Research Service, Veteran Affairs Western New York Healthcare System, 3495 Bailey Avenue, Buffalo, NY 14215, USA; Department of Ophthalmology, The State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Ross Eye Institute, 1176 Main Street, Buffalo, NY 14209, USA.
Exp Eye Res. 2013 Oct;115:239-45. doi: 10.1016/j.exer.2013.06.024. Epub 2013 Jul 3.
Corneal endothelial cells form a leaky barrier on the posterior surface of the cornea, allowing influx of nutrient-carrying aqueous humor through the paracellular space and efflux of excess fluid. Corneal edema arises when the density of these non-proliferative endothelial cells declines from endothelial disease or intraocular surgery. The cellular changes occurring at low densities are ill-defined. We therefore investigated the paracellular pathway of corneal endothelial cell monolayers of varying density to determine alterations occurring in paracellular permeability and monolayer morphology. Primary cultures of bovine corneal endothelial cells (BCECs) were passaged onto permeable supports under varying culture conditions to obtain confluent monolayers of <1000, 1000-1999 and >2000 cells/mm(2). Culture growth was monitored by transendothelial electrical resistance measurements. Diffusional permeability to sodium fluorescein, FITC-dextran MW 4000 or FITC-dextran MW 20,000 was measured. Confluent cultures were also analyzed by immunofluorescence localization of the tight junction protein ZO-1 and by transmission electron microscopy. For comparison, we evaluated ZO-1 for low and high density human corneal endothelium. Our results showed that all BCEC cultures grew to the same final transendothelial electrical resistance regardless of final density. In the diffusional permeability assay, permeability increased significantly only for the smallest tracer molecule (sodium fluorescein) in the lowest density monolayers (<1000 cells/mm(2)). ZO-1 immunofluorescence distinctly localized to intercellular junctions in high density BCEC cultures but had more diffuse localization at lower densities. Transmission electron microscopy imaging revealed cells with thinner cross-sectional profiles and longer overlapping intercellular processes at low density relative to high density cultures. Low density human corneal endothelium lacked the diffuse ZO-1 distribution seen in BCECs. Our data supports the hypothesis that barrier integrity is the primary function disrupted in low density corneal endothelial monolayers and contradicts the idea of a linear decline in barrier function with decreasing cell density.
角膜内皮细胞在角膜后表面形成一个渗漏屏障，允许富含营养的房水通过细胞旁间隙流入，并排出多余的液体。当这些非增殖性内皮细胞的密度由于内皮疾病或眼内手术而下降时，就会出现角膜水肿。在低密度下发生的细胞变化尚未明确界定。因此，我们研究了不同密度的角膜内皮细胞单层的细胞旁途径，以确定细胞旁通透性和单层形态发生的变化。将牛角膜内皮细胞（BCEC）原代培养物在不同的培养条件下传代到可渗透的载体上，以获得小于 1000、1000-1999 和大于 2000 个细胞/mm(2)的细胞密度的细胞。通过跨内皮电阻测量监测细胞培养生长情况。测量了对钠荧光素、FITC-葡聚糖 MW 4000 或 FITC-葡聚糖 MW 20000 的扩散渗透性。还通过紧密连接蛋白 ZO-1 的免疫荧光定位和透射电子显微镜分析了融合培养物。为了比较，我们还评估了低和高密度人角膜内皮中的 ZO-1。我们的结果表明，无论最终密度如何，所有 BCEC 培养物都生长到相同的最终跨内皮电阻。在扩散渗透性测定中，仅在最低密度单层（<1000 个细胞/mm(2)）中最小的示踪分子（钠荧光素）的渗透性才显著增加。高密 BCC 培养物中 ZO-1 的免疫荧光明显定位于细胞间连接处，但在较低密度下定位更为弥散。透射电子显微镜成像显示，与高密培养物相比，低密培养物中的细胞具有更薄的横截面轮廓和更长的重叠细胞间过程。低密人角膜内皮缺乏在 BCEC 中所见的弥散 ZO-1 分布。我们的数据支持这样的假设，即屏障完整性是低密角膜内皮单层中首先受到破坏的功能，并且与细胞密度降低导致屏障功能线性下降的观点相矛盾。
