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Ionic components of electric current at rat corneal wounds.

Vieira AC, Reid B, Cao L, Mannis MJ, Schwab IR, Zhao M - PLoS ONE (2011)

Bottom Line: After wounding, Ca(2+) efflux increased steadily whereas K(+) showed an initial large efflux which rapidly decreased.Pharmacological agents which stimulate ion transport significantly increased flux of Cl(-), Na(+) and K(+).Injury to the cornea caused significant changes in distribution and expression of Cl(-) channel CLC2.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, 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, Ca(2+) 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 Cl(-), 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 Cl(-), Na(+) and K(+). Injury to the cornea caused significant changes in distribution and expression of Cl(-) 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. Ca(2+) and Cl(-) 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.

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Chloride flux at corneal wounds A.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).
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pone-0017411-g006: Chloride flux at corneal wounds A.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).

Mentions: Ion-selective micro-electrode data are expressed two ways. First we show the concentration difference between the reference position in the bath solution, and the measuring position close to the cornea surface or wound edge (see Fig. 2C). For example, Figure 3A shows calcium concentration at unwounded cornea (time 0) and at wound edge (wound edge has a relatively higher concentration). We then present data of actual ion flux, calculated from data recorded with the electrode in self-referencing mode as it oscillates close to the cornea surface (see Fig. 2D, E). For example, Figure 3B shows calcium flux before and after wounding, showing a steady increase of calcium efflux (outward ion flow) after wounding. For each ion measured, the concentration difference data, and ion flux data, correlated. For example, higher ion concentration at wound edge = ion efflux (outward flow). Note that in all timelapse graphs (Figs. 3–7), the measurements made at intact cornea surface before wounding are shown at time zero.


Ionic components of electric current at rat corneal wounds.

Vieira AC, Reid B, Cao L, Mannis MJ, Schwab IR, Zhao M - PLoS ONE (2011)

Chloride flux at corneal wounds A.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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045448&req=5

pone-0017411-g006: Chloride flux at corneal wounds A.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).
Mentions: Ion-selective micro-electrode data are expressed two ways. First we show the concentration difference between the reference position in the bath solution, and the measuring position close to the cornea surface or wound edge (see Fig. 2C). For example, Figure 3A shows calcium concentration at unwounded cornea (time 0) and at wound edge (wound edge has a relatively higher concentration). We then present data of actual ion flux, calculated from data recorded with the electrode in self-referencing mode as it oscillates close to the cornea surface (see Fig. 2D, E). For example, Figure 3B shows calcium flux before and after wounding, showing a steady increase of calcium efflux (outward ion flow) after wounding. For each ion measured, the concentration difference data, and ion flux data, correlated. For example, higher ion concentration at wound edge = ion efflux (outward flow). Note that in all timelapse graphs (Figs. 3–7), the measurements made at intact cornea surface before wounding are shown at time zero.

Bottom Line: After wounding, Ca(2+) efflux increased steadily whereas K(+) showed an initial large efflux which rapidly decreased.Pharmacological agents which stimulate ion transport significantly increased flux of Cl(-), Na(+) and K(+).Injury to the cornea caused significant changes in distribution and expression of Cl(-) channel CLC2.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, 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, Ca(2+) 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 Cl(-), 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 Cl(-), Na(+) and K(+). Injury to the cornea caused significant changes in distribution and expression of Cl(-) 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. Ca(2+) and Cl(-) 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.

Show MeSH
Related in: MedlinePlus