We report here the first measurement, to the best of our knowledge, of the electrical potential generated by the ocular surface epithelium in human subjects, offering a new approach to study ocular surface function and health. This approach was motivated by the experimental use of nasal PD measurements to assess CFTR function in humans with CF
12,17,18 and the development of OSPD in our lab as applied to mice
3,9 and rabbits.
11 Measurement of OSPD in human subjects is technically straightforward. As discussed further below, the baseline OSPD provides a composite measure of the activities of membrane transport proteins in corneal and conjunctival epithelium. The responses to drugs and ion substitution isolate the activities of specific transport processes.
The technical methods used herein are based largely on prior nasal potential difference measurements in humans and OSPD measurements in small animals, although notable additional developments were necessary for OSPD measurement in human subjects. As done for nasal potential difference measurements in humans, an electrical recording system was used that produces accurate OSPD information without significant artifacts, such as junction potentials, and without causing electrical shock. Also, sterile perfusate solutions were used that contain clinical-grade compounds and approved drugs. Electrical contact with the ocular surface was accomplished by everting the lower eyelid to create a small fluid pool into which the tip of a soft, flexible perfusion catheter was immersed under direct slit-lamp visualization, as opposed to in nasal potential difference studies where the perfusion catheter is blindly inserted into the nostril, and the site of contact with the nasal epithelium cannot be directly visualized, thus causing variable electrical tracings. The perfusion catheter tip used herein both delivered specified perfusate solutions and maintained electrical contact with the ocular surface. Fluid overflow created by the continuous perfusion was collected using an absorbent gauze secured to the cheek. Various future adaptations and advances are possible, such as development of a custom perfused contact lens system to study cornea versus conjunctiva selectively and eliminate the need for external positioning of the perfusion catheter tip.
The design of OSPD experiments and the interpretation of data rely on an understanding of the origin of the PD. We previously reported a mathematical model to define quantitatively the influence of the various ion transport processes and paracellular conductance on the OSPD, as well as the effects of perfusate ion substitution maneuvers.
3 The baseline OSPD, which is exterior negative when referenced against the corneal stroma, is a consequence of the active Na/K ATPase at the basolateral membrane of ocular surface epithelial cells. The positive current from the cell interior to the corneal stroma (by exchange of three sodium ions for two potassium ions) produces, under open-circuit conditions, the exterior negative potential. The magnitude of the OSPD is affected by the various passive ion transport processes and paracellular resistance. Ion substitution creates a chemical driven force to bias OSPD values to focus on particular sets of ion transport pathways. For example, the low chloride maneuver used herein, together with ENaC inhibition, produces OSPD values that provide further information on chloride transport pathways, allowing interpretation of the isoproterenol effect in terms of CFTR activation. Although much can be learned by semiquantitative and comparative OSPD measurements, as has been done for nasal PD measurements, quantitative modeling of the OSPD can enhance data interpretation and identify mechanisms that may not be otherwise apparent.
The studies reported here represent assessment of the ion transport function at the ocular surface in live human subjects. Our OSPD measurements are open-circuit recordings of the physiological potential differences generated by ocular surface epithelial tissues. An alternative informative approach, although not possible in vivo, is measurement of short-circuit current across isolated epithelia. Short-circuit current reports the quantity of current that is exogenously driven across an epithelium in order to maintain a zero transepithelial potential difference. Short-circuit current has been measured in various preparations of isolated cornea
19,20 and conjunctiva
11,21,22 from rabbits and amphibia, as well as in corresponding epithelial cell cultures,
23 and has been informative in identifying sodium and chloride transporting pathways. Although electrophysiological measurements in isolated tissue allow precise specification of the composition of solutions bathing the apical (tear-facing) and basolateral surfaces of the epithelium, they do not preserve the in vivo architecture and hormonal/neural environment in live subjects.
The OSPD data implicate CFTR as a major prosecretory mechanism in human ocular surface epithelia. A robust average hyperpolarization of 15.6 mV was seen in response to isoproterenol in a zero chloride solution, which was absent in two CF subjects lacking functional CFTR. This cAMP-dependent OSPD hyperpolarization is similar to that seen in human nasal potential difference measurements
24,25 and in OSPD studies in mice and rabbits.
9,11 In the animal studies, CFTR-selective inhibitors were also used to confirm that the OSPD hyperpolarization reflects CFTR function, although at present no CFTR inhibitor has been approved for human use. The significant role of CFTR as a prosecretory mechanism at the ocular surface supports the use of CFTR activators as potential therapy for dry eye disorders. A triazine small-molecule CFTR activator that is in preclinical development has been shown to prevent and reverse dry eye pathology in experimental animal models.
26,27
An interesting and perhaps unexpected observation was the minimal effect of amiloride, a blocker of proabsorptive sodium channel ENaC, on OSPD, with only a 1.7-mV depolarization produced by a high concentration of amiloride. In similar nasal potential difference measurements in humans, amiloride generally produces a >10-mV depolarization,
16 and in mouse and rabbit OSPD measurements amiloride produced 6-mV and 5-mV depolarizations, respectively.
3,11 The simplest interpretation of this finding is that ENaC plays a minor role as a proabsorptive mechanism in human ocular surface, which would suggest that blockers of ENaC, which have been evaluated for dry eye disorders,
28 may have limited efficacy. However, the amiloride data should be interpreted with caution given our incomplete knowledge of the full repertoire of ion transporters in human cornea and conjunctiva.
Measurement of OSPD in human subjects has a number of potential applications in studying basic ocular physiology, evaluating disease status, monitoring epithelial health, and testing drug candidates. Changes in OSPD in response to selective modulators of transport and signaling mechanisms, together with ion substitution, are informative in defining transport mechanisms and their regulation, as done here for the investigation of ENaC and CFTR. Potassium channels, for example, might be investigated using selective channel modulators and studying effects of potassium ion substitution in the perfusate. OSPD measurements should be informative in quantifying the regulation of ion transport processes in response to disease conditions. For example, whether the expression or function of CFTR is altered in dry eye disorders can be studied, as can potential compensatory upregulation of other prosecretory mechanisms. An intriguing potential application of OSPD is following the recovery of corneal barrier disruption from a variety of conditions, including trauma, ocular prosthetic devices, infection, and neurotrophic keratopathy. Finally, measurement of OSPD can provide a quantitative surrogate measure of the efficacy and pharmacodynamics of drug candidates that target ion transport mechanisms, such as chloride or potassium channel activators and sodium channel inhibitors.