Ionic Components of Electric Current at Rat Corneal Wounds Ana Carolina Vieira1,2., Brian Reid3*., Lin Cao 3 , Mark J. Mannis1, Ivan R. Schwab 1 , Min Zhao1,3 1 Department of Ophthalmology, University of California Davis, Davis, California, United States of America, 2 Department of Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil, 3 Department of Dermatology, University of California Davis, Davis, California, United States of America Abstract Background: Endogenous electric fields and currents occur naturally at wounds and are a strong signal guiding cell migration into the wound to promote healing. Many cells involved in wound healing respond to small physiological electric fields in vitro. It has long been assumed that wound electric fields are produced by passive ion leakage from damaged tissue. Could these fields be actively maintained and regulated as an active wound response? What are the molecular, ionic and cellular mechanisms underlying the wound electric currents? Methodology/Principal Findings: Using rat cornea wounds as a model, we measured the dynamic timecourses of individual ion fluxes with ion-selective probes. We also examined chloride channel expression before and after wounding. After wounding, Ca2+ efflux increased steadily whereas K+ showed an initial large efflux which rapidly decreased. Surprisingly, Na+ flux at wounds was inward. A most significant observation was a persistent large influx of Cl2, which had a time course similar to the net wound electric currents we have measured previously. Fixation of the tissues abolished ion fluxes. Pharmacological agents which stimulate ion transport significantly increased flux of Cl2, Na+ and K+. Injury to the cornea caused significant changes in distribution and expression of Cl2 channel CLC2. Conclusions/Significance: These data suggest that the outward electric currents occurring naturally at corneal wounds are carried mainly by a large influx of chloride ions, and in part by effluxes of calcium and potassium ions. Ca2+ and Cl2 fluxes appear to be mainly actively regulated, while K+ flux appears to be largely due to leakage. The dynamic changes of electric currents and specific ion fluxes after wounding suggest that electrical signaling is an active response to injury and offers potential novel approaches to modulate wound healing, for example eye-drops targeting ion transport to aid in the challenging management of non-healing corneal ulcers. Citation: Vieira AC, Reid B, Cao L, Mannis MJ, Schwab IR, et al. (2011) Ionic Components of Electric Current at Rat Corneal Wounds. PLoS ONE 6(2): e17411. doi:10.1371/journal.pone.0017411 Editor: Che Connon, University of Reading, United Kingdom Received November 9, 2010; Accepted January 31, 2011; Published February 25, 2011 Copyright: 2011 Vieira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health National Eye Institute grant 1R01EY019101 (to MZ and BR). The authors thank the Wellcome Trust for continuous support (068012). This work was also supported in part by Research to Prevent Blindness, Inc., an NSFC grant (30628026), and UC Davis Dermatology Department developmental fund. MZ is also supported by grants from the California Institute of Regenerative Medicine RB1-01417, NSF MCB- 0951199. The funders had no role in study design, data collection/analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: brireid@ucdavis.edu . These authors contributed equally to this work. Introduction At human skin wounds, naturally occurring electric fields and currents were detected many years ago [1–4]. Modern techniques have confirmed and significantly extended our understanding of these endogenous electric fields. Electric currents of ,10–100 mA/ cm2 and fields of ,40–177 mV/mm at wounds in humans and various animals have been measured in recent years with vibrating probes, micro-glass electrodes, micro-needle arrays, and bioelec- tric imagers [5–9]. Endogenously induced electric fields at wounds have emerged as an important signal that plays a critical role in wound healing [10–13]. Chemotaxis, haptotaxis (response to surface-bound molecules), contact guidance, free edges and other factors at wounds are directional cues for cell migration and wound healing. Recently, we demonstrated that electric signals in cell migration during wound healing are far more important than previously thought since they override other directional cues [6,10,14]. Applied electric fields have been shown to stimulate division, migration and differentiation of many types of cells: corneal epithelial cells [15–18], neuronal cells [19–21], fibroblasts [22] and skin epithelial cells [23–24] in a process called galvanotaxis or electrotaxis. The mechanisms of how the electric currents are generated and maintained however remain very poorly understood and largely unstudied. It is generally assumed that the wound electric currents are caused by a short-circuit of the trans-epithelial potential difference (TEPD) at the wound site. In another words, a passive leakage of ions and other charged molecules. The TEPD is a common feature of most epithelia, including skin and corneal epithelia. In the cornea, ions are constantly being transported across the corneal epithelium, which generates and maintains the TEPD with the outer side negative [25]. Sodium ions are actively transported from the tears to the aqueous humor while chloride PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e17411