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First published online September 15, 2004
doi: 10.1242/10.1242/jcs.01341

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Nerve regeneration and wound healing are stimulated and directed by an endogenous electrical field in vivo

College of Life Sciences and Medicine, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK

View larger version (50K): [in a new window]   Fig. 1. The origin and effects of the wound-induced EF in rat cornea. (A) Simplified schematic representing the stratified rat corneal epithelium as a single layer of cells resting on a basement membrane and covered with a tear film. Cells are connected by tight junctions (brown squares), which form the major electrical resistive barrier of the epithelium. Intact mammalian corneal epithelium transports Na+ inwards (pink) and Cl- outwards (blue) and this separation of charge establishes a transcorneal potential difference (TCPD) of around +40 mV (internally positive). At a wound (asterisk), ionic currents flow underneath the cell layers, pass through the lesion and have return paths in the tear fluid layer. This short-circuits the TCPD, which drops to 0 mV at the wound, but the TCPD remains at normal values 500 µm from the wound. This establishes a steady, laterally oriented EF (red arrows) directed towards the wound (Chiang et al., 1992). (B) The TCPD varies as a function of distance from the wound edge and we manipulated this pharmacologically. Control plot (red) represents directly measured data, with 100% of the normal TCPD present 500 µm from the wound edge (Chiang et al., 1992). The effects of various drugs are shown. For example, prostaglandin E2 (PGE2) increased the TCPD more than fourfold (425%). The drop off with distance to the wound is inferred by comparison to the no drug control graph. The x axis maps onto the diagrams in (A) and (C). PGE2 enhances chloride efflux, aminophylline and ascorbic acid inhibit phosphodiesterase breakdown of cAMP, which also enhances Cl- efflux, AgNO3 increases both early Na+ uptake and later Cl- efflux. Ouabain inhibits the Na+/K+ ATPase and furosemide inhibits the active Cl- efflux. (C) Detail of the wound in (A). The EF vector is defined as the flow of positive charge (red arrow). Current flows out the wound (asterisk), which acts as a cathode. Several cell behaviours close to the wound are directed by the wound-induced EF. Cell migration and nerve sprouting (green) are directed cathodally, towards the wound edge and mitotic spindles (green) align parallel to the EF. Cell division therefore also is oriented by the naturally occurring EF (Song et al., 2002).

View larger version (53K): [in a new window]   Fig. 2. The wound-induced EF controlled wound-healing rates. (A) Circular lesions in rat cornea (3.5 mm in diameter at 0 hour) are labeled yellow with fluorescein and outlined with dots and shown at 10, 20 and 30 hours after wounding. Control (top row) healing is incomplete by 30 hours. Enhancing the wound-generated EF with PGE2 (second row), caused wound closure before 30 hours. Aminophylline, AgNO3 and ascorbic acid also enhanced wound-healing rates (not illustrated). Collapsing the EF with ouabain (bottom row) or furosemide (not shown) slowed wound healing which was incomplete by 30 hours. Neomycin also enhanced the wound-healing rate up to 30 hours after a wound (third row). (B) Corneal epithelial wound-healing rates varied with the wound-generated EF. Enhancing (with ascorbic acid, AgNO3, aminophylline and PGE2) or reducing (with ouabain and furosemide) the TCPD and therefore the steady wound-induced EF pharmacologically (x-axis), respectively enhanced and reduced the wound healing rate (µm/hour, mean±s.e.m. of 0-10 hours post wound). Regression formula for the correlation between TCPD and wound healing rate is y=34+0.12x-0.0002x2, Pearson correlation=0.91, correlation is significant at 0.01 level. A minimum of four experiments was performed in each group. Neomycin did not affect the TCPD, but enhanced the wound-healing rate significantly.

View larger version (178K): [in a new window]   Fig. 3. Early response of corneal nerves during wound healing. Anti-ßIII tubulin staining for corneal nerves at 0 hour (A) and 24 hours (B) after wounding. There were fewer nerves detectable at 0 hour and most were randomly distributed. At 24 hours after wounding, nerve numbers were increased dramatically. Nerve sprouts run parallel to each other and are oriented perpendicularly toward the wound edge (bottom). Bars, 50 µm.

View larger version (98K): [in a new window]   Fig. 4. Nerves grow perpendicularly towards a wound edge. (A-I) Enhancing or reducing the wound-induced EF increased or decreased perpendicular sprouting. FITC anti-GAP-43 and propidium iodide staining for whole-mounted, wounded cornea. By 24 hours, the frequency of perpendicular nerve sprouts doubled in untreated corneas (compare A and D). Enhancing the wound-generated EF with PGE2 (E) and aminophylline (F), more than doubled the frequency of perpendicular nerve sprouts at 16 hours compared to 16 hour control (C). At 24 hours, perpendicular nerve sprouting was abolished in corneas treated with ouabain, d-tubocurare and neomycin (G, H and I, respectively). All images are representative projections from three separate experiments. The corneal wound edge is at the bottom margin. (J) Perpendicular nerve sprouting towards a wound edge is proportionate to the wound-induced EF. Enhancing or reducing the TCPD and therefore the steady wound-induced EF pharmacologically (x axis), respectively enhanced and reduced the polarization index of nerve sprouting angles (y axis). On the x axis, control TCPD is shown as 100%, and pharmacologically modified TCPDs are compared with this. On the y axis, the PI for controls is shown as 100%, and the PIs for other treatment groups are scaled accordingly. Regression formula for the correlation between TCPD and proportion of perpendicular nerves is y=-3.7+1.3x-0.0017x2, Pearson correlation=0.93, correlation is significant at 0.05 level (two-sided, P=0.02). The number of wounded, whole-mounted corneas was between four and eight for each treatment and the total number of nerve sprouts was between 46 and 186. Bars, 100 µm.

View larger version (171K): [in a new window]   Fig. 5. Classification of four types of new nerve sprouts. (A-D) Nerves in whole-mounted, wounded corneas are green (FITC anti-GAP-43) against a red propidium iodide-stained background. Four types of new nerve sprouts were seen: (A) New growing sprouts from intact nerves; (B) New growth from basal layers; (C) New growth from a cut stump (arrowhead shows the cut end of the nerve); (D) complex nerves with no clear orientation. New sprouts from intact existing nerves formed the majority of the total new nerve population and were the only type of nerve changing dramatically with time and different drug treatment (A). Arrows in (A,C), cut edge at 0 hours. Bars, 50 µm.

View larger version (24K): [in a new window]   Fig. 6. Directed nerve sprouting as a function of time and distance from the wound (EF strength). The polarization index of nerve sprouting angles was calculated for new sprouts from intact nerves. From 16 to 24 hours, new sprouts oriented significantly with time (compare 16 and 24 hour control PI values, P<0.01). In control corneas, new sprouts within 0-250 µm of wound edge were oriented more perpendicularly than those at 250 µm-1 mm, where the EF strength drops off (P<0.01 for 16 hours; P<0.05 for 24 hours). At 16 hours, all four drugs that increased the EF (PGE2, aminophylline, AgNO3 and ascorbic acid) enhanced perpendicular orientation up to twofold (compare 16 hour control PI with 16 hour PGE2 and aminophylline PI values, both P<0.01). At 24 hours, reducing EF with ouabain or furosemide significantly reduced nerve orientation (compare 24 hour control PI with 24 hour ouabain or furosemide PI values, P<0.01 or P<0.05 respectively), and this was more obvious at 0-250 µm (compare 24 hour ouabain 0-250 µm PI with 250 µm-1 mm PI, P<0.05). Tubocurare and neomycin also significantly reduced nerve orientation (compare 24 hour control PI with 24 hour tubocurare and neomycin PI values, P<0.05 and 0.01, respectively).

View larger version (64K): [in a new window]   Fig. 7. Percentage of curved nerves from intact branches 0-16 hours after wounding. (A) All drugs that increased the EF (PGE2, aminophylline, AgNO3 and ascorbic acid) enhanced the nerve turning response in the period 0-16 hours after a wound (compare 16 hours control value with percentages in the four treatments at 16 hours, P<0.05). This nerve turning response roughly doubled during the 16-24 hour period after wounding in controls (compare 16 hour control value with 24 hour control value, P<0.01). Reducing the EF with ouabain or furosemide, significantly suppressed the nerve turning response at 24 hours (compare 24 hour control value with 24 hour ouabain or furosemide value, P<0.01). Preventing transduction of the EF with tubocurare or neomycin also significantly suppressed this turning response at 24 hours (P<0.01, and P<0.05 respectively). (B) Representative photomicrograph of nerve turning. New sprouts that turned through more than 10° to project toward the wound edge were counted at either 16 hours or 24 hours after wounding. The image is a projection from three separate experiments and shows that nerves turned in either direction to come to project perpendicular to the wound edge along the EF vector. Arrow shows the wound edge at 0 hour. Bar, 50 µm.

View larger version (64K): [in a new window]   Fig. 8. The number of new nerve sprouts was increased by the wound-induced EF. From 16 to 24 hours after wounding, new sprouts grew from the intact nerves (compare 16 and 24 hour control values, P<0.01). Pharmacologically enhancing the endogenous EFs also significantly increased new sprouts as early as 16 hours (compare 16 hour control values with values for all other treatments at 16 hours, P<0.01). Ouabain, furosemide, neomycin and d-tubocurare all reduced nerve sprout numbers 24 hours after wounding (when compared with 24 hour control value).

View larger version (71K): [in a new window]   Fig. 9. The number of new sprouts is a function of time and distance from the wound (EF strength). (A) At the wound edge (0-250 µm), new sprouts more than doubled in control corneas over the period from 16 hours (D) to 24 hours (E) (P<0.01), indicating that the EF effect was most potent at the leading edge between 16 and 24 hours. (B) At 500 µm-1 mm from the wound edge, there was no change in sprout number between 16 hour (F) and 24 hour (G) control values (P>0.05). Pharmacologically enhancing the endogenous EF, significantly increased nerve sprouts at 16 hours, for instance, compare the 16 hour control cornea (H) with the 16 hour aminophylline-treated cornea (I), (P<0.01), indicating that for all four drug-enhanced EFs the effects had penetrated to a depth of 1 mm between 0 and 16 hours. (C) At 250-500 µm from the wound edge, suppressing the endogenous EF with ouabain (K) or furosemide and preventing EF transduction with neomycin or tubocurare all significantly reduced nerve sprout numbers at 24 hours compared to 24 hour control values (J) (P<0.01), indicating that the EF effect had penetrated to a depth of at least 500 µm. Bars, 50 µm.