Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye
Fig 2
Topical CLU protects the ocular surface barrier via an all-or-none mechanism.
The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Dose response experiment. The desiccating stress (DS) protocol was applied for 5 days while also treating with (Left) recombinant human CLU (rhCLU) at the indicated 10-fold dilutions (n = 6), (Middle) recombinant human CLU (rhCLU) at 0.1, 0.3, 0.6, or 1 ug/mL (n = 6), or (Right) recombinant mouse CLU (rmCLU) at 0.3, 0.6, and 1 ug/mL (n = 4). *P<0.0001. (B) Experiment comparing CLU with BSA. The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant human CLU (rhCLU) and BSA, individually or in combination, as indicated. *P<0.0001 (n = 4). (C) Stress reduction experiment. The standard desiccating stress (DS) protocol was applied for 5 days while eyes were also treated with recombinant human CLU (rhCLU) at 0.01, 0.1, and 1 ug/mL. Using a subset (n = 4) of each treatment group the effect of each rhCLU dose on integrity of the ocular surface barrier was confirmed by the fluorescein uptake test at day 5. Then the rest of the mice in each treatment group were subjected for two more days to a more moderate desiccating stress by continuing with the air draft and heat, but omitting scopolamine and CLU treatments. The fluorescein uptake test was then performed on these remaining mice. *P = 0.004 (n = 4); **P = 0.05 (n = 4)