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 (Cl2, 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. Influx of sodium at wounds Unwounded corneas had a slightly higher sodium concentration than background concentration. Surprisingly, after wounding, sodium concentration decreased and became lower than back- ground (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). Large and steady chloride influx We then focused on Cl2 because our previous data [6] indicated a significant role for Cl2 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 Cl2 flux at wounds showed a large influx of Cl2, 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 Cl2 influx thus represents electric current (flow of positive charge) flowing out of the wound. In normal artificial tear solution, Cl2 flux quickly reached over 100 nmol/ cm2/sec (5 min after wounding). The Cl2 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 Cl2 concen- tration at the wound. Ion Currents at Cornea Wounds We then tested the effects of aminophylline and ascorbic acid on Cl2 flux, because we showed previously that both drugs significantly increased wound electric currents and wound healing [6]. Aminophyline treatment (10 mM) significantly increased Cl2 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 mM broad-spectrum chloride channel blocker DIDS (4,49-diisothiocyanatostilbene-2,29-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). Hydrogen efflux at wounds 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 0.60560.04 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. Relative contributions of different ion fluxes to the overall wound electric current 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 mainte- nance 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 Cl2 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 (4,49-diisothiocyanatostilbene- 2,29-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. Wounding redistributes and upregulates chloride channel CLC2 at corneal wounds We then investigated the distribution and expression of Cl2 channel CLC2, because it is expressed abundantly and specifically in corneal epithelial tissue [39]. In unwounded corneas, CLC2 PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e17411