Ion Currents at Cornea Wounds Figure 5. Sodium flux at corneal wounds. A. Na+ concentration. Unwounded cornea had a slighly higher Na+ concentration than background. However, upon wounding, the Na+ concentration at the wound edge was lower than background (negative values). B. Na+ flux. Intact cornea showed a small Na+ efflux. On wounding this became a sodium influx which was maintained for 90 min. Both aminophylline and ascorbic acid induced a large Na+ efflux at later timepoints (5 min and later; *P,0.01). doi:10.1371/journal.pone.0017411.g005 needle (WPI; cat # SSM33A70) with a platinum ball electroplated to the tip. The probe, mounted on a 3-dimensional micromanip- ulator (model H; Line Tool Co., Allentown PA, USA), is vibrated in solution about 50 mm from the cornea surface by a piezoelectric bender (made in-house) at a precise frequency (usually ,150– 200 Hz). If an electric current is present due to ion flux, the charge on the platinum ball fluctuates in proportion to the size of the current. The probe is connected to a lock-in amplifier (model SR530; Stanford Research Systems, Sunnyvale, CA, USA) that locks on to the probe’s specific frequency signal. The probe is calibrated in a current density of 1.5 mA/cm2 at the start and end of experiments. Eyes were treated as above for 20 min in drug at room temperature before measurements. DIDS (4,49-diisothiocya- natostilbene-2,29-disulfonic acid disodium salt hydrate) was obtained from Sigma-Aldrich (cat # D3514). Statistics Data analysis, graphs, linear trendlines and statistical calcula- tions were done with Microsoft Excel. Data are expressed as mean 6 standard error of the mean (SEM). Differences between mean values were compared using Student’s t test. Results We present the data from ion-selective measurement in two ways: 1) as the ion concentration compared to background: higher Figure 6. Chloride flux at corneal wounds A. Cl2 concentration. Unwounded cornea had a slighly higher Cl2 concentration than background. Upon wounding, the Cl2 concentration at the wound edge transiently increased, (5 min) and then decreased, becoming lower than background (negative values) from 10 min onwards. B. Cl2 flux. Unwounded cornea had a small chloride efflux. Wounding induced a large, sustained influx which increased with time. Ascorbic acid reversed the chloride influx, giving a small efflux (**P,0.01), but aminophylline significantly enhanced chloride influx at time-points 5– 60 min (*P,0.04). C. Wound electric current was significantly reduced in the presence of 200 mM broad-spectrum chloride channel blocker DIDS (#P,0.03). doi:10.1371/journal.pone.0017411.g006 or lower at the cornea surface or wound edge; and 2) as ion flux, calculated from data recorded with the probe in self-referencing mode oscillating close to the wound edge. Gradual and persistent increase in calcium efflux at corneal wounds We first measured calcium concentration at corneal wounds. Calcium concentration at unwounded cornea surface was only slightly higher than background level in the artificial tear solution PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e17411