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Katelyn Keefer, Sanjay Anant Mahadik, arushi goyal, Ramya Ravindran, Prema Padmanabhan, Sudhir Rachapalli, sudhir ranganath, Uday B Kompella, Sangly P Srinivas; Measurement of Corneal Epithelial Permeability to Fluorescein in Humans by Sequential Multi-Drop Method. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4357.
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Fluorescein permeability (P) of the corneal epithelium is affected in dry eye disease, aging, and diabetes. P is measured by variations of the “fluorometric single-drop” method. This approach suffers from poor repeatability as the measurements are influenced by inner filter effects (IFE) and sensitivity at higher and lower concentrations of the dye, respectively. The objective of this study, therefore, is to establish an improved method for measurement of P based on sequential instillation of topical fluorescein.
First, one drop of 0.35% fluorescein is instilled and its subsequent dynamics in the tears is followed by a custom-built ocular fluorometer. Next, 2 drops of 2% of fluorescein are instilled 10 min apart. 15 minutes later, the ocular surface is washed. Finally, fluorescence in the stroma (Fs) is measured 4 times. Assuming negligible loss into a/c, we derived an equation for permeability: P = (Q * Fs)/ (2*AUC); Q is the stromal thickness and AUC = (2/0.35) * F0/ke. F0 and ke are estimated based on fluorescence vs. time profile following the first drop of 0.35% fluorescein. Proof-of-principle of the different aspects of the approach has been investigated in a cohort of healthy volunteers (n >10).
The fluorescein dynamics in the tears following 0.35% drop could be followed with a high S/N ratio, without IFE, and without overloading the fluorometer (n>10). Analysis of the decay profile showed expected inter-individual as well as drop-to-drop variability in ke and F0. At 15 min following 0.35% drop, Fs was close to the measured background. After multiple drops of the dye at 2%, Fs increased to at least 10-20x the background and could be measured very accurately. Based on average values of ke and F0 and assuming stromal thickness of 475 µm, the calculated range of P varied from 5-15 nm/sec (n=5).
The inherent problem of IFE limits the use of fluorescein at high concentrations. Accordingly, we chose the low concentration drop (i.e., 0.35%) to circumvent IFE and determine fluorescein dynamics in the tears. The high concentration drops (i.e., 2%) surmounted the problem of poor sensitivity and allowed Fs measurements at high precision. The measured values of P, albeit much higher than previous reports, concurs with permeability of many solutes reported for the corneal epithelium.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Protocol for multi-drop approach to determine corneal epithelial permeability
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