Measurement of endothelial permeability to fluorescein with a Spot Fluorometer

Abstract

Purpose : The corneal endothelium (CE) is dysfunctional in Fuchs Endothelial Corneal Dystrophy. CE restricts fluid influx into the stroma from the a/c (referred to as the barrier function). It also overcomes the residual fluid leak by an active fluid transport from stroma into the a/c. In this study, we have assessed a protocol for measurement of the barrier function of the endothelium using a new ocular fluorometer in human subjects

Methods : Two % fluorescein drops were instilled on the conjunctiva 3-4 times 10 min apart at 11 AM. Subsequently, the loss of the dye from the stroma was measured. Specifically, we measured fluorescence from the stroma and a/c every 30 min over a period of 3 hours using a custom-built ocular fluorometer. These data were then used to calculate to permeability to fluorescein (Pf) as a measure of barrier function of the CE using the formula: Pf = qcm * alpha * Fs/(rac * Fs –Fa), where qcm = mean corneal thickness (measured by OCT; = 1.25 * CCT), alpha is the decay rate constant obtained by fitting stromal fluorescence vs. time curve into a single exponential decay model, Fs is the log mean of stromal fluorescence over 2 hrs of decline, Fa is the log mean of a/c fluorescence over 2 hrs of decline, and rac is the ratio of concentration of fluorescein in the a/c to that of cornea when they are in equilibrium (assumed to be 0.9).

Results : The new fluorometer, which is based on a slit lamp, is sensitive and enables measurement of fluorescence from any spot within or on the surface of the eye with a depth resolution < 300 µm. Following topical fluorescein, the stromal and a/c fluorescence showed a significant increase initially but was followed by a continuous decline over 2-3 hours. The a/c measurements were relatively noisy but could be used to calculate Pf. In normal subjects, the calculated Pf was 19.2 x10^-4 + 9.77 x10^-4 cm/min (n = 10). We also observed that advanced FECD patients show have much higher permeability than previously reported.

Conclusions : The fluorometer is suitable for measurement of Pf in healthy and FECD patients. The method is based on the assumption that fluorescein loss from the stroma occurs largely across the CE into the a/c. This is an acceptable assumption if the superficial epithelium is a barrier for fluorescein but we have that in severe FECD epithelial barrier property is compromised. Hence, the method should be used with caution in advanced FECD

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.