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 Cl2 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). Wounding increased and redistributed Cl2 channel expression Corneal epithelium has multiple chloride channels (CLCs) and transporters: the CLC gene family, CLCas (Ca2+-activated Cl2 channels), CFTR (cystic fibrosis transmembrane conductance regulator), and Cl2 transporters such as Na+-K+-2Cl2 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 develop- ment 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]. Ion Currents at Cornea Wounds Pharmacological regulation of fluxes of specific ions Aminophylline significantly enhanced chloride influx, and stimulated a sodium efflux (Fig. 5, 6). Aminophylline is a nonspecific phosphodiesterase inhibitor and enhances cAMP levels, increasing Cl2 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 Cl2 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 phosphodiaster- ase, 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+, Cl2 and Ca2+ concen- trations 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 (Cl2, 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 Cl2 transport to enhance wound healing. In summary, increased active transport of Cl2 is a rapid response to injury and forms a significant portion of the wound electric current. Wounding cornea induced fluxes of Ca2+, K + , Na+, Cl2, and H+ with distinct time courses, which can be modulated by fixation or pharmacological treatment. Increased Cl2 flux after wounding correlated with up-regulation and re-distribution of Cl2 channels. Electric signaling at wounds appears to be an active signaling mechanism which may be exploited to induce and accelerate wound healing. Defining the PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e17411