Electrical Stimulation to Enhance Axon Regeneration After Peripheral Nerve Injuries in Animal Models and Humans
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Injured peripheral nerves regenerate their lost axons but functional recovery in humans is frequently disappointing. This is so particularly when injuries require regeneration over long distances and/or over long time periods. Fat replacement of chronically denervated muscles, a commonly accepted explanation, does not account for poor functional recovery. Rather, the basis for the poor nerve regeneration is the transient expression of growth-associated genes that accounts for declining regenerative capacity of neurons and the regenerative support of Schwann cells over time. Brief low-frequency electrical stimulation accelerates motor and sensory axon outgrowth across injury sites that, even after delayed surgical repair of injured nerves in animal models and patients, enhances nerve regeneration and target reinnervation. The stimulation elevates neuronal cyclic adenosine monophosphate and, in turn, the expression of neurotrophic factors and other growth-associated genes, including cytoskeletal proteins. Electrical stimulation of denervated muscles immediately after nerve transection and surgical repair also accelerates muscle reinnervation but, at this time, how the daily requirement of long-duration electrical pulses can be delivered to muscles remains a practical issue prior to translation to patients. Finally, the technique of inserting autologous nerve grafts that bridge between a donor nerve and an adjacent recipient denervated nerve stump significantly improves nerve regeneration after delayed nerve repair, the donor nerves sustaining the capacity of the denervated Schwann cells to support nerve regeneration. These reviewed methods to promote nerve regeneration and, in turn, to enhance functional recovery after nerve injury and surgical repair are sufficiently promising for early translation to the clinic.
KeywordsPeripheral nerve regeneration Peripheral nerve injury Electrical stimulation Delayed nerve repair Side-to-side crossbridges
Injured nerves in the peripheral nervous system (PNS) regenerate their lost axons in contrast to those nerves in the central nervous system (CNS) that cannot. The Schwann cells within the denervated distal nerve stump provide the essential support for the regeneration of the PNS nerve fibers in contrast to the analogous glial cells of the CNS, the oligodendrocytes . Yet, the recovery of function after human peripheral nerve injuries is frequently disappointing. This is the case particularly for injuries that are sustained at some distance from the denervated targets, requiring that the injured nerves regenerate over long distances and over long periods of time at the established rate of ~1 mm/day .
The basis for this poor regeneration is explored in this review before presenting the evidence that brief electrical stimulation is effective in accelerating axon outgrowth across injury sites [3, 4] that, even after delayed surgical repair of injured peripheral nerves, functional recovery is enhanced . Associated with this enhancement, the electrical stimulation upregulates the expression of neurotrophic factors and, in turn, growth-associated genes [6, 7]. Although exogenous application of growth factors is effective in promoting nerve regeneration after delayed nerve repair or through nerve grafts that connect transected nerve stumps [8, 9, 10, 11], and even though developments have been made in the delivery of these factors , the question remains as to whether the stimulation of endogenous sources of these factors and other as yet unknown mediators of nerve regeneration is more appropriate. Additionally, recent work has indicated an accelerating effect on target reinnervation when denervated muscle is electrically stimulated immediately after nerve transection but, how the daily requirement of long-duration electrical pulses can be delivered to muscles remains a practical question that needs to be addressed prior to clinical application .
Finally, a relatively new technique has been introduced that has the potential to prevent the progressive deterioration of the Schwann cell support of regenerating nerves. The technique of inserting nerve autografts through perineurial windows in peripheral nerves to bridge between a donor nerve and a recipient denervated distal nerve stump, encourages the regeneration of axons from the donor nerve into the recipient denervated nerve stump and allows limited axon regeneration proximal and distal to the nerve autografts that bridge the two nerves, the side-to-side cross-bridges. In turn, these cross-bridges allow for greatly improved nerve regeneration through the chronically denervated distal nerve stump when the proximal nerve stump is surgically united with the distal “protected” nerve stump [13, 14, 15]. Even when the surgical coaptation of the transected nerve stumps is performed immediately after injury, the long distance of nerve regeneration would otherwise allow for the deterioration of the growth support of the chronically denervated distal nerve stumps. The occupation of some of the denervated endoneurial tubes by the regenerated axons from the donor nerve sustains the growth permissive environment. The distal growth of the axons toward the denervated targets may also prevent the rapid denervation atrophy that normally occurs.
The Window of Opportunity for Nerve Regeneration is Restricted
The question remains as to why functional outcomes are so poor after delayed nerve surgeries or injuries suffered where long periods of time pass before target reinnervation would be expected. The answer to the question is generally assumed to be that regenerating axons fail to reinnervate target muscles due to irreversible denervation atrophy with ultimate fat replacement [2, 25, 28]. Experimentally, we asked the question of the consequences of the long periods of isolation of the injured neurons that regenerate their lost axons from their denervated targets (chronic axotomy of the neurons), the long periods of denervation of the Schwann cells in the distal nerve stumps (chronic Schwann cell denervation), and, finally, the chronic denervation of the muscles (chronic muscle denervation). Each of these conditions feature in human patients after delayed nerve repair. They also feature after immediate nerve repair for peripheral nerves that are injured close to the spinal cord and the dorsal root ganglia.
Brief Electrical Stimulation Accelerates Axon Outgrowth Across the Nerve Injury Site After Both Immediate and Delayed Nerve Repair
Electrical stimulation of the transected nerve proximal to the site of transection and surgical repair reduces the staggering of the axons as they regenerate across the suture site: a 2-week period of continuous stimulation at 20 Hz or even 1 h of stimulation promoted the outgrowth of regenerating axons across the suture line with the result that all motoneurons regenerated their axons over the 25-mm distance within 3 weeks (Fig. 4c, d). The electrical stimulation did not affect the rate of axonal transport as determined by injecting radiolabeled thymidine into the neurons . The rationale for stimulating the nerve for 2 weeks was that this period of electrical stimulation after a crush injury of the nerve to the soleus muscle of the rabbit accelerated recovery of contractile forces in the muscle . The finding that electrical stimulation accelerated the recovery of muscle contractile force indicated a positive effect of electrical stimulation without localizing the site of action of the electrical stimulation. Moreover, later findings by Pockett and Gavin  demonstrated the return of reflex contractions of ankle extensor muscles after electrical stimulation of the crushed sciatic nerve. Again, though, the site of action of the electrical stimulation was unknown. These investigators reduced the time period of electrical stimulation to record positive effects for durations as short as 15 min. In our study, we found that a 1-h period of electrical stimulation was effective in accelerating axon regeneration, this finding being serendipitous because longer periods of electrical stimulation were ineffective for sensory neurons whose axon outgrowth was also accelerated by 1-h of electrical stimulation but not by electrical stimulation for longer periods of time . Most importantly, we established that the action potentials, generated by the brief electrical stimulation of the axons proximal to the lesion site and transmitted back to the soma of the neurons, were essential for the efficacy of the accelerated axon outgrowth in response to the electrical stimulation. This was because a tetrodotoxin blockade of these potentials obliterated the effect of the electrical stimulation .
The findings of accelerated nerve outgrowth after immediate nerve repair have been replicated many times for several different nerves, including the sciatic nerve and its main tributary nerve branches [60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91]. Presently, neither the frequency nor the duration of electrical stimulation have been considered in detail, although the crushed facial nerve was stimulated daily until functional recovery at 20 Hz for 30 min/day, the stimulation commencing a day after the crush injury [67, 81, 82]; the transected sciatic nerve was stimulated for only 20 min after delayed nerve repair , and for 10 min after a transection injury that was repaired via a silicone tube filled with a collagen gel .
The Effect of Electrical Stimulation on Nerve Regeneration is Mimicked by Pharmacological Elevation of Cyclic Adenosine Monophosphate
Brief Electrical Stimulation Accelerates Axon Outgrowth Within the CNS
The analogous growth promoting effects of brief low-frequency electrical stimulation and rolipram-induced elevation of neuronal cAMP in the PNS suggested that the electrical stimulation paradigm might also promote nerve outgrowth in the CNS. Indeed, Woolf and colleagues had demonstrated that elevated cAMP in dorsal root ganglion neurons was responsible for the efficacy of a conditioning lesion to the sensory axons in the PNS in promoted outgrowth from transected central axons . The efficacy of the conditioning lesion in promoting regeneration of CNS axons was first demonstrated in 1984 . Because sensory neurons discharge at rates up to 200 Hz, we compared the efficacy of a 1-h period of continuous 200- and 20-Hz electrical stimulation applied to the sciatic nerve immediately after cutting the central axons at the level of T8 in the spinal cord . The 20-Hz electrical stimulation paradigm but not the 200-Hz electrical stimulation paradigm promoted the outgrowth of CNS sensory axons at the level of T8, although to a lesser extent than the conditioning lesion (Fig. 8). This outgrowth was associated with a significant elevation in cAMP in the neurons that were stimulated at 20 Hz but not at 200 Hz, the elevation being the same as after the conditioning lesion. The discrepancy between the elevated cAMP and the efficacy of the electrical stimulation in promoting axon outgrowth suggested additional mechanisms to account for the greater effect of the conditioning lesion than the electrical stimulation .
The Role of Neurotrophic Factors and Androgens in the Efficacy of Electrical Stimulation, and Daily Exercise Programs in Promoting Axon Regeneration in the PNS
Although GDNF has been shown to be efficacious when applied in microspheres around the suture site of injured peripheral nerves with and without an intervening acellular conduit [112, 113, 114, 115, 116, 117, 118], the difficulties of titrating effective doses of BDNF illustrate some of the difficulties that may be encountered with exogenous sources of the neurotrophic factors. The biological modulation of the neurotrophic factors is difficult to replicate and problems of excess administration have been described, as, for example, the administration of excess doses of GDNF using retroviral vectors in rats where the findings of axonal coils was attributed to too much GDNF [119, 120] .
While electrical stimulation upregulates the expression of neurotrophic factors and their receptors, this expression is transient, declining within days . The administration of androgens in conjunction with electrical stimulation, however, sustains their upregulation [81, 82]. Over the course of evaluating the extent of activity-mediated axonal regeneration, Sabatier et al.  and Wood et al.  discovered a sex difference in the effectiveness of treadmill training when comparing 2 treadmill training paradigms, continuous and interval training. A daily slow training protocol at 10 m/min for 1 h resulted in a marked increase in the length of regenerating axons in male mice 2 weeks after nerve transection and repair, but the protocol had no effect on regeneration in female mice [85, 122]. On the other hand, when the female mice were exposed to a faster 20 m/min training protocol for 2-min intervals 4 times daily, an impressive enhancement in axonal regeneration was seen. No enhancement was appreciated in the male mice exposed to the same interval training protocol. The studies from English’s laboratory provide evidence that sex steroid hormones, particularly testosterone, mediate this sex difference in exercised mice . They also demonstrate that androgens are also critical for the efficacy of low-frequency electrical stimulation in promoting axon outgrowth . In the former case, castration of male rats eliminated the enhanced axon regeneration by daily interval training, and treating unexercised female mice with an aromatase inhibitor, anastrozole, to block the conversion of testosterone or its precursors into estradiol, enhanced axonal regeneration . Subsequent experiments demonstrated that treating mice with flutamide, an androgen receptor blocker, inhibited the effect of both exercise and electrical stimulation in both sexes . As treadmill training can be readily applied to humans, the translational potential of this modality in combination with electrical stimulation in peripheral nerve injuries is promising, while activity-dependent therapies also simultaneously empower patients to assume responsibility for their own recovery.
The sluggish crossing of regenerating axons across injury sites, whether or not the endoneurial tubes are disrupted by transection of peripheral nerves, compounds the problems of the slow rate of axon regeneration of 1 mm/day in humans and of 3 mm/day in animals [3, 123]. As a result, functional recovery after nerve injuries is commonly recognized to be disappointing . The efficacy of brief low-frequency electrical stimulation in accelerating axon outgrowth across the injury site in both animal and human studies results in accerelated and improved functional recovery. This improvement was demonstrated in a number of published and ongoing studies of human nerve injuries [94, 125, and Chan, unpublished data]. Importantly, the electrical stimulation regimen is effective after delayed nerve repair in animals and humans [5, 94]. These very promising findings anticipate further studies that may form the basis for the adoption of technique of intraoperative brief electrical stimulation at the time of surgical repair of injured nerves to become the standard of practice in management of peripheral nerve injuries. The ability of training programs after surgical repair of peripheral nerves to accelerate nerve regeneration in animal studies also holds considerable promise for the management of peripheral nerve injuries [85, 122]. Whilst movement is usually restricted after surgical repair of injured nerves, possibilities such as the adoption of imagined movement in the early stages of recovery followed by adoption of active programs of activity may be explored in the future.
Finally, there are several other surgical and pharmacological strategies that are being explored to promote regeneration and to counteract the negative effects of chronic nerve injuries . These include the placement of end-to-side or side-to-side nerve autografts between a donor nerve and a recipient denervated distal nerve stump [14, 127], and the localized administration of FK506 and neurotrophic factors to the surgical site . In the former case, the ingrowth of axons into a denervated nerve stump that proceeds both proximal and distal to the insertion site of the autograft into the recipient denervated stump significantly improves the regeneration of axons after surgical coaptation of the proximal and distal nerve stumps through the distal nerve stump into which the donor axons had grown. Local administration of FK506 to the coaptation site of a transected nerve was also very effective in promoting nerve regeneration. Hence, a combinational approach to surgical repair of transected nerves has enormous potential for greatly improving nerve regeneration and, in turn, functional recovery after peripheral nerve injuries.
I thank all my colleagues who contributed to the several published papers that are included in this review, and the Canadian Institutes of Research who provided the grant funding to carry out the work published in the papers.
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