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Aditi Bauskar, M Elizabeth Fini, Shinwu Jeong; Clusterin (CLU) Ameliorates Pre-existing Ocular Surface Barrier Damage in a Mouse Model of Human Dry Eye. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):304.
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© ARVO (1962-2015); The Authors (2016-present)
The ocular surface (OCS) barrier, which prevents penetration by noxious agents, is disrupted in human dry eye (DE). Using a mouse model for desiccating stress (DS) that mimics DE, we showed that loss of MMP9 activity in knockout mice protects the barrier, as evidenced by reversal of staining with ophthalmic dyes and protection of the barrier against proteolysis (Dursen et al., 2002, IOVS). CLU is a secreted glycoprotein that inhibits the proteolytic activity of MMP9, as well as MMP9-mediated damage to an epithelial barrier (Jeong et al., 2012, Am J Path). CLU expression is reduced at the OCS in the mouse DS model. We showed that topical CLU protects the barrier against DS (ARVO 2014). Here we examine effects of CLU on pre-existing barrier damage.
The secreted form of recombinant human CLU was purchased from R&D Systems and formulated in PBS at 2 ug/ml. To create DS, 6-week old female C57BL/6J mice (n=6 eyes/group) were kept in a room at 800F with <40% humidity and an air draft was applied by placing blowers on either side of each mouse cage. Mice were also treated 4 times a day for 5 days with scopolamine hydrobromide (0.5 mg/0.2ml in PBS) by subcutaneous injection. Mice kept under ambient conditions without scopolamine injection served as baseline (BL) controls. OCS barrier damage was quantified by measuring permeability to FLUORESOFT®-0.35% (FS), an ophthalmic vital dye. Statistical comparisons were done using generalized estimating equations.
5-day DS protocol was applied to create DS and barrier damage was assayed in 2 eyes of each group. BL mice exhibited low FS uptake of 25±3 fluorescence units (FU). In contrast DS-induced mice exhibited high FS uptake of 280±12 FU. The mice were then treated topically, 4 times a day for the next 5 days, with 1 ul of either CLU or PBS control (n=4 eyes/group). Following treatment, OCS barrier damage was quantified by measuring permeability to FS. Eyes stressed for 5 days and treated with PBS alone exhibited FS uptake of 225±14 FU, similar to FS uptake of DS-induced mice, whereas CLU treated mice exhibited FS uptake of 19±11 FU, a drastic reduction compared to PBS treated group (p<0.0001).
We show that topical CLU ameliorates pre-existing OCS barrier damage in a mouse model of human DE. This suggests that CLU might be an effective therapeutic for the treatment of human DE.
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