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Results: 8

1.
Figure 5

Figure 5. Sodium flux at corneal wounds.. From: Ionic Components of Electric Current at Rat 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).

Ana Carolina Vieira, et al. PLoS One. 2011;6(2):e17411.
2.
Figure 7

Figure 7. Relative contributions of ions to wound current.. From: Ionic Components of Electric Current at Rat Corneal Wounds.

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).

Ana Carolina Vieira, et al. PLoS One. 2011;6(2):e17411.
3.
Figure 8

Figure 8. Distribution and expression of calcium-activated chloride channel-2 (CLC2).. From: Ionic Components of Electric Current at Rat Corneal Wounds.

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 µm. C. In human corneal epithelial cell monolayer, scratch wounding induced increased expression of CLC2 channel mRNA (*P<0.05).

Ana Carolina Vieira, et al. PLoS One. 2011;6(2):e17411.
4.
Figure 1

Figure 1. Which ions contribute to the wound electric current?. From: Ionic Components of Electric Current at Rat Corneal Wounds.

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.

Ana Carolina Vieira, et al. PLoS One. 2011;6(2):e17411.
5.
Figure 4

Figure 4. Potassium flux at corneal wounds.. From: Ionic Components of Electric Current at Rat Corneal Wounds.

A. K+ concentration. Immediately after wounding there was a rapid transient increase of K+ concentration. This initial high concentration decreased over 5–20 min after wounding, reaching a stable lower value. B. K+ flux. There was a large K+ efflux immediately after wounding, which dropped after 20 min. Aminophylline had no effect on the initial peak of efflux, but appeared to enhance efflux at later time points (60–90 min; *P<0.03). C. K+ concentration in high [K+]. In hi-K+ solution (20 mM) the initial peak of K+ concentration was absent and the wound showed a lower K+ concentration (negative values), indicating K+ influx.

Ana Carolina Vieira, et al. PLoS One. 2011;6(2):e17411.
6.
Figure 3

Figure 3. Calcium flux at corneal wounds.. From: Ionic Components of Electric Current at Rat Corneal Wounds.

A. Ca2+ concentration. Calcium concentration at unwounded cornea was slightly above background (0.015 mM). After wounding, Ca2+ concentraion increased until 20 min then plateaued. B. Ca2+ flux. Unwounded cornea showed a small Ca2+ efflux. After wounding, calcium efflux at the wound edge increased to reach a maximum value after 20 minutes. This efflux was maintained for up to 90 minutes. Aminophylline had no effect on Ca2+ flux. C. Fixation. Calcium concentration was measured for 30 minutes to confirm normal efflux. The eye was then fixed. Subsequent measurements showed a drop in calcium concentration to almost zero, even lower than unwounded values.

Ana Carolina Vieira, et al. PLoS One. 2011;6(2):e17411.
7.
Figure 6

Figure 6. Chloride flux at corneal wounds A.. From: Ionic Components of Electric Current at Rat Corneal Wounds.

Cl concentration. Unwounded cornea had a slighly higher Cl concentration than background. Upon wounding, the Cl concentration at the wound edge transiently increased, (5 min) and then decreased, becoming lower than background (negative values) from 10 min onwards. B. Cl 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 µM broad-spectrum chloride channel blocker DIDS (#P<0.03).

Ana Carolina Vieira, et al. PLoS One. 2011;6(2):e17411.
8.
Figure 2

Figure 2. Measuring flux of specific ions at corneal wounds.. From: Ionic Components of Electric Current at Rat Corneal Wounds.

A. Mounting eyes for wounding and probe measurements. A custom-made dish with wire loops holds the eye, leaving the cornea free for measurements and wounding (scale bar 2 cm). Right panel shown wound on cornea stained with fluorescein. B. For measurements, the cornea faces the electrode in the center of the dish. The eye can be rotated and/or the electrode moved to measure at different positions. C. Schematic showing wounded rat cornea with ion selective microelectrode in reference position (‘ref’) and measuring position (‘meas’) at the wound edge. Insert on right shows raw data recording trace (see Methods for details). D, E. Self-referencing mode. D. The electrode moves at low frequency (0.3 Hz) between two points (‘near’ and ‘far’) 30 µm apart at the wound edge. E. Recording trace which has been converted into ion concentration using the calibration trace (see below). F, G. Calibration. F. Calibration of the probe in standard solutions. G. A linear trendline obtained by plotting the amplifier output in mV vs. the logarithm of the ion concentration yields a formula (shown in red) used to calculate ion concentration and, in turn, the actual ion flux.

Ana Carolina Vieira, et al. PLoS One. 2011;6(2):e17411.

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