Ionic components of electric current at rat corneal wounds

To determine if the initial large potassium efflux was due to ion leakage or active transport, we measured K+ efflux after two type of treatment: 1) aminophyline drug, and 2) artificial tear solution with a high potassium concentration (20 mM instead of the normal 5 mM). Aminophylline (10 mM) had no effect on the initial peak, but significantly increased potassium efflux at later time-points (60, 90 min; P<0.03), suggesting that the initial large efflux was leakage, while the later smaller potassium efflux (after 20 min) was due to active transport ( Fig. 4B ). In high K+ artificial tear solution (20 mM), unwounded cornea surface had virtually the same K+ concentration as that in the background bathing solution. However, immediately after wounding, the initial increase in potassium concentration (indicating efflux) seen in normal solution was absent. Instead, we measured a large decrease in K+ concentration, reaching a peak (nearly 3 mM lower than the background concentration) at the wound with the same time scale (∼5 min) as the increase of K+ in normal artificial tear solution. This decrease in K+ concentration indicated an influx of potassium ( Fig. 4C ; negative values). The influx was maintained up to 20 min (the longest time we measured). These results suggest that the initial large potassium efflux was leakage from damaged cells/tissue, which can be proportionally reversed by increasing the external potassium concentration. Also, we would note that leakage of other ions (Cl−, Ca2+, Na+) from damaged cells was presumably negligable due to their low intracellular concentrations (more than ten times less than K+), and so was not detected by our measurements.

Unwounded corneas had a slightly higher sodium concentration than background concentration. Surprisingly, after wounding, sodium concentration decreased and became lower than background (negative values) at the wound edge, reaching the lowest level (1.5 mM) within 5 min ( Fig. 5A ). The sodium concentration difference between the wound and the background started to gradually decrease, but remained significantly lower than background for at least 90 min after wounding.

We then determined the flux of Na+ ( Fig. 5B ). Consistent with the changes in Na+ concentration at wounds, wounding induced significant sodium influx (15 nmol/cm2/sec), which quickly reached a peak in 5 min, and was maintained at the same level for up to 90 min ( Fig. 5B ; negative values show influx).

Because the Na+ influx is a very unexpected finding, we tested the effects of two drugs that we have shown previously to increase wound electric currents [6]. Treatment with either aminophylline or ascorbic acid induced significant Na+ efflux at the wound. Aminophyline treatment resulted in a significantly increased Na+ efflux of 15–20 nmol/cm2/sec (P<0.01, when compared to non-drug treated Na+ flux). Treatment with ascorbic acid resulted in even higher Na+ efflux of 30–50 nmol/cm2/sec (P<0.01). The Na+ efflux persisted up to 90 min ( Fig. 5B ). In separate experiments, we confirmed these results with concentration measurements: wounds in ascorbic acid had an increased sodium concentration rather than the decreased concentration seen in normal solution, confirming the reversal of sodium flux (data not shown).

We then focused on Cl− because our previous data [6] indicated a significant role for Cl− in generating cornea wound electric currents. Intact cornea surface showed the same chloride concentration as background. After wounding there was a very slow but steady decrease in chloride concentration at the wound edge ( Fig. 6A ).

Measurement of the Cl− flux at wounds showed a large influx of Cl−, largest amongst all the ions we measured. By convention, flux of anion equals electrically the same amount of cation flux in the opposite direction. The Cl− influx thus represents electric current (flow of positive charge) flowing out of the wound. In normal artificial tear solution, Cl− flux quickly reached over 100 nmol/cm2/sec (5 min after wounding). The Cl− influx kept rising, reaching a peak of 300 nmol/cm2/sec 60 min after wounding and maintained at this level up to 90 min ( Fig. 6B ). The measurement of chloride influx thus correlated with the decrease of Cl− concentration at the wound.

We then tested the effects of aminophylline and ascorbic acid on Cl− flux, because we showed previously that both drugs significantly increased wound electric currents and wound healing [6]. Aminophyline treatment (10 mM) significantly increased Cl− flux from 100–150 nmol/cm2/sec to over 400 nmol/cm2/sec at 5, 20 and 60 min after wounding (P<0.03) ( Fig. 6B ). Ascorbic acid however appeared to inhibit the chloride influx, although it significantly increased the Na+ efflux (see Fig. 5B , 6B ). To investigate the role of chloride channels in generating cornea wound electric current via active chloride flux, we measured wound electric current with a vibrating probe [7], [37] in the presence of 200 µM broad-spectrum chloride channel blocker DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate). DIDS almost halved the wound current (P<0.03) indicating a large contribution of chloride flux to the overall cornea wound electric current ( Fig. 6C ).

We found a small efflux of protons at unwounded corneas, resulting in a decrease of pH of 0.102 pH units at the surface (data not shown). Immediately after wounding there was a significant increase in proton efflux (P<0.02) causing a reduction of pH at the wound edge of 0.605±0.04 pH units. The pH did not change significantly up to 90 min after wounding (P>0.40), staying at about 0.534 pH units, showing a steady and maintained efflux of protons after wounding.

An influx of negative ions generates an outward flow of positive electric current. Therefore, the progressive increase in ion flux (Cl, Ca2+) found in our time lapse experiments is consistent with our published data that showed a progressive increase and maintenance of a large outward electric current at cornea wounds [6]. Overlaying the compiled ion flux data (all ions) with the wound electric current shows the correlation between ion flux increase and increase of electric current after wounding ( Fig. 7A ).

Comparing the relative contributions of different ion fluxes to the overall wound electric current showed a predominant contribution of Cl− influx, and negligible contributions of Ca2+, K+ or Na+ ( Fig. 7B ). We chose the 5 min timepoint as this is close to the time that the wound electric current data in Reid et al. [6] was measured. Chloride influx was converted from negative to positive, as influx of a negative ion will produce an outward electric current. Sodium influx data was left negative. We can see in Figure 7B that chloride ion flux contributes most to the normal (‘control’) wound electric current. Enhancement of wound current with aminophylline appears to be mainly by stimulation of chloride flux, but also to a lesser extent by reversal of sodium flux (inward to outward). We also showed that broad-spectrum chloride channel blocker DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate) significantly reduced the overall wound electric current, suggesting that chloride channels, and flux of chloride ions, contribute significantly to the generation of cornea wound electric currents. ( Fig. 6C ). Thus we confirm our hypothesis that active transport of chloride ions is the major contributer to cornea wound electric current.

We then investigated the distribution and expression of Cl− channel CLC2, because it is expressed abundantly and specifically in corneal epithelial tissue [39]. In unwounded corneas, CLC2 channels were seen predominantly in the apical layers of the corneal epithelium ( Fig. 8A ). One hour after wounding (scraping away 2 mm2 of epithelium), fluorescence was increased throughout the entire thickness of the epithelium, showing re-distribution and increased concentration of CLC2 channels close to the wound ( Fig. 8B ). Interestingly, the timecourse of up-regulation correlates with the increase of chloride flux, which reached its maximum value one hour after wounding (see Fig. 6B ). We also used quantitative PCR (qPCR) to detect CLC2 channel mRNA in human corneal epithelial cell monolayer before and after wounding. After scratch wounding, CLC2 mRNA expression was significantly increased (P<0.05) ( Fig. 8C ).

We report here the ionic components of naturally-occurring wound electric currents. Our results challenge the conventional assumption that wound electric currents are merely ion leakage due to breakdown of the epithelial barrier that short-circuits the trans-epithelial potential at the wound site. Using ion selective self-referencing microelectrodes, we demonstrate that wounding the cornea resulted in: 1) a Ca2+ efflux that reached a peak of 5 pmol/cm2/sec 20 min after wounding and persisted thereafter; 2) a rapid rise in K+ efflux to 150–200 pmol/cm2/sec that quickly decreased after 20 min following wounding; 3) a surprising influx of Na+ of ∼15 nmol/cm2/sec; 4) a large and rising Cl− influx reaching 300 nmol/cm2/sec that lasted for at least 90 min after wounding. Importantly, the fluxes of different ions had very distinctive time courses, and were abolished or significantly affected by chemical fixation or by pharmacological agents that modulate ion transport. Wounding the cornea resulted in significant re-distribution and up-regulation of CLC2 Cl− channels in the corneal epithelium.

Epithelia generate electric potentials. The healthy cornea maintains a trans-epithelial electrical potential difference (TEPD) of approximately 25–45 mV, with the tear side negative. Similar TEPs have been found in skin, airway epithelium, gut epithelium, kidney epithelium and other types of epithelia that form tight junctions and transport ions [40]–[46]. Ion transport is required to generate the TEPD. In the cornea, for example, chloride ions are transported from the aqueous to the tear side while sodium is transported in the opposite direction (see Fig. 1 ). Tight junctions between epithelial cells form a barrier of high electrical resistance which helps to maintain the TEPD. Damage or injury to the corneal epithelium breaks this barrier, causing a ‘short-circuit’ at the wound, which produces large and maintained electrical currents [6]. Current flow through adjacent tissue and out of the wound generates endogenous electric fields orientated towards the wound center which stimulate cells to migrate into the wound to promote wound healing [10]. Accumulating experimental evidence suggests strongly that this endogenous electric field guides cells at the wound edge to migrate into the wound center to heal the wound [4], [21], [24], [25], [47]–[49]. Electric fields of physiological strength have very strong guidance effects that override other directional cues in guiding cell migration during wound healing [6], [10].

The specific ionic fluxes that account for ion transport in corneal epithelia vary among species. For example, in amphibians ion transport in corneal epithelial cells is essentially chloride flux [50], [51]. In mammals, it is accounted for by approximately equal contributions of inwardly directed sodium transport towards the stroma and outwardly directed chloride transport into the tears [30]. We previously postulated that the cornea wound electric current might consist primarily of sodium ions [6], since tears contain high levels of sodium (80–170 mM) [52]. However, in this study we found a small efflux of sodium in unwounded corneas and an influx of sodium after wounding. This may suggest that, in rats, sodium ions do not significantly contribute to the net electric current at cornea wounds. The corneas of different species have different mechanisms of ion transport. In rabbits, for example, sodium is transported inward, which is surprising since this does not seem to favor water removal from the stroma [53], [54].

Wounding initiates many signaling cascades. The most well studied include release of growth factors and cytokines and reorganization of extracellular matrix. Cells at the wound actively regulate the release of growth factors and modify the extracellular matrix. With a vibrating probe, we have measured large outward electric currents at cornea wounds, which had a dynamic time course and could be affected by drugs that regulate ion transport, suggesting that the wound electric fields and currents are an active response of cells and tissues to injury [6], [34].

In this study we used ion selective microelectrode measurements to demonstrate that individual ion species, Ca2+, K+ and Na+, also have distinct dynamic time courses at corneal wounds. Calcium efflux started small and progressively increased until reaching a stable value after around 20 minutes. This stable value lasted at least 90 minutes ( Fig. 3A, B ). Chemical fixation eliminated the calcium flux ( Fig. 3C ). These results suggest that calcium flux is likely to be actively-regulated after wounding. In contrast, potassium ions had a large transient efflux immediately after wounding which dropped after the first 20 minutes ( Fig. 4A, B ). The intracellular concentration of potassium is approximately 140 mM, while the concentration of potassium in the bathing artificial tear solution (BSS+) is only 5 mM, and in tears has been variously estimated at 6–42 mM [52]. In high potassium solution (20 mM), the initial peak seen in normal BSS was absent and the flux direction was reversed, suggesting that the initial large potassium efflux is due to leakage from damaged cells at the wound edge.

An unexpected finding is the influx of sodium ( Fig. 5 ). It had been previously been assumed that Na+ accumulation at the basal side is a major component generating the transepithelial potential difference. Drug treatment with aminiophylline or ascorbic acid reversed the direction of Na+ flux, which may contribute partially to the effects of this drug on wound electric current and healing rate that we have seen previously [6]. In the corneal epithelium, pH can affect barrier function and ion transport [55]. Immediately after wounding, we saw an increase in proton efflux and a subsequent decrease in pH at the wound edge. Decreased extracellular pH has been shown to slow epithelial chloride secretion from stroma to tears [56]. This might partly explain the influx of chloride we saw that progressively increased after wounding.

The large influx of chloride at the wound edge represents a most significant finding ( Fig. 6A, B ). There are four novel features: 1) the Cl− influx increased progressively after wounding with a different pattern than Ca2+, K+ and Na+; 2) the flux is the largest of all the ions measured: ∼300 nmol/cm2/sec, very much larger than the flux of Na, Ca2+ or K+; 3) aminophylline, which we have shown to increase wound electric current and wound healing [6], significantly enhanced chloride influx; and 4) broad-spectrum chloride channel blocker DIDS significantly reduced wound electric current ( Fig. 6C ).

Corneal epithelium has multiple chloride channels (CLCs) and transporters: the CLC gene family, CLCas (Ca2+-activated Cl− channels), CFTR (cystic fibrosis transmembrane conductance regulator), and Cl− transporters such as Na+-K+-2Cl− co-transporter [57], [58]. Some CLCs have been characterized in both rat and rabbit corneal epithelial cells, and more recently in human cornea [39]. CLCa2 plays a role in human corneal epithelial stratification and cell adhesion. During epithelial stratification, hCLCa2 expression level increases, suggesting a role in the growth of multi-layered corneal epithelia during both natural development and tissue cultivation [57], [59]. Here we saw that after wounding CLC2 expression was re-distributed and up-regulated at cornea wound edges ( Fig. 8 ). Re-distribution and up-regulation of CLC2 expression and an increase in chloride transport imply that modulation of chloride ion transport is an active response to wounding that plays a role in cellular guidance and migration as well as tissue stratification, which occurs later in the wound healing process, after epithelial cells have covered the entire wound bed [29].

Aminophylline significantly enhanced chloride influx, and stimulated a sodium efflux ( Fig. 5 , 6 ). Aminophylline is a nonspecific phosphodiesterase inhibitor and enhances cAMP levels, increasing Cl− transport. Cyclic AMP has been implicated in the active transport of chloride in frog [60] and rabbit corneal epithelia [30]. The transparency of frog corneas is influenced by aminophylline, suggesting the participation of chloride in corneal deturgescence [60]. Aminophylline also causes a significant increase in outward electric current in wounded cornea, and significantly increases the rate of corneal wound healing [6]. The effects of aminophyline on Cl− influx and Na+ efflux seen here correlate well with our previous observations of aminophylline-enhanced cornea wound currents and wound healing [6].

The corneal epithelium concentrates ascorbic acid to a level that is higher than in any other tissues [61]. Ascorbate is transported into the posterior chamber by the ciliary body epithelium, enters the aqueous humor, diffuses through the corneal endothelium into the stroma and is finally concentrated by the epithelial cells [62], [63]. Ascorbic acid absorbs UV radiation and may provide photo protection to the cornea, lens and retina [53], [64]. Here, ascorbic acid inhibited chloride influx, but caused a large sodium efflux ( Fig. 5 , 6 ). The decrease in chloride influx might be compensated for by an increase in sodium efflux to maintain the TEP. Ascorbic acid stimulates active transport of both sodium and chloride across amphibian cornea, probably by inhibiting phosphodiasterase activity and thus increasing the cyclic AMP content of the corneal epithelium [62], [65]. Ascorbic acid is known to aid in healing and significantly decreases the incidence of corneal ulcerations and perforations in alkali burned eyes [66]. We demonstrated that ascorbic acid enhances the transcorneal potential difference, wound electric currents and wound healing rate in the cornea [6]. In addition to the effect on phosphodiasterase, ascorbic acid may increase Na+ efflux through mechanisms yet to be defined.

These results suggest a very important aspect in wound electric signaling, that is active transport of ions rather than passive flux or leakage. Mechanistically, they point to specific ionic mechanisms. Leakages of ions, into or out of tissues, following breakdown of the epithelial barrier and cell membranes, is undoubtedly an important initial response after wounding. This leakage is however very short-lived, rising to peak in a couple of minutes and returned to almost baseline 20 minutes after wounding. The leakage of K+ from damages cells is driven by concentration gradients. Intracellular K+ concentration is about 30 time higher than that outside the cell, while intracellular Na+, Cl− and Ca2+ concentrations are much lower than in the extracellular space. The subsequent concentration gradients dictate that the majority of leakage from damaged cells is K+. In contrast, we saw flux of other ions (Cl−, Ca2+) increasing steadily after wounding, suggesting that active transport of these ions is dynamically regulated as part of the wound response. Clinically, these results point to better targeted manipulation of the long term wound electric signaling through enhancement of Ca2+ and Cl− transport to enhance wound healing.

In summary, increased active transport of Cl− is a rapid response to injury and forms a significant portion of the wound electric current. Wounding cornea induced fluxes of Ca2+, K+, Na+, Cl−, and H+ with distinct time courses, which can be modulated by fixation or pharmacological treatment. Increased Cl− flux after wounding correlated with up-regulation and re-distribution of Cl− channels. Electric signaling at wounds appears to be an active signaling mechanism which may be exploited to induce and accelerate wound healing. Defining the ionic and molecular foundations of wound electric field generation offers potential novel approaches to modulate wound healing, e.g. to develop eye-drops targeting ion transport to aid in the challenging management of non-healing corneal ulcers.