Nature's Electric Potential: A Systematic Review of the Role of Bioelectricity in Wound Healing and Regenerative Processes in Animals, Humans, and Plants
In injured peripheral nerves, recovery is often disappointing, due to poor regenerative capacity of neurons. Promisingly however, electrical stimulation enhances nerve regeneration, targets re-innervation and improves functional recovery in animal and human models (Gordon, 2016). 3. Post-stroke rehabilitation. In stroke treatment, neuromuscular electrical stimulation (NMES) is applied to peripheral nerves or motor points of target muscles in order to restore motor function. With NMES application recovery is improved compared with controls in numerous (although not all) studies involving upper and lower limb and hand rehabilitation (reviewed by Knutson et al., 2015, 2016).
Myocardial infarction results in wounding to heart tissue, and is a leading cause of death globally (Bui et al., 2011). Strategies for regeneration of the heart include stimulating the remaining live cells in the heart, and cardiac tissue engineering to provide replacements for diseased or damaged tissues (Ptaszek et al., 2012; Thavandiran et al., 2013). Electrical stimulation has been demonstrated to promote a remarkable ultrastructural organization of cardiomyocytes in animal models (Radisic et al., 2004). However, the effects of electrical field stimulation in human cardiac tissue engineering remain unreported in the literature and recapitulation in vivo of the key signals that influence cells to develop appropriate structure and associated function remains elusive (Thavandiran et al., 2013).
A unique pattern of electric currents is found in the eye lens, with large outward currents (20–40 μA/cm2) at the lens equator and inward currents at the anterior and posterior poles (Robinson and Patterson, 1982). This may have a basis in the spatial distribution of Na+/K+-ATPase activities in the lens epithelial cells (LECs; Candia and Zamudio, 2002). Following cataract extraction, an artificial lens is often implanted, on the premise that the human lens has no capacity for regeneration. Unfortunately, after cataract surgery, aberrant proliferation sometimes results in posterior capsule opacification (PCO; Zhao et al., 2012).
However, when the anterior capsule is preserved, the lens does indeed regenerate. LECs in vivo migrate either toward the equator, or toward the front of the lens. Given that applied EFs direct the migration and differentiation of cultured LECs, this suggests that the endogenous lens EF may be directing the LEC migration and differentiation in vivo. Moreover, LECs differ in their response according to their location in relation to the EF vector. PCO occurs due to inappropriate proliferation and migration of LECs, possibly as a result of the normal electric control of migration and proliferation being disrupted following surgical lens removal. It has been proposed that preservation of the whole capsule with its attached anterior lens preserves the electrical signals which may be critical to lens regeneration (reviewed by Zhao et al., 2012).
A TEP also exists in the retinal pigment epithelium (Miller et al., 1978; Griff et al., 1985; Quinn and Miller, 1992). Degenerative diseases of the retina, such as macular degeneration, exhibit numerous channelopathies, in which there is a reduced number or malfunction of membrane ion channels (Wimmers et al., 2007; Zhao et al., 2012). The importance of the retinal TEP is suggested by inactivation of a gene that abolishes the TEP in mice, which leads to a phenotype similar to retinitis pigmentosa in humans (Bosl et al., 2001).
The wound-generated voltage changes are transduced into second-messenger cascades and changes of gene expression via, for instance, activation of voltage gated ion channels, and voltage regulation of phosphorylation, which lead to functional changes in proteins (Okamura and Dixon, 2011; reviewed by Tseng and Levin, 2013). The first genes involved in this signal transduction have been identified by Zhao et al. (2006). The directional migratory response of cells depends on the opposing, “compass sensing” activity of two enzymes: PIP3 kinase (Phosphoinositide 3-kinase) is polarized to the leading edge and the lipid phosphatase PTEN (phosphatase and tensin homolog) to the trailing edge (Stewart et al., 2007). Genetic disruption of PIP3 impairs the KC electrotactic migration, and deletion of the PTEN gene enhances it (Zhao et al., 2006).
Since both bioelectric and pharmacological interventions have shown a measure of success in clinical wound healing applications, promising future therapies could employ combined electric-pharmacological strategies (Martin-Granados and McCaig, 2014). For instance, discovery of the genes (encoding PI3K and PTEN) that regulate electrically driven wound healing have led to the possibility of PTEN inhibition as a pharmacological target to enhance wound healing (Zhao, 2007). Other combined interventions are also promising. For instance, ES can contribute to improved healing of pressure ulcers compared with standard wound care, but may be most effective in combination with other techniques such as vacuum-assisted closure (VAC) therapy and the application of platelet-rich plasma (PRP; Kuffler, 2015).
A number of pilot studies feature promising ultrasound applications. For instance, non-healing diabetic foot ulcers treated with ultrasound three times per week exhibited significant wound area reduction (Yao et al., 2014), by reducing inflammatory cytokines and facilitating tissue regeneration. In venous stasis ulcerations, ultrasound was found to improve wound healing by stimulating cell proliferation (Samuels et al., 2013). Improved wound healing using ultrasound is reported for critical limb ischemia (Kavros et al., 2007). An ultrasound variant involving surface acoustic wave (SAW) patch therapy led to improved tissue oxygenation in ischaemic feet (Rosenblum et al., 2014). Treatments involving combined ultrasound and electric field stimulation (CUSEFS) appear to complement and supplement one other (Avrahami et al., 2015), leading to improved closure of intransigent diabetic foot ulcers and venous leg ulcers. The ultrasound stimulates fibroblasts to create collagen, and collagen deposition becomes more organized, providing an improved weave for epithelialization. The electric stimulation pulls the fibroblasts and collagen together, resulting in a tighter and more ordered collagen weave overall. However, further high quality trials are required, as indicated by the mixed success (based on only low quality evidence to date) of electrotherapy and ultrasound rotator cuff disease treatments (Page et al., 2016). Moreover, the success rate of treatments such as PEMF appears to vary dramatically due to differing parametric settings and treatment strategies (Shi et al., 2013).
In relation to wound healing technologies, application of an electric field in clinical situations increases the rate and success of wound repair, with the largest evidence base in favor of unidirectional ES (Koel and Houghton, 2014). However, the underlying mechanisms are still not well-understood (Koppes et al., 2014). Regarding regeneration, although electrical field stimulation invokes regeneration in animal models, its application for human regenerative tissue technologies remains in its infancy, hindered by the elusive nature of the key regenerative mechanisms and pathways which could be translated into clinical outcomes (Eming et al., 2014). Moreover, knowledge that has been acquired of genetic and protein data implicated in regeneration remains to be coupled to how regenerative growth emerges from cellular activities (Lobo et al., 2012). In relation to this, three findings emerge as promising but little studied areas of research to shed light on these problems, as follows.
Bioelectric parameters (such as Vmem and electric fields; Section Bioelectric Mechanisms of Wound Healing and Regenerative Processes in Animals and Plants—An Overview) predict and correlate both with growth/patterning and wound healing/regeneration. Yet large gaps remain. For instance, what is the relationship between the two, viz. the electric fields associated with external morphology and those associated with wounding and regeneration? For instance, voltage patterns conform to complex tree morphology (Rosene and Lund, 1953); and yet the bioelectric parameters of tree wound calli (which appear to regulate their development in relation to the natural form of the tree) remain unknown.
A hypothesis emerging from this data is that these bioelectric features bear instructive information (Levin, 2013) which are involved in ultimately conforming the wound tissues to the morphology of the organism. This is consistent with the hypothesis that electric currents play a role in Xenopus tadpole tail regeneration (Reid et al., 2009). Similarly, in earthworm regeneration, each segment has a specific electric potential. Segments are added by regeneration until the total endogenous field potential is that of a normal full-sized worm (Kurtz and Schrank, 1955; Levin, 2009), suggesting that bioelectric cues are essential to the regeneration process, which fundamentally recapitulates the generation of body form. Secondly, if electric fields are indeed implicated in regeneration processes, voltages imposed within the physiological range can be expected to stimulate regeneration. This is indeed the case. Applied fields drive regeneration of limb morphology in amphibians, birds and mammals (Section The Role of Ionic Flux Changes in Highly Regenerating Animals). With electric field stimulation, crop plant cell cultures exhibit improved growth rate and shoot formation (Goldsworthy and Rathore, 1985), including plants recalcitrant toward regeneration (Gill et al., 1987). Applied MFs stimulate kiwi shoot regeneration from callus (Rugini et al., 1991). Pulsed magnetic field (PMF) applied to soybean seedlings stimulate a higher frequency of shoot and root regeneration (Radhakrishnan and Kumari, 2013).
Magnetic fields (MFs) applied to the commercially important Paulownia explants display increased regeneration percentages, shoot numbers, and chlorophyll content, with the authors speculating that MFs directly interact with Ca2+- channel proteins and the movement of Ca2+- into the cytosol, which in turn may lead to the production of cytokinin, an adenine-derived plant hormone which stimulates protein synthesis and cell division (Çelik et al., 2008). From these evidences it appears that bioelectric signals are indeed providing instructive information in the initiation, proliferation, differentiation and integration of new tissues, and the orchestration of the morphogenetic form appropriate for the wound region and characteristic to the organism.
The discovery of ion channels and pumps such as V-ATPases, found ubiquitously in animals and plants, and their role in activating downstream morphogenetic cascades is particularly salient. Since bioelectric determinants of morphology are largely invisible to modern molecular profiling techniques, Levin and colleagues advocate mapping between spatiotemporal ionic profile patterns and tissue patterning outcomes (Levin, 2012b). This same general rationale can be applied not only for morphogenesis, but also for regeneration. This is a strategy aimed at discovering the precise identification of the bioelectric information that activates the regeneration processes.
However, the generative processes underlying morphogenesis are still not well-characterized, and remain elusive, with numerous reviews focusing on this problem (e.g., Wardlaw, 1970; Raff and Kaufmann, 1983; Goodwin, 1985, 2000; Gordon and Parkinson, 2005; Levin, 2012a; Newman and Linde-Medina, 2013; Tyler, 2014). Voltage mapping provides some of the most tractable and exciting methodologies to test the above hypothesis, i.e., if there is indeed a relationship between the bioelectric features of wounds and the morphology of the organism, and potentially provides a handle on any mechanisms in common. Moreover, a recent application of voltage mapping indicates its promise for clinical interventions. On the basis that transmembrane potential (Vmem) disruptions lead to brain malformations in amphibian models, which can be rescued by voltage modulations (Pai et al., 2015; Figure 3), such knowledge could be translated into procedures to apply ion channel drugs. Such drugs, already approved for human use, could promote the required changes in Vmem properties to improve the wound healing response. There is growing evidence to show the relevance for such voltage modulation as a therapeutic target, based on comparison of animal and human model systems. For instance, a microarray genome-wide analysis combined with pharmacologically induced ion channel depolarization leads to numerous transcriptional responses, which are conserved between diverse model systems (frog, axolotl, and human), in in vivo and in vitro contexts, and affecting various disease networks in common, suggesting a conserved set of responses (Pai et al., 2016).
However, currently only ES applications are in clinical use. The clinical application of voltage modulations remain in their infancy, applied to elucidating bioelectric pathways only in the animal model system. Such studies hold great promise for informing a much more effective control of regenerative growth in biomedical settings (Adams and Levin, 2013).
What causes the current switch from positive to a negative polarity in highly regenerating animals? Although, the molecular basis for this reversal is unknown, such reversals can be found elsewhere in the animal and plant kingdoms, allowing its basis to be explored in more accessible model systems, such as the plant root tip. Thus, another testable hypothesis is that this phenomenon in regenerating animals may have mechanisms in common with the plant root apex transition zone. The electric current peaks and oscillates in this zone (Masi et al., 2009) and is also synchronous with oscillating gene expression patterns (Moreno-Risueno and Benfey, 2011). It would be interesting, therefore, to further probe the relationship between the current reversals and various gene-expression patterns and their place in the instructional pathways guiding growth in this region.
Given the link between underlying bioelectric processes common to morphogenesis, wound healing and regeneration demonstrated above, another relevant model system to probe the basis for ion flux changes is the limb bud, in which there is a current switch from inward to outward three stages before limb bud formation, which predicts the site of the limb-forming region (Altizer et al., 2001).
In recalcitrant wounds, it has been suggested that the endogenous EFs are askew, compromised or absent (Kloth, 2014; Fraccalvieri et al., 2015). Moreover, diabetic skin has a lower TEP and therefore smaller wound currents (Ionescu-Tírgovişte et al., 1985). The TEP in normal skin also declines with age (Nuccitelli et al., 2011), which could be a contributory factor in age-related delayed wound healing and impaired microcirculation. These ideas are summarized in a model (Figure 4). The figure shows the field lines have a directionality indicated by arrows, and the current also flows in the direction of the field lines. Compared with normal skin wounds (Figure 4A) the diabetic skin, aged normal skin and recalcitrant wounds (Figure 4B) exhibit a diminished electric field and associated wound current, which could explain the impaired wound healing in these various patients. Further research is required to experimentally verify this model which is still largely unexplored. This is in spite of promising findings, such as in aged skin, in which electrotherapy (Section Clinical Applications) can promote wound closure and reverse the ischaemia (reviewed by Gould et al., 2015). This is potentially of great importance if the endogenous currents are indeed aberrant, because this could be a focus for more informed therapeutic interventions. For instance, Cl− and Ca2+ fluxes are dynamically regulated during the wound response in normal epithelium (Vieira et al., 2011) but have not yet been elucidated in non-healing ulcers. Therapeutic interventions could then target and normalize impaired specific ion fluxes that underlie effects such as diminished cell migration into the wound bed. This rationale is further confirmed by empirical findings from a diabetic mouse model, in which the cornea expresses weaker wound currents.
This correlates with impaired wound healing and, importantly, is attributed at least in part to diminished flux of Cl− ions and lower expression of anion transporters (Shen et al., 2016).
This kind of knowledge could also inform the design of next generation electroceutical wound care devices. These could incorporate graphene which, with its extreme electrical conductivity and sensititivity to magnetic fields (Salcman, 2014), may have numerous applications for non-contact electric stimulation (Mattei and Rehman, 2014), by providing sufficient field strength to control cell interactions (Heo et al., 2011).
More generally, in wound healing endogenous currents are known to activate several major signaling cascades, promote the directional migration of many cell types, and are involved in all three stages of wound healing (namely inflammation, new tissue formation, and remodeling; Martin-Granados and McCaig, 2014). Thus, it still remains to be explored precisely which of these processes are impaired in pathologies exhibiting aberrant voltage patterns, and their causal relationship in either generating or reflecting the pathologies. Moreover, although the human ability to regenerate finger tips correlates with the existence of an endogenous current emerging from the stump, the role of this current still remains to be discovered. The ability to regenerate digit skeletal structures in humans is restricted to the terminal phalangeal bone (Neufeld and Zhao, 1995) and, at present, there remain insurmountable technical problems for regrowing human digits and limbs in the laboratory (Shieh and Cheng, 2015). The closest experimental counterpart is the rodent model. However, although the anatomical and molecular events of digit regeneration in mice are becoming increasingly understood (Han et al., 2008), the role of bioelectric cues in these processes still largely awaits to be investigated, even in this mammal model. Such studies could help to close the gap between the clinical need and the basic science.
Plants and plant extracts appear to have therapeutic potential, with 21,000 plants so listed by the World Health Organization (WHO). Many of these are targeted to wound healing or neuroprotection (Ghosh and Rangan, 2012; Sharma et al., 2013. For instance, Alpinia oxyphylla fruits afford neuroprotective effects against glutamate-induced wounding to cortical neurons Yu et al., 2003). Most studies to date are applied on animal models such as mice and rats, and further human studies are required to determine their target modes of action and effectiveness. A third testable hypothesis is proposed, that such phytopharmaceuticals act by augmenting or restoring aberrant wound currents in recalcitrant wounds.
Certain organisms have extremely long lifespans, such as multi-centennial rougheye rockfish, Sebastes aleutianus (lifespan ~200 years; Cailliet et al., 2001); the Greenland shark (Somniosus microcephalus; at least 272 years; Nielsen et al., 2016); and certain bivalve molluscs such the clam Arctica islandica, with a lifespan of up to 507 years (Butler et al., 2013). In such organisms virtually all of their bodily functions ranging from brain to heart and immune system are exquisitely preserved (Austad, 2010), and thus their study may help to unravel the primary causes and molecular patterns of age-related cellular degeneration (Philipp and Abele, 2009). Moreover, wound healing and regenerative capacities are known to be extraordinarily elevated in such animals (Philipp and Abele, 2009), so it would be interesting to ascertain whether this can be attributed to any bioelectric characteristics (such as Vmem). These are as yet undocumented. Such data could also be contrasted with that of aberrant wound patterns (Section Aberrant and Quiescent Voltage Patterns) to seek clues as to mechanisms which in aberrant patterns are impaired, and in exceptionally long lifespans may be augmented.
Longevity in plants can exceed even that of extremely long-lived animals, with Pinus longaeva known to live from 4,713 to 5,062 years (Lanner and Connor, 2001; http://www.rmtrr.org/oldlist.htm) However, compromised regeneration in aged trees is a serious problem in horticulture, limiting the propagation of elite cultivars (Ikeuchi et al., 2016). Reduced root regenerative capacity has been attributed to loss of auxin responsiveness in peas (Pisum sativa; Rasmussen et al., 2015), whilst application of auxin improves regeneration ability in aged plants of Arabidopsis (Chen et al., 2014). Moreover, regeneration frequency is increased with an imposed electric field, and this correlates with an increase of auxin concentration (Kral et al., 2016). Wound-induced gene expression is tightly correlated with wound-induced electrical signal transmission in Arabidopsis (Mousavi et al., 2013; Forde and Roberts, 2014), and these signals lead to the upregulation of the jasmonate pathway (Mousavi et al., 2013; Section Bioelectric Mechanisms of Wound Healing and Regenerative Processes in Animals and Plants—An Overview). It is also recognized that jasmonate and auxin signaling pathways interact at various levels (Larrieu and Vernoux, 2016), whereby jasmonate can promote auxin biosynthesis, and auxin can induce the expression of jasmonate biosynthetic genes (Zhang et al., 2016). Auxin is both a versatile messenger in cell interactions, and provides patterning information (reviewed by Berleth and Sachs, 2001). Activation of auxin signal transduction genes leads to downstream cascades, which activate auxin responsive elements, which finally mediate the gene regulation involved in cell division and expansion (De Vos et al., 2012).
Thus, taken together these findings suggest that it would be productive to probe the characteristics of wound-induced electric signals in aged plants, to establish if any bioelectric factors contribute to the diminished regeneration capacity. This is not only of potential horticultural importance but may be of translational relevance if there is a disruption of common upstream signals and affected physiological processes, such as aberrant membrane voltages.
This analysis of literature has demonstrated that bioelectric characteristics are crucial elements in normal development, wound healing and regeneration and occur ubiquitously throughout the animal and plant kingdoms. The nature of the molecular pathways involved in wound healing is fundamentally different between the kingdoms. However, common elements include electric field changes following wounding, and the involvement of various membrane ion channels and pumps. A central feature during regeneration is the re-instigation of the natural electric field patterns previously evident during morphogenesis. Importantly, applied electric fields are one of the very few strategies that have met with proven success in improving the healing rate of recalcitrant wounds, and in coaxing regeneration in organisms which do not normally regenerate. A recent conference on the molecular and cellular basis of regeneration and tissue repair aimed to provide an integrative platform for scientists using a wide variety of models, including plants (Galliot et al., 2017). Evidence was provided, for instance, of an electric field applied to Arabidopsis cut roots significantly increasing the regeneration rate (Kral et al., 2016). This example shows that plant studies are a contemporary focus of interest for regeneration studies, along with numerous animal models (Galliot et al., 2017), which together provide a growing evidence base of a role for bioelectricity which is of prospective translational relevance.
It is becoming increasingly recognized that the programme of development resides not only in the information of gene sequences within the transcriptional code (a combinatorial code of transcription factors), but also requires the involvement of other combinatorial sequence codes. These include the histone code (which multiplies the informational capacity of the genes); cell surface code (residing in cell surface glycoproteins); cytoskeleton code; apoptosis code and ubiquitin code (reviewed by Barbieri, 2016). Moreover, the concept of a simple, linear flow of information of gene sequences is giving way to new model of development involving biological networks, with a multi-directional flow of information moving between hierarchical tiers (Franklin and Vondriska, 2011). It is thus all the more remarkable that regeneration of body form and complex structures such as muscle and cartilage can be induced in the amputated limbs of animals that do not regenerate them, simply by the application of an electric field or a H+ efflux. This suggests that the bioelectric application has a master regulatory effect (Levin, 2009) to reactivate the developmental coding networks involved in regeneration.
Thus, the mapping of voltage patterns in normal/impaired healing/regenerating limbs, and discerning the cell and molecular signatures associated with them may translate into discovering the key players and signals activating these processes. Since we still do not know how the right molecules are orchestrated to be in the right place at the right time in either development or the shaping of wound healing and regeneration, the aberrant voltage pattern and the patterns of elevated longevity provide model systems to probe these mysteries. This can bring vital knowledge for future regenerative therapies.
The author confirms being the sole contributor of this work and approved it for publication.
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.