨大空泡和孔隙的流动，但这些结构如何形成以及它们如何影响眼内压（IOP）的调节尚不清楚。在这项研究中，我们开发了一种在受控压力降（2 或 6mmHg）下沿基底到顶端方向（即体内流动穿过内壁的方向）灌注人施莱姆管内皮细胞（HSCEC）的体外巨大空泡形成模型。该系统安装在共聚焦显微镜上进行延时实时成像，并用荧光活细胞染料 calcein 对细胞进行染色。在灌注开始时，在原本均匀染色的细胞质内出现椭圆形的空区，三维重建显示这些空区是细胞的穹顶状外突，形成巨大空泡样结构或 GVL，重现了真正巨大空泡的经典“印章戒指”外观。从 2mmHg 增加到 6mmHg 的压力降增加了 GVL 高度（14 ± 4 对 21 ± 7μm，p < 0.0001）和内皮水力传导系数（1.15 ± 0.04 对 2.11 ± 0.49μl min⁻¹mmHg⁻¹cm⁻²；p < 0.001），但不同供体来源的细胞系之间对压力的 GVL 反应存在显著差异。在灌注过程中，GVL 被观察到“迁移”并聚集在细胞层周围，尽管保持相同的压力降，但它们经常塌陷。在人脐静脉和猪主动脉内皮细胞中也观察到 GVL 形成，表明巨大空泡形成不是施莱姆管细胞的独特特性。然而，在这些其他细胞类型中，在灌注过程中很少观察到 GVL“迁移”或收缩，这表明施莱姆管内皮细胞可能更适应承受基底到顶端的压力梯度。总之，我们建立了一个体外模型系统来研究巨大空泡动力学，并证明该系统再现了巨大空泡形态和行为的关键方面。该模型为研究内皮细胞生物力学在正常和青光眼眼中眼压调节中的作用提供了有前途的机会。
