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Richard Zhang, Mijeong Park, Alex Richardson, Nicodemus Tedla, Cintia S De Paiva, Stephanie L Watson, Denis Wakefield, Nick Di Girolamo; An optimized model of dry eye disease using benzalkonium chloride in C57BL/6 mice: effects on the ocular surface. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1411.
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© ARVO (1962-2015); The Authors (2016-present)
Inclusion of the preservative benzalkonium chloride (BAC) in ophthalmic solutions is prevalent, despite the noted potential for inducing dry eye disease (DED). While mouse models of DED that incorporate BAC have assessed its’ effects on the ocular surface, the impact on limbal epithelial stem cells (LESC) is under-investigated. Our investigation aimed to address this knowledge-gap by generating an optimized murine model of BAC-induced DED.
C57BL/6J mice (n=60) were administered topical BAC at concentrations ranging 0.05-0.2% in a time-dependent manner, over 7 days. Clinical and histopathological evaluations included fluorescein staining, corneal smoothness index, and H&E and PAS staining for tissue architecture and goblet cell (GC) density. The effect of a single 10 min BAC exposure (0.00001-0.1%) on cultivated mouse primary corneo-limbal epithelial cells (CLEC) (n=6) was examined using morphological and functional assays including; lactate-dehydrogenase (LDH) release, annexin V/propidium iodide staining and colony forming efficiency (CFE).
Topical 0.1% BAC, dispensed once daily over 7 days, induced punctate fluorescein staining and had no major effect on corneal smoothness. H&E staining revealed disorganized basal and suprabasal corneal epithelial cells with enlarged cytoplasmic halos. Furthermore, PAS+ conjunctiva-derived GCs were decreased by 1.8-fold compared to naive mice (23.41 ± 5.17 vs 41.40 ± 4.01 GC/mm, p<0.0001). Applying BAC at 0.05% had minimal effects on the ocular surface, while 0.2% BAC induced severe corneal epithelial defects. No significant sex-related differences were observed. In cultured mouse CLEC, increasing concentrations of BAC triggered cell contraction, vacuolation and detachment. At 0.01% BAC, LDH release increased after 24 hrs compared to control cells (16.07% ± 3.32%, p=0.017), and elevated necrosis by 4.1-fold immediately after exposure (9.14% ± 5.90% vs 37.93% ± 7.51%, p=0.0062). Finally, concentrations of BAC as low as 0.0001% decreased CFE compared to controls (0.01% ± 0.01% vs 1.27% ± 0.31%, p<0.0001).
This study describes a murine model of DED with clinicopathological features comparable to human disease, and therefore provides the foundations to explore the short and long-term consequences of BAC on the ocular surface, particularly on LESCs, which are critical for maintaining corneal health and vision.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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