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Darren Knight, Bryan Winn; A Multi-step Approach to Evaluating the Validity of a Novel Animal Model for Dry Eye Disease. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):350.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye disease causes tremendous morbidity in the United States and the most prevalent animal models for research use scopolamine to induce dry eye conditions. There are many dry eye models existing in the literature but for any given lab, one must evaluate the model for robustness before attempting experimentation. This can be completed by using quantitative PCR, automated western blot analysis, and polymorphonucleur (PMN) density calculations simultaneously. We hypothesize that a sample model, using 2,4,6-trinitrobenzenesulfonic acid (TNBS), will demonstrate a consistent pattern of increased inflammation at all levels of the measured pathways.
Two Sprague Dawley rats were treated with scopolamine implants, 3 left untreated to serve as controls, and 10 were given a regimen of TNBS. 15 mg of scopolamine were administered daily via implantable osmotic pumps. TNBS was directly applied to the rat’s lids 3 times daily for 7 consecutive days. 6 treated eyes had their conjunctivas and corneas dissected for Hoechst stain and PMN density calculations. Treated eyes had their cytokine expression (IL-1β, IL-6, IL-17, TGFβ, and TNFα) measured using both quantitative real-time PCR and automated quantitative protein identification. The simple Western machine combined capillary electrophoresis as well as immunoassay technology to quantitate target proteins.
In comparison to the untreated rat eyes, conjunctiva PMN density was increased in 2/3 of samples (P=0.0024, 0.0345). TNBS treated corneas demonstrated increased PMN density in 2/3 samples (P= 0.0006, <0.0001), although only 1 sample corresponded with the density-increased conjunctiva. Quantitative PCR demonstrated increased quantities of IL-1β and IL-17 in the rat conjunctivas. TNBS treated rat corneas demonstrated increased expression of TGFβ by real time PCR. Using the automated Western protein analysis, IL-6 was moderately increased in the cornea after treatment with TNBS (P=0.038). Scopolamine did not demonstrate these findings. For both the TNBS and scopolamine models, no significant difference in cytokines was observed in conjunctival tissues.
The TNBS model did not demonstrate a consistent pattern of inflammation on the tested ocular surfaces according to this multi-pronged technique. Future research for novel dry eye disease treatments can utilize this three-pronged approach to evaluate future models for dry eye disease.
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