Ion Currents at Cornea Wounds Figure 7. Relative contributions of ions to wound current. A. Timelapse. Overlaying wound electric current data (red) with compiled ion flux data (flux of all ions combined; blue) shows that electric current increase after wounding is due to an increasing ion flux (mainly chloride). Wound electric current data from Reid et al. [6]. B. Combined ion flux. We compiled ion flux data at 5 min after wounding. Chloride is the major ion contributing to the normal (‘Control’; blue) wound current. Enhancement of wound current by aminophylline (‘Amin.’; red) is mostly due to stimulation of chloride flux, but also partly by reversal of sodium flux (inward to outward). doi:10.1371/journal.pone.0017411.g007 (0.01560.002 mM). After wounding, the calcium concentration increased within the first 20 min. The level then remained stable at around 0.07 mM (Fig. 3A, showing only up to 90 minutes). In longer experiments, this value was maintained, remaining significantly higher than the unwounded value for up to six hours after wounding (P,0.04). We then determined calcium flux at corneal wounds using the electrode in self-referencing mode to calculate the actual calcium ion flux. We saw a small calcium efflux in unwounded corneas. After wounding, the calcium efflux increased steadily until 20 min, stabilizing at a maximum value of 5 pmol/cm2/sec, that was significantly greater than unwounded flux (P,0.02) (Fig. 3B). We previously showed that pharmacological drug aminophyl- line significantly increased cornea wound current and enhanced wound healing [6]. Here, aminophylline (10 mM) had no effect on calcium flux (Fig. 3B), suggesting that aminophylline-induced enhancement of cornea electric current is not due to increased calcium flux. Amiloride, which inhibits the Na+/Ca+ exchanger and voltage-gated Ca+2 channels [38], also had no effect on calcium flux (data not shown). To confirm that calcium efflux was due to active transport, we measured calcium concentration for 30 minutes to confirm normal efflux and then fixed the cornea for 30 min in 4% paraformaldehyde. After fixation and washing, the calcium concentration had dropped to virtually zero (Fig. 3C). Sharp rise and rapid decrease in potassium efflux Potassium concentration at unwounded cornea surface was virtually the same as the background level in the artificial tear solution. However, immediately after wounding there was a rapid and large increase of potassium concentration at the wound edge from ,0 mM to over 1 mM (Fig. 4A). This high level of potassium then dropped quickly, reaching a lower, stable value at ,0.5 mM from 20–90 min after wounding which remained significantly higher than unwounded values. Similarly, wounding also induced a rapid increase in K+ efflux of 150–200 pmol/cm2/ sec. This large efflux of K+ however was short-lived, starting to decrease 20 min after wounding, and reached a significantly lower level of ,50 pmol/cm2/sec (Fig. 4B). To determine if the initial large potassium efflux was due to ion leakage or active transport, we measured K+ efflux after two type of treatment: 1) aminophyline drug, and 2) artificial tear solution with a high potassium concentration (20 mM instead of the normal 5 mM). Aminophylline (10 mM) had no effect on the initial peak, but significantly increased potassium efflux at later time-points (60, 90 min; P,0.03), suggesting that the initial large efflux was leakage, while the later smaller potassium efflux (after 20 min) was due to active transport (Fig. 4B). In high K+ artificial tear solution (20 mM), unwounded cornea surface had virtually the same K+ concentration as that in the background bathing solution. However, immediately after wounding, the initial PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e17411