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 through- out 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). Discussion 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 Figure 8. Distribution and expression of calcium-activated chloride channel-2 (CLC2). A. In unwounded cornea, CLC2 channels were concentrated in the superficial epithelial cells (arrow). B. One hour after wounding, fluorescence was present throughout the entire thickness of the epithelium, showing re-distribution and increased concentration of CLC2 channels. Scale bars 50 mm. C. In human corneal epithelial cell monolayer, scratch wounding induced increased expres- sion of CLC2 channel mRNA (*P,0.05). doi:10.1371/journal.pone.0017411.g008 Ion Currents at Cornea Wounds 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 Cl2 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 Cl2 channels in the corneal epithelium. Trans-epithelial potential difference, wound electric fields and directional signals for cell migration 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 evi- dence 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]. Cl2 influx constitutes the major part of wound electric currents 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, PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e17411