Department of Ophthalmology, University of Rostock, Doberaner Strasse 140, Rostock, Germany.
Curr Eye Res. 2012 Nov;37(11):982-9. doi: 10.3109/02713683.2012.700754. Epub 2012 Jun 25.
Trehalose has been shown to protect epithelial cells from desiccation damage in cell culture and the murine dry eye model. The present study evaluates the protective role of trehalose in reconstructed human corneal epithelium (3D-HCE) during desiccation.
The morphology of 3D-HCE was examined using in vivo an ex vivo confocal laser-scanning microscopy (CLSM). The 3D-HCE was desiccated with or without pre-treatment with trehalose. Evaluation of protective role of trehalose was conducted using different in vitro cell viability assays and CLSM. Tissue thickness for each condition was determined by optical coherence tomography (OCT).
3D-HCE tissue revealed similar features with human cornea at histological level. After desiccation the percentage of living cells was only 32% in 3D-HCE tissue without pre-incubation and 98% in trehalose-pre-incubated tissue, as shown by a cell viability assay. These findings were confirmed by using a Live-Dead assay. Also, the confocal immunofluorescence analysis revealed much better preservation of tight junctions in trehalose-pre-treated tissue.
CLSM and an in vitro cell viability assays could be successfully used for the characterization of 3D-HCE tissue. We demonstrated the protective role of trehalose using reconstructed corneal epithelium (3D-HCE), which mimics HCE and has the potential to become a valuable model in ophthalmic research.
海藻糖已被证明可在细胞培养和小鼠干眼症模型中保护上皮细胞免受干燥损伤。本研究评估了海藻糖在重建的人角膜上皮（3D-HCE）干燥过程中的保护作用。
使用体内和体外共聚焦激光扫描显微镜（CLSM）检查 3D-HCE 的形态。在有或没有海藻糖预处理的情况下对 3D-HCE 进行干燥。使用不同的体外细胞活力测定和 CLSM 评估海藻糖的保护作用。通过光学相干断层扫描（OCT）确定每种条件下的组织厚度。
3D-HCE 组织在组织学水平上显示出与人角膜相似的特征。干燥后，未经预孵育的 3D-HCE 组织中活细胞的百分比仅为 32%，而经海藻糖预孵育的组织中为 98%，这是通过细胞活力测定得出的。使用 Live-Dead 测定也证实了这一发现。此外，共聚焦免疫荧光分析显示，海藻糖预处理组织中紧密连接的保存情况要好得多。
CLSM 和体外细胞活力测定可成功用于 3D-HCE 组织的表征。我们使用重建的角膜上皮（3D-HCE）证明了海藻糖的保护作用，该组织模拟了 HCE，有可能成为眼科研究中的有价值模型。
