Wound healing is essential for maintaining the integrity of multicellular organisms. In every species studied, disruption of an epithelial layer instantaneously generates endogenous electric fields, which have been proposed to be important in wound healing. The identity of signalling pathways that guide both cell migration to electric cues and electric-field-induced wound healing have not been elucidated at a genetic level. Here we show that electric fields, of a strength equal to those detected endogenously, direct cell migration during wound healing as a prime directional cue. Manipulation of endogenous wound electric fields affects wound healing in vivo. Electric stimulation triggers activation of Src and inositol-phospholipid signalling, which polarizes in the direction of cell migration. Notably, genetic disruption of phosphatidylinositol-3-OH kinase-gamma (PI(3)Kgamma) decreases electric-field-induced signalling and abolishes directed movements of healing epithelium in response to electric signals. Deletion of the tumour suppressor phosphatase and tensin homolog (PTEN) enhances signalling and electrotactic responses. These data identify genes essential for electrical-signal-induced wound healing and show that PI(3)Kgamma and PTEN control electrotaxis.

Direct-current (DC) electric fields are present in all developing and regenerating animal tissues, yet their existence and potential impact on tissue repair and development are largely ignored. This is primarily due to ignorance of the phenomenon by most researchers, some technically poor early studies of the effects of applied fields on cells, and widespread misunderstanding of the fundamental concepts that underlie bioelectricity. This review aims to resolve these issues by describing: 1) the historical context of bioelectricity, 2) the fundamental principles of physics and physiology responsible for DC electric fields within cells and tissues, 3) the cellular mechanisms for the effects of small electric fields on cell behavior, and 4) the clinical potential for electric field treatment of damaged tissues such as epithelia and the nervous system.

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It has long been known that cells can be induced to migrate by the application of small d.c. electric fields (EFs), a phenomenon referred to as galvanotaxis. We recently reported some significant effects of electric signals of physiological strength in guiding cell migration and wound healing. We present here protocols to apply an EF to cells or tissues cultured in an electrotactic chamber. The chamber can be built to allow controlled medium flow to prevent the potential development of chemical gradients generated by the EFs. It can accommodate cells on planar culture or tissues in 3D gels. Mounted on an inverted microscope, this setup allows close and well-controlled observation of cellular responses to electric signals. As similar EFs are widely present during development and wound healing, this experimental system can be used to simulate and study cellular and molecular responses to electric signals in these events.

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.

Controlling cell division is fundamental. One environmental cue that exerts profound control over both the orientation and frequency of cell division in vivo is a naturally occurring, woundinduced electric field (EF). Wounds in rat corneas generate endogenous EFs in the plane of the epithelial sheet because the transcorneal potential difference (TCPD; ؉40 mV internally positive) collapses at the wound edge, but is maintained at normal levels at 0.5 mm back from the wound. We manipulated the endogenous EF this creates by using drugs with differing actions. The wound-induced EF controlled the orientation of cell division; most epithelial cells divided with a cleavage plane parallel to the wound edge and perpendicular to the EF vector. Increasing or decreasing the EF pharmacologically, respectively increased or decreased the extent of oriented cell division. In addition, cells closest to the wound edge, where the EF was highest, were oriented most strongly by the EF. Remarkably, an endogenous EF also enhanced the frequency of cell division. This also was regulated by enhancing or suppressing the EF pharmacologically. Because the endogenous EF also regulated the wound healing rate, it may act as one control of the interplay between cell migration and cell division during healing.cleavage plane ͉ electric fields ͉ cell migration ͉ corneal epithelium

Endogenous electric currents generated instantly at skin wounds direct migration of epithelial cells and are likely to be important in wound healing. Migration of fibroblasts is critical in wound healing. It remains unclear how wound electric fields guide migration of dermal fibroblasts. We report here that mouse skin wounds generated endogenous electric currents for many hours. Human dermal fibroblasts of both primary and cell-line cultures migrated directionally but slowly toward the anode in an electric field of 50–100 mV mm−1. This is different from keratinocytes, which migrate quickly to the cathode. It took more than 1 hour for dermal fibroblasts to manifest detectable directional migration. Larger field strength (400 mV mm−1) was required to induce directional migration within 1 hour after onset of the field. Phosphatidylinositol-3-OH kinase (PI3 kinase) mediates cathode-directed migration of keratinocytes. We tested the role of PI3 kinase in anode-directed migration of fibroblasts. An applied electric field activated PI3 kinase/Akt in dermal fibroblasts. Dermal fibroblasts from p110γ (a PI3 kinase catalytic subunit) null mice showed significantly decreased directional migration. These results suggest that physiological electric fields may regulate motility of dermal fibroblasts and keratinocytes differently, albeit using similar PI3 kinase-dependent mechanisms.

Correct guidance of the migration of neural progenitor cells (NPCs) is essential for the development and repair of the central nervous system (CNS). Electric field (EF)-guided migration, electrotaxis, has been observed in many cell types. We report here that, in applied EFs of physiological magnitude, embryonic and adult NPCs show marked electrotaxis, which is dependent on the PI3K/Akt pathway. The electrotaxis was also evidenced by ex vivo investigation that transplanted NPCs migrated directionally towards cathode in organotypic spinal cord slice model when treated with EFs. Genetic disruption or pharmacological inhibition of phosphoinositide 3-kinase (PI3K) impaired electrotaxis, whereas EF exposure increased Akt phosphorylation in a growth factor-dependent manner and increased phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels. EF treatments also induced asymmetric redistribution of PIP3, growth factor receptors, and actin cytoskeleton. Electrotaxis in both embryonic and adult NPCs requires epidermal growth factor (EGF) and fibroblast growth factor (FGF). Our results demonstrate the importance of the PI3K/Akt pathway in directed migration of NPCs driven by EFs and growth factors and highlight the potential of EFs to enhance the guidance of various NPC populations in CNS repair therapies.