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Barbara De Servi, Celine Olmiere, Marisa Meloni, VitroScreen; Hyperosmolar dryness stress on 3D HCE model: a new tool for pre-clinical assessment of tear substitutes. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6031. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Dryness stress model has already been established in 3D human corneal epithelium cell culture (3D HCE). In order to better mimic Tear Dysfunction Syndrome, a new stress model including both physical dryness stress and hyper-osmolarity has been developped in 3D HCE. Relevance and predictability of this model were verified with reference tear substitutes.
The 3D HCE tissues were transferred in controlled conditions (R.H. <40%, T=40°C and sorbitol 0.6M in the medium) during 16h in order to induce stress. At the end of the stress period, products (30μl) were added during 24h on stressed HCE. Different tear substitutes, iso-osmolar or hypo-osmolar, were tested (hyaluronate, trehalose, HP-Guar and carboxymethylcellulose [CMC]). After a 24h-incubation with products, following endpoints have been quantified: Trans-Epithelial Electrical Resistance (TEER), LDH release and gene expression of TNF-α, MMP-9, MUC-4, AQP-3, OCLN and ZO-1. Western blot and immunohistochemistry analysis have been performed on selected samples.
A significant LDH release has been observed in this stressed 3D HCE model, demonstrating an alteration of epithelial barrier functionality and fence properties. After the 24h-incubation, all products reduced the LDH release suggesting a strong efficacy in reducing epithelium damage. Different defence or protective mechanisms can be underlined according to the expression level of the selected genes. A significant up-regulation of AQP3 (water loss blocking), MUC4 (improve mucoadhesion), ZO-1 (ocular surface homeostasis) and LDH release and TEER (barrier function restoring) and decrease in TNF-α (anti-inflammatory action) were observed with trehalose, hypoosmolar hyaluronate and HP-Guar. No effect was observed with CMC on ZO-1 and AQP3.
Thanks to a multiple endpoint approach, a reproducible stress model condition mimicking hyperosmolarity and dryness of corneal epithelium had been established: effects were more pronounced with trehalose and also with hypotonic hyaluronate and HP-Guar. The results obtained with the different tear substitutes confirmed literature data regarding their specific mechanism of action in protecting corneal epithelium surface from hyperosmolarity and dryness induced damages.
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