Corneal epithelial permeability to fluorescein in humans by a multi-drop method

Abstract

PurposeThe permeability of the corneal epithelium to fluorescein Pdc is an indicator of the health of the ocular surface. It can be measured in a clinical setting by determining the accumulation of fluorescein in the stroma following administration of the dye on the ocular surface. Here we demonstrate a new multi-drop method for the measurement of Pdc by a spot fluorometer.MethodsTwenty-nine healthy participants were recruited for this study. First, a probe-drop of fluorescein (0.35%, 2 μL) was instilled on the conjunctiva. The clearance of the dye from the tears was immediately measured using the fluorometer. Following this, two loading drops (2%; 6 μL each) were administered 10 min apart. Fifteen minutes later, the ocular surface was washed and fluorescence from the stroma Fs was measured. Permeability was calculated using Pdc = (Q x Fs)/ (2 x AUC), where Q is the stromal thickness and AUC is the area under the fluorescence vs. time curve for the loading drops.ResultsAfter the probe drop, the tear fluorescence followed an exponential decay (elimination rate constant; kd = 0.41 ± 0.28 per min; 49 eyes of 29 subjects), but the increase in Fs was negligible. However, after the loading drops, the measured Fs was ~ 20-fold higher than the autofluorescence and could be recorded at a high signal to noise ratio (SNR > 40). The intra-subject variability of kd was insignificant. Since fluorescein undergoes concentration quenching at > 0.5%, the value of AUC for the loading drops was estimated by scaling the AUC of the probe drop. The calculated Pdc was 0.54 ± 0.54 nm/sec (n = 49). A Monte Carlo simulation of the model for the multi-drop protocol confirmed the robustness of the estimated Pdc.ConclusionsThe new multi-drop method can be used in place of the single-drop approach. It can overcome a lack of sensitivity in fluorometers of high axial resolution. The Pdc estimated by the multi-drop method is ~ 11-fold higher than previously reported but closer to the value reported for other drugs with equivalent octanol/water partition coefficient.

Roles unveiled for membrane-associated mucins at the ocular surface using a Muc4 knockout mouse model

“…2 . First, we used fluorescein staining, which primarily measures superficial punctate keratopathy 38 , i.e., damage to individual epithelial cells and the tight junctions between them 39 , 40 . No significant difference in fluorescein staining was observed in Muc4 −/− mice as compared to WT littermates (Fig.…”

Ocular Spot Fluorometer Equipped With a Lock-In Amplifier for Measurement of Aqueous Flare

“…The fluorometer, which is built around a typical slit lamp, was developed to assess tear dynamics, epithelial permeability, endothelial permeability, and aqueous humor dynamics using fluorescein. 7 The instrument can measure fluorescence from any desired spot in the anterior segment of the human eye. The illumination source is a white light-emitting diode (LED; 10 W).…”

Corneal Imaging Techniques for Dry Eye Disease

“…• Corneal epithelial permeability: It can measure this parameter by calculating the rate or extent of fluorescein penetration across the cornea [106]. This can reflect the degree of epithelial injury or dysfunction in DED patients.…”

“…• Tear turnover rate: This can reflect tear production and drainage imbalances, which may contribute to ocular surface irritation and inflammation in DED patients [106,107].…”