ions are pumped in the opposite direction, towards the tear side (Fig. 1) [26–28]. Tight junctions between epithelial cells form a high resistance barrier to minimize ion leakage and maintain the TEPD. Damage to the corneal epithelium disrupts the tight junctional barrier and the TEPD collapses at the wound. The electrically positive TEPD under the surrounding intact epitheli- um thus drives a laterally-oriented electric field running from the intact cornea across the wound edge into the wound center, with the wound the negatively-charged cathode (Fig. 1). Human corneal epithelial cells migrate to the cathode in vitro [15–18] and increasing cornea wound current pharmacologically enhances wound healing [6]. Our own and other studies have shown that wound electric currents have a dynamic time course and appear to be actively regulated [6–8]. We thus hypothesize that wound electric currents are an actively modulated wound response mechanism, i.e. wound tissues actively generate and regulate the wound electric currents. We set to investigate the ionic and molecular mechanisms of the endogenous wound electric currents using a cornea wound model. The corneal epithelium has multiple functions including mechan- ical protection from pathogens and refraction of light onto the lens and retina [29]. Appropriate optical refraction is dependent on a smooth corneal surface and the maintenance of appropriate stromal thickness. As well as the endothelium, the corneal epithelium also contributes to the maintenance of corneal transparency by preventing stromal swelling [30]. The integrity of the epithelial surface is therefore essential for normal vision. Most corneal epithelial wounds are repaired promptly by the migration of epithelial cells at the wound margin towards the center of the wound [31–33]. The initial response in corneal wound healing is the migration of surrounding epithelial cells into the wound bed, which is likely to be guided by the electrical signal [6,10]. Here, we used ion-selective probes to define the ionic mechanisms of wound electric currents. We used pharmacological manipulation of ion transport to alter ion flux, and also fixed the tissues to make sure the fluxes happen only in live tissues. We also determined changes in distribution and expression of chloride transport molecules following wounding. By defining the electro- physiological properties of the cornea we will be able to understand the mechanisms of generation of the endogenous wound electric fields and offer new approaches to manage wound Figure 1. Which ions contribute to the wound electric current? The corneal epithelium transports ions to generate and maintain a transepithelial potential difference (TEPD) of ,25–45 mV. Injury breaks the epithelial barrier and collapses the potential at the wound (left). The positive potential in the surrounding intact epithlium drives ion current flow out of the wound (blue arrows) and forms laterally-orientated wound electric fields (red arrow) with the wound the cathode. doi:10.1371/journal.pone.0017411.g001 Ion Currents at Cornea Wounds healing and corneal injury. Therapeutic modulation of the corneal wound ion flux in order to enhance the endogenous electric field might represent a promising new strategy towards promoting wound healing in patients with chronic non-healing lesions. Methods Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were approved by the University of California, Davis, Institutional Animal Care and Use Committee (protocol # 07- 13078). Eyes and custom-made chamber Using ex vivo corneas allows for easier pharmacological manipulation and ion substitution of the bathing solution. We used this model as the predominant experimental system for comparision with our extensive previous data on rat and human donor cornea wound electric currents [6,34]. The rats (Sprague- Dawley, male, weight 200–224 g) were euthanized by CO2 inhalation and cervical dislocation. The eyes were enucleated and kept in cold BSS until use. A chamber was manufactured to hold isolated eyes for wounding and ion flux measurements. This was a 9 cm Petri dish (Falcon; BD Biosciences, San Jose, CA, USA) with two loops of nickel-chromium wire glued in so that the eye is held immobile but can be rotated and/or tilted to give full access to the whole cornea (Fig. 2A, B). The diameter of each loop is 5–8 mm. The loops hold the eyeball in place so the cornea can be accessed from different angles. Using a scalpel or a trephine, a wound can be made while the cornea is viewed under a dissecting microscope at 620–640. After taking measurements of the unwounded cornea, a wound was made under a dissecting microscope (model SMZ-10, Nikon Instruments Inc., Melville, NY, USA) by scraping the cornea with an ophthalmologic scalpel (Beaver, 22.5u, 4 mm; BD Co., Franklin Lakes, NJ, USA) to remove approximately 2 mm2 of corneal epithelium. Preparation of ion selective microelectrodes Borosilicate glass capillaries without filament (10 cm long, 1.5 mm outer diameter, 1.12 mm inner diameter) were purchased from World Precision Instruments (WPI, Inc., Sarasota, FL, USA; cat #TW150-4). Silanization solution I (cat # 85126), sodium ionophore II cocktail A (cat # 71178), chloride ionophore I cocktail A (cat # 24902), calcium ionophore I cocktail A (cat # 21048), potassium ionophore I cocktail A (cat # 60031) and hydrogen ionophore I cocktail A (cat # 95291) were obtained from Sigma- Aldrich (St. Louis, MO, USA). During measurements, corneas were immersed in an artificial tear solution (BSS+; Alcon Laboratories, Inc., Fort Worth, TX, USA) which contained (mM): 122.18 NaCl, 5.1KCl, 1.05 CaCl2.2H2O, 0.98 MgCl2.6H2O, 2.96 Na2HPO4, 25 NaHCO3, 5.11 D-Glucose and 0.3 glutathione disulphide. Borosilicate glass capillaries were heat-pulled using a Sutter P- 97 electrode puller (Sutter Instrument Company, Novato, CA, USA) with the following settings: heat 470, pull 13, velocity 15, delay 1, to give tips 3–4 mm in diameter. Electrodes were heated in an oven (Model 10; Quincy Lab Inc., Chicago, IL, USA) at 200uC overnight to dehydrate and then rendered hydrophobic by addition dropwise of about 3 ml of silanization solution. Electrodes were kept in the oven until all the silanization solution had vaporized. After cooling, electrodes were stored in an electrode storage jar (WPI; cat # E215) inside a glass dessicator (Fisher Scientific, Pittsburgh, PA, USA; cat # 08-595D) containing PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e17411