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ARUSHI GOYAL, Deepti Talele, Prema Padmanabhan, RR Sudhir, Sanjay Mahadik, Pavani Murthy, Uday B Kompella, Sudhir Ranganath, Sangly P Srinivas; Reevaluation of the epithelial permeability to fluorescein by the multi-drop method. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2614.
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Previously we had proposed a novel multi-drop method for measurement of epithelial permeability to fluorescein (Pdc). The method, developed for a spot fluorometer (non-scanning), had revealed a much higher Pdc than previously reported. Therefore, we have examined the method with more subjects to reevaluate the key variabilities of the protocol.
One drop of 0.35% fluorescein (2 µL; probe drop) is instilled on the conjunctiva, and subsequent clearance from the tears is measured using the fluorometer. Subsequently, two drops of 2% fluorescein (6 µL; loading drops) are administered 15 min apart. Fifteen minutes after the second loading drop, the ocular surface is washed with saline and stromal fluorescence (Fs) is measured. Assuming negligible loss of fluorescein into the anterior chamber from the stroma, we estimate permeability by Pdc = (Q×Fs)/(2×AUC), where Q is the stromal thickness and AUC is the area under the fluorescence vs. time curve for the loading drops. AUC is obtained by scaling exponential decay of the tear fluorescence following the probe drop. Q is measured by OCT. Experiments have been performed in a cohort of human subjects with no known factors affecting the health of their corneal epithelium.
After administration of the probe drop, fluorescein clearance in tears followed a single exponential decay. Repeat measurements of the rate of fluorescein clearance and fluorescence at time t = 0 did not show significant variability. At 15 minutes after the probe drop, fluorescence in the stroma was close to baseline measurements. However, after the two loading drops, measured fluorescence in the stroma (i.e., Fs) was significantly greater than the baseline. Corneal staining of the epithelium was also not noticed. In the cohort of 21 subjects (36 eyes), the mean value of Pdc was 1.81 + 1.30 nm/sec. The results of a Monte Carlo simulation, assuming a normal distribution of measured parameters, also confirmed the robustness of our estimated value of Pdc.
Estimated Pdc by the multi-drop method is at least ~30x times greater than values reported based on the conventional single-drop method. This new estimate of Pdc, however, is consistent with the permeability of a wide variety of solutes reported for the corneal epithelium in humans and animal models.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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