Wound healing in rat cornea: the role of electric currents

Abstract

Human corneal epithelial cells respond rapidly following injury to restore the integrity of the ocular surface. What stimulates and guides cells to move into the wound to heal? One candidate is the wound-induced electric field. Using vibrating probe techniques, we provide detailed temporal and spatial mapping of endogenous electric currents at rat corneal wounds. We find Cl- and Na+ are the major components of electric currents in rat corneal wounds. Na+ is the major component of ionic transport in the resting (nonwounded) rat cornea and of the wound center leakage current, whereas Cl- is a more important component of the endogenous electrical current at the wound edges. Enhancing or decreasing Cl- flow with clinically approved pharmacological agents such as aminophylline, ascorbic acid, or furosemide increased or decreased endogenous wound electric currents, respectively. These changes in wound currents correlated directly with the rate of wound healing in vivo. Thus, pharmacologically enhancing or decreasing wound-induced electric currents increased and decreased wound healing rate, respectively. This may have wide-reaching and novel therapeutic potential in the management of wound healing and may help explain some mechanistic aspects of the effects of some clinically used agents.

Figure 1

The corneal epithelium is a battery. Active pumping of Na⁺ from the apical (tear) side of the cornea across the epithelium and stroma to the basal side (aqueous humor) and of Cl⁻ from basal to apical side establishes a transepithelial potential difference; in mammalian corneal epithelium this is ~25 mV across ~50 μm, with the inside positive. Disruption of the epithelial layer (i.e., wounding) disrupts tight junctions that normally maintain high resistance and separate outside from inside. This generates a short circuit at the wound site, which drains high concentrations of Na⁺ and K⁺ toward the wound from surrounding tissue, and Cl⁻ flows through the low resistance wound site into the basal side. This generates a net flow of positive charge from intact surrounding tissues into the wound site and out of the wound into the tear solution, i.e., an endogenous wound electric current (dashed arrows). This wound electric current can be measured, and pharmacological manipulation by increasing or decreasing Na⁺, K⁺, and Cl⁻ transport would be expected to enhance or decrease the capacity of the batteries, and therefore the wound electric current. If these currents play a significant role in epithelial wound healing, then pharmacological enhancement or depression of them ought to increase or decrease wound healing.

Figure 2

Wounding the corneal epithelium generates significant and persistent outward electric currents at the wound site. A) Positions of the vibrating probe when measuring intact and wounded cornea. Measurements were made at five positions across the cornea, ~1 mm apart (see Materials and Methods for details; scale bar 1 mm). B) Currents measured at intact (open circles, n=8) and wounded (open squares, n=6) cornea. Wound edge currents for individual cornea were derived by taking the average of the left and right edge measurements. Measurements were made shortly (~5–10 min) after wounding. Wound currents were significantly greater than current at normal, intact cornea. In addition, current at the wound edge was significantly greater than at the wound center, indicating substantial ion pumping or leakage at the cut edge of corneal epithelium (*P<0.02; **P<0.003). C) Typical example of the time course of a wound current. Measurements were made at the wound edge, perpendicular to the corneal surface. The current before wounding (0.24 μA/cm²) is shown at time zero (filled circle).

Figure 3

Flows of Na⁺ and Cl⁻ are major components of endogenous electric fields (A–D) and wound edges drive strong currents directed into the wound (E–G). A) Electrical current in normal, intact cornea in sodium-free solution (filled circles, n=10) was significantly less than normal, and in most regions reversed to become an inward current (*P<0.02). B) The current at the wound center in sodium-free tear solution (filled squares, n=8) was significantly increased but at the wound edge significantly reduced (*P<0.03; **P<0.0001). C) Electrical current in normal cornea in chloride-free tear solution (filled circles, n=10) was not significantly different from normal (P>0.1). D) Wound current in chloride-free tear solution (filled squares, n=12) was significantly increased at wound edge and center (*P<0.001). E) Measurements made perpendicular to the corneal surface across wounds (diameter 2 mm, n=4) at ~300 μm intervals. There is a peak of current at the wound edge, but more variable here than inside or outside the wound. F) Measurements made at the center (C1–C3) and edge (E1–E10) of wounds (diameter 2 mm, n=6) at different angles to the corneal surface. Arrows in G show the different current directions measured; thus C1, E3, E8 perpendicular (~90°) to surface, C2, C3, E2, E4, E7, E9 (~60°), E1, E5, E6, E10 (~30°). The largest currents were recorded when the probe was oriented to measure current flowing out of the cut edge of the corneal epithelium (positions E5 and E6; *P<0.03; **P<0.04).

Figure 4

Pharmacological manipulation of cornea epithelial transportation of Na⁺ and Cl⁻ significantly enhances (A–D) or decreases (E, F) endogenous wound electric currents and TCPD (G). A, B) Aminophylline (filled circles, n=7) significantly increased electrical current at normal cornea (*P<0.04) and caused increased current at corneal wounds (filled squares, n=6; *P<0.04.). C, D) Ascorbic acid (filled squares, n=10) induced significantly increased current at wounds. A larger increase was seen at the wound edge (80%) than at the wound center (54%; *P<0.03; **P<0.01). E, F) Furosemide (filled circles, n=8) significantly decreased electrical current in the normal cornea, actually causing a reversal from the normal outward current (open circles) to a small inward current (*P<0.004). Similarly, furosemide (filled square, n=10) significantly decreased current at the wound edge, but had no effect on the current at the wound center (P>0.5; *P<0.05; **P<0.01). G) These drugs had a similar effect on the transcorneal potential difference (TCPD). Control cornea had a PD of ~0.40 ± 0.02 mV (inside positive). After 2 h treatment with aminophylline or ascorbic acid, TCPD was increased significantly (amin: 188%, 1.15±0.1 mV; asc acid: 92%, 0.75±0.05 mV; P<0.01). Furosemide significantly reduced TCPD after 2 h (by 72%; 0.11±0.02 mV, n=4, P<0.01). *P<0.01. H) There was a good correlation (R²+0.984) between healing rate and TCPD

Figure 5

Modulating wound-generated currents pharmacologically can control wound healing rates. A) Healing of circular lesions in the cornea is shown over time. Circular keratectomy (φ=3.5 mm) was performed on corneas at 0 h. Lesions were labeled yellow with fluorescein and are shown here outlined with dots. Increasing the wound current with aminophylline or ascorbic acid increased wound healing rates significantly, whereas reducing the wound current with furosemide significantly decreased the healing rate. B) There was a good correlation (99% significant) between wound current and healing rate. Increasing (with ascorbic acid or aminophylline) or decreasing (with furosemide) the wound edge current pharmacologically (X axis) increased or decreased, respectively, the wound healing rate (Y axis). Regression formula for the correlation between current and healing rate: Y = −1.27X² + 17.55X−9.16, R² = 0.988, Pearson correlation = 0.93. Correlation is significant at 0.01 level. A minimum of 4 experiments was performed in each group. Compared with 30 h drug treatment, applying drugs for only the first 2 h of the 30 h experiment had a smaller effect, though still significantly different from control (P<0.05).