Purpose

Inclusion of the preservative benzalkonium chloride (BAC) in ophthalmic solutions is prevalent, despite the noted potential for exacerbating dry eye disease (DED). Whilst studies incorporating BAC have assessed its’ effects as a mouse model of DED, the impact on limbal epithelia is under-studied. Our investigation aimed to comprehensively assess the impact of different BAC dosing regimens and their suitability as a mouse model of DED.

Methods

C57BL/6J mice (n = 72) were administered topical BAC (0.05–0.2%) over 7 days. Fluorescein staining, corneal smoothness index, and immuno-histological analyses were applied to determine architectural and cellular changes on the ocular surface following BAC treatment. The effect of BAC (0.0001–0.01%) on cultivated primary mouse corneo-limbal epithelial cells (CLECs) (n = 6) was examined using morphological and functional assays.

Results

Whilst 0.2% BAC induced severe corneal epithelial defects, 0.1% BAC dispensed once daily over 7 days, induced punctate fluorescein staining without detriment to corneal smoothness. Histochemical staining revealed disorganized basal corneal epithelial cells with enlarged cytoplasmic halos. Furthermore, PAS⁺ goblet cells were decreased. BAC treatment also modulated K14 expression and distribution within the limbus. In cultured CLEC, BAC triggered cell contraction and vacuolation, increased LDH release and elevated cell necrosis by 4.1-fold. Concentrations of BAC as low as 0.0001% decreased colony formation.

Conclusions

This study describes how exposing C57BL/6 mice to BAC induce some clinicopathological features of DED seen in humans, and therefore provides the foundations to explore the consequences on the ocular surface, particularly on limbal epithelia and its’ stem cells.

中文翻译：

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剂量依赖性苯扎氯铵的毒性赋予眼表上皮变化以干眼病的特征。

目的

尽管在眼科溶液中可能会加重干眼病（DED），但在眼科溶液中普遍包含防腐剂苯扎氯铵（BAC）。虽然结合BAC的研究已评估了其作为DED小鼠模型的作用，但对角膜缘上皮细胞的影响的研究仍不足。我们的研究旨在全面评估不同BAC给药方案的影响及其作为DED小鼠模型的适用性。

方法

C57BL / 6J小鼠（n = 72）在7天内接受了局部BAC（0.05–0.2％）。BAC治疗后，应用荧光素染色，角膜平滑度指数和免疫组织学分析来确定眼表的结构和细胞变化。使用形态学和功能分析检查了BAC（0.0001-0.01％）对培养的原代小鼠角膜-上皮细胞（CLEC）（n = 6）的影响。

结果

0.2％BAC导致严重的角膜上皮缺损，而0.1％BAC在7天内每天分配一次，可诱导点状荧光素染色而不损害角膜平滑度。组织化学染色显示基底角膜上皮细胞杂乱无章，细胞质晕增大。此外，PAS ⁺杯状细胞减少。BAC治疗还调节角膜缘内K14的表达和分布。在培养的CLEC中，BAC触发细胞收缩和空泡化，增加LDH释放，并使细胞坏死升高4.1倍。BAC的浓度低至0.0001％可减少菌落形成。

结论

这项研究描述了如何将C57BL / 6小鼠暴露于BAC诱导人中所见的DED的某些临床病理特征，从而为探讨对眼表，尤其是对角膜缘上皮及其干细胞的后果提供了基础。