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Purpose: Dry eye disease (DED) is characterized by loss of tear film stability that becomes self-sustaining in a vicious cycle of pathophysiological events. Currently, desiccation stress (DS) is the dominant procedure for inducing DED in mice, however its' effect on limbal epithelial stem cells (LESCs) has been overlooked. This study aimed to establish a DS model via the use of a novel hardware to investigate the impact on the ocular surface including LESCs., Methods: A mouse transporter unit was customized to generate a dehumidified environment. C57BL/6J mice were exposed to mild DS and injected with scopolamine hydrobromide (SH) or remained untreated (UT) under standard vivarium conditions for 10 consecutive days (n = 28/group). Clinical assessments included phenol red tear-thread test, fluorescein staining and optical coherence tomography assessments. Histopathological and immunofluorescence was used to evaluate tissue architecture, goblet cell (GC) status, lacrimal gland (LG) inflammation and epithelial phenotype on the ocular surface. Whole flat-mounted corneas were immunostained for keratin-14 (K14), then imaged by confocal microscopy and analyzed computationally to investigate the effect of DS on LESCs., Results: Custom modifications made to the animal transporter unit resulted in dehumidified cage relative humidity (RH) of 43.5 ± 4.79% compared to the vivarium 53.9 ± 1.8% (p = 0.0243). Under these conditions, aqueous tear production in mice was suppressed whilst corneal permeability and corneal irregularity significantly increased. H&E staining indicated stressed corneal basal epithelial cells and increased desquamation. DS-exposed mice had reduced GC density (41.0 ± 5.10 GC/mm vs 46.9 ± 3.88 GC/mm, p = 0.0482) and LGs from these mice exhibited elevated CD4 + cell infiltration compared to controls. DS elicited K14 + epithelial cell displacement, as indicated by increased fluorescence signal at a distance of 50-100 μm radially inwards from the limbus [0.63 ± 0.053% (DS) vs 0.54 ± 0.060% (UT), p = 0.0317]., Conclusions: Application of mild DS using customized hardware and SH injections generated features of DED in mice. Following DS, ocular surface epithelial cell health decreased and LESCs appeared stressed. This suggested that potential downstream effects of DS on corneal homeostasis are present, a phenomenon that is currently under-investigated. The method used to induce DED in this study enables the development of a chronic model which more closely resembles disease seen in the clinic., (Copyright © 2021. Published by Elsevier Inc.)

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., Competing Interests: Declaration of competing interest None of the authors have conflicts of interest to declare., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)