1 School of Optometry and Vision Science, University of Waterloo , Waterloo, Ontario, Canada .
J Ocul Pharmacol Ther. 2013 Nov;29(9):794-802. doi: 10.1089/jop.2013.0002. Epub 2013 Aug 1.
To investigate the effect of differently preserved ophthalmic solutions on the viability and barrier function of human corneal epithelial cells (HCEC) using fluorescent dyes.
HCEC monolayers were exposed to the ophthalmic solutions containing benzalkonium chloride (BAK), edetate disodium, polyquad, stabilized oxychloro complex (Purite), sodium perborate, or sorbic acid for 5 min, 15 min, and 1 h. At 24 h after exposure, the cultures were assessed for metabolic activity using alamarBlue. The enzyme activity, membrane integrity, and apoptosis were evaluated using confocal microscopy. Barrier function was assessed using sodium fluorescein.
The metabolic assay showed that the BAK-preserved ophthalmic solutions significantly reduced cell viability after a 5-min exposure compared to the phosphate buffered saline treated control (P<0.05). Using confocal microscopy, the micrographs showed that BAK caused a reduction in the enzyme activity, increased membrane permeability, and decreased the number of viable cells. Ophthalmic solutions with new preservatives had varying time-dependent adverse effects on cell viability, and the preservative-free solution had the least effect on HCEC. Sodium fluorescein permeability showed that HCEC monolayers treated with BAK-preserved solutions were more permeable to sodium fluorescein than those treated by the other ophthalmic solutions (P<0.05).
BAK-preserved solutions had greater adverse effects on metabolic activity, enzyme activity, membrane integrity, cell viability, and barrier function than the solutions that were not preserved with BAK. Our study suggests that BAK-free especially, preservative-free ophthalmic solutions are safer alternatives to BAK-preserved ones.
使用荧光染料研究不同保存方式的眼科溶液对人角膜上皮细胞（HCEC）活力和屏障功能的影响。
将 HCEC 单层暴露于含有苯扎氯铵（BAK）、乙二胺四乙酸二钠、聚季铵盐、稳定氧氯复合物（Purite）、过硼酸钠或山梨酸的眼科溶液中 5 分钟、15 分钟和 1 小时。暴露后 24 小时，使用 alamarBlue 评估细胞代谢活性。使用共聚焦显微镜评估酶活性、膜完整性和细胞凋亡。使用荧光素钠评估屏障功能。
代谢测定表明，与磷酸盐缓冲盐水处理的对照组相比，BAK 保存的眼科溶液在 5 分钟暴露后显著降低了细胞活力（P<0.05）。共聚焦显微镜图像显示，BAK 导致酶活性降低、膜通透性增加和活细胞数量减少。具有新型防腐剂的眼科溶液对细胞活力具有不同时间依赖性的不良影响，而无防腐剂的溶液对 HCEC 的影响最小。荧光素钠通透性表明，用 BAK 保存溶液处理的 HCEC 单层对荧光素钠的通透性大于用其他眼科溶液处理的 HCEC 单层（P<0.05）。
与未用 BAK 保存的溶液相比，BAK 保存的溶液对代谢活性、酶活性、膜完整性、细胞活力和屏障功能的不良影响更大。我们的研究表明，不含 BAK 的溶液，特别是不含防腐剂的溶液，是 BAK 保存溶液的更安全替代品。
