University of Houston, College of Optometry, The Ocular Surface Institute (TOSI), United States.
University of Houston, College of Optometry, The Ocular Surface Institute (TOSI), United States; University of Texas Health Science Center at Houston, Center for Clinical and Translational Sciences (CCTS), United States.
Cont Lens Anterior Eye. 2024 Oct;47(5):102187. doi: 10.1016/j.clae.2024.102187. Epub 2024 May 17.
Midday fogging (MDF) occurs when particulate material accumulates in the fluid reservoir (FR) beneath scleral lenses (SL), and its impact on epithelial cells is unknown. This study examines the in vitro pro-inflammatory effect of the FR on human corneal epithelial cells in varying degrees of MDF.
Normal SL neophytes were recruited to wear SL 8 h daily for 4 days. Following 8 h on days 1 and 4, optical coherence tomography (OCT) images were acquired for MDF quantification using ImageJ, and the FR was collected. FR samples from the same eye were later pooled, diluted 2-fold and applied on human telomerase-immortalized corneal epithelial (hTCEpi) cells cultured on Terasaki microwell plates. Tumor necrosis factor (TNF)-α and culture media were used as positive and negative controls, respectively. After a 30-minute treatment, the nuclear factor-kappa B (NF-κB) pathway was measured by NF-κB-p65 immunofluorescence and images were analyzed with ImageJ. Pearson's correlation was conducted to determine the association between median nuclear fluorescence and MDF.
Fourteen FR samples with a mean volume of 22 ± 16 µl were tested. Mean MDF severity following 8 h of SL wear was 25 ± 17 units (range 7 - 64). The median nuclear fluorescence (NF-κB-p65 translocation) in cultured hTCEpi cells ranged from 31.43 to 45.16 while the negative and positive controls were 44.71 ± 1.72 and 108.77 ± 68.38, respectively. Although a potential positive trend between MDF and median nuclear fluorescence was observed, Pearson's correlation analysis revealed no significant association (r = +0.48, P = 0.09).
The results suggest that the FR can trigger NF-κB-p65 translocation in hTCEpi cells, which may be associated with MDF severity. This study introduces the use of Terasaki microwell plates for immunofluorescence studies of the FR. The technique is simple, minimizes sample usage, and does not require expensive instrumentation.
当颗粒物质在巩膜镜片（SL）下方的液体储器（FR）中积聚时，会发生中午起雾（MDF），但其对上皮细胞的影响尚不清楚。本研究检查了 FR 对不同程度 MDF 的人角膜上皮细胞的体外促炎作用。
招募正常 SL 新佩戴者，每天佩戴 SL 8 小时，持续 4 天。在第 1 天和第 4 天的 8 小时后，使用 ImageJ 采集光学相干断层扫描（OCT）图像以量化 MDF，收集 FR。随后将来自同一只眼的 FR 样本汇集、稀释 2 倍并应用于 Terasaki 微孔板上培养的人端粒酶永生化角膜上皮细胞（hTCEpi）。肿瘤坏死因子（TNF）-α和培养基分别用作阳性和阴性对照。治疗 30 分钟后，通过核因子-κB（NF-κB）-p65 免疫荧光测量 NF-κB 途径，并使用 ImageJ 分析图像。进行皮尔逊相关性分析，以确定中位数核荧光与 MDF 之间的关联。
共测试了 14 个 FR 样本，平均体积为 22 ± 16 µl。佩戴 SL 8 小时后，MDF 严重程度的平均值为 25 ± 17 个单位（范围 7-64）。培养的 hTCEpi 细胞中的中位数核荧光（NF-κB-p65 易位）范围为 31.43 至 45.16，而阴性和阳性对照分别为 44.71 ± 1.72 和 108.77 ± 68.38。尽管观察到 MDF 与中位数核荧光之间存在潜在的正趋势，但 Pearson 相关性分析显示无显著关联（r = +0.48，P = 0.09）。
结果表明，FR 可以触发 hTCEpi 细胞中 NF-κB-p65 的易位，这可能与 MDF 严重程度有关。本研究介绍了使用 Terasaki 微孔板进行 FR 的免疫荧光研究。该技术简单，最大限度地减少了样本用量，且不需要昂贵的仪器。
