A Perspective on Electrical Stimulation and Sympathetic Regeneration in Peripheral Nerve Injuries

Peripheral nerve injuries (PNIs) are common and devastating. The current standard of care relies on the slow and inefficient process of nerve regeneration after surgical intervention. Electrical stimulation (ES) has been shown to both experimentally and clinically result in improved regeneration and functional recovery after PNI for motor and sensory neurons; however, its effects on sympathetic regeneration have never been studied. Sympathetic neurons are responsible for a myriad of homeostatic processes that include, but are not limited to, blood pressure, immune response, sweating, and the structural integrity of the neuromuscular junction. Almost one quarter of the axons in the sciatic nerve are from sympathetic neurons, and their importance in bodily homeostasis and the pathogenesis of neuropathic pain should not be underestimated. Therefore, as ES continues to make its way into patient care, it is not only important to understand its impact on all neuron subtypes, but also to ensure that potential adverse effects are minimized. This piece gives an overview of the effects of ES in animals models and in humans while offering a perspective on the potential effects of ES on sympathetic axon regeneration.


Introduction: Electrical Stimulation in Animal Models
Peripheral nerve injuries (PNIs) are common and have a yearly incidence of >500,000 1 ; however, adjunctive treatment options improving the inefficient innate regenerative ability of peripheral nerves are limited. 2rrently, there are no U.S. Food and Drug Administration (FDA)-approved treatment options for improving peripheral nerve regeneration post-PNI, although activity-based therapies, such as electrical stimulation (ES), have received decades worth of pre-clinical research to support its potential as a complementary therapeutic option.][5] The interest in ES as a potential method for reducing the functional decline exhibited in peripheral targets after PNI dates back as far as the early 1800s.For example, in 1841, denervated muscles of frogs that had experienced sciatic nerve injuries were found to contract with the application of ES and subsequently found to exhibit slower muscle atrophy. 6In 1945, ES was also found to slow the muscle atrophy of rat skeletal muscle after sciatic nerve injury. 7ince then, ES has been extensively studied in animal models with rodent models rising to the forefront.][10][11][12] This improved regeneration has also been observed with the use of nerve autografts, which entails two coaptation sites and larger nerve defects. 135][16] Interestingly, enhancement was not observed when delayed nerve repair involved the use of a biodegradable conduit. 179][20][21] Additional sessions of ES performed for 3-4 weeks after the initial ES session have not been found to further enhance regeneration in both mice and rats. 13,18On the contrary, it is worthy to note that daily 1-h ES sessions for 1 week post-repair resulted in improved motor axon regeneration compared to a single application of ES at the time of nerve repair in rats, 22 so the ideal paradigm appears to be unclear.
4][25][26][27][28][29] Conditioning ES has emerged as a potential method for improving outcomes in nerve transfer surgeries, which are relevant in cases such as brachial plexus injuries, proximal nerve injuries, spinal cord injury, and stroke.The idea of conditioning a nerve with ES stems from a conditioning lesion, which is a commonly used experimental technique that involves injuring the nerve with a ''conditioning'' lesion preceding a ''test'' lesion.Although still theoretical, a conditioning ES would allow for the donor nerve to be stimulated percutaneously, and non-injuriously, in the clinic at a specified time before the actual nerve transfer surgery to improve the effi-ciency and regenerative capacity of the donor nerve.Conditioning ES has displayed excellent results in the rat model, with motor and sensory recovery even surpassing those observed with perioperative ES 24,25 ; however, more research is needed to solidify the feasibility and superiority of conditioning ES over perioperative ES given that this comparison has only been explored by a single lab group.

Electrical Stimulation in Humans for Peripheral Nerve Injury
With the success of ES in accelerating axon regeneration and functional distal target reinnervation in animal models, several pilot clinical studies have been conducted in various patient populations (Fig. 1).Perioperative ES has shown promising results in clinical trials with studies showing improvement in both motor and sensory recovery.In one study, patients with severe carpal tunnel syndrome received 20 Hz of ES for 1 h with the stimulation intensity titrated to their maximal tolerance limit to the affected median nerve after carpal tunnel decompression surgery. 30This study found that ES resulted in more rapid improvement of motor and sensory function through nerve conduction studies as well as patient-reported outcome measures.This indicates that ES can be efficacious even after long-standing nerve injury present with severe carpal tunnel syndrome.
2][33][34][35][36] A randomized controlled study with patients diagnosed with mild-to-moderate Bell's palsy found that the addition of ES alongside prednisolone and/or acyclovir resulted in a significantly better recovery rate compared to those treated with prednisolone and/or acyclovir alone. 31Additionally, stimulationinduced blinking in patients with facial nerve palsy was found to reduce the negative ocular symptoms associated with facial nerve palsy in a preliminary study; however, the efficacy was likely limited to participants with less severe palsy. 33Three weeks of daily ES applied 4 weeks after facial nerve palsy onset was also found to improve functional facial movements and electrophysiological measures at the 3-month follow-up visit. 34On the other hand, the use of ES in the treatment of Bell's palsy in the acute phase (<30 days since onset) did not result in a statistically significant difference in terms of clinical effects.
Currently, the use of ES in humans is limited by the availability of FDA-approved intraoperative nerve stimulators.8][39][40][41] With external stimulators, stimulation protocols proceeded for 1 h after the patient awakened from anesthesia, so the level of stimulation could be titrated to patient comfort. 30Implantable stimulators, however, have been developed for pain modulation.Over time, nerve stimulators have continued to improve in their ease of implantation in more hard-to-reach coaptation sites and their ability to minimize patient complications associated with an implanted stimulator. 42 absorbable hydrogel scaffold was recently developed and tested in rats. 42This hydrogel can also be formulated as a grounded gel, which can be used for percutaneous and open surgical approaches, with the formulation injected at the site of the injured nerve.This formulation stabilizes the electrodes, conforms to the surrounding anatomical features, and establishes a conductive path without electrode-nerve contact, making this method potentially attractive for the stimulation of nerves in anatomically challenging locations.The hydrogel led to improved motor and sensory recovery, increased muscle mass, and increased axonogenesis.
In light of these studies, the utility of ES to complement nerve repair in humans holds great potential for FIG. 1. Cycle of how clinical scenarios inform basic science research, which then informs future standards of care for patients.The current standard of care of the treatment of peripheral nerve injuries relies on the slow and inefficient process of intrinsic axon regeneration with very few patients achieving full functional recovery.Therefore, in rats, 1 h of electrical stimulation (ES) of the proximal stump after surgical repair was found to be a viable method to promote motor axon regeneration into the distal stump. 8A clinical trial of ES in patients with severe carpal tunnel syndrome requiring surgical decompression resulted in improved functional recovery. 30On the basic science side, researchers continue to study different nerve injury scenarios and seek to understand and improve adjunctive therapeutic methods that will inform the future of patient care.Created with Biorender.com.
improving sensory and motor outcomes after PNI.[45][46][47] Anatomy of Peripheral Nerves When it comes to peripheral nerves, it is important to note that most of the axons are unmyelinated.Of the *27,000 axons in the rat sciatic nerve, only 29% are myelinated, consisting of 6% motor axons and 23% sensory axons. 48The rest of the nerve consists of 48% unmyelinated sensory axons and 23% unmyelinated sympathetic axons (Fig. 2).
Post-ganglionic parasympathetic axons, another important part of the autonomic nervous system that consists of unmyelinated axons, do not travel through the lumbar-level sciatic nerve. 49The remaining sections of this perspective piece will focus primarily on the unmyelinated sympathetic axons found nearly ubiquitously in peripheral nerves.

Perspective on Electrical Stimulation for Sympathetic Axon Regeneration
Current research on the use of ES have been mostly focused on motor and sensory axon regeneration and their functional recovery.However, the paradigm that is widely used (20-Hz stimulation for 1 h at a stimulation intensity titrated to 1.5 • the intensity to elicit a muscle twitch in animal models and to patient comfort in humans) may not be at a high enough intensity to recruit sympathetic neurons into activity.Because of the small caliber and unmyelinated nature of sympathetic axons (in addition to other unmyelinated sensory axons), they will be recruited last into activity by ES.Larger caliber, myelinated axons are recruited first  48 because of their lower input resistance attributed to having more parallel ion channels and nodes of Ranvier. 50ome experimental designs use stimulus intensities as high as 50 V to stimulate sympathetic neurons. 51,52n comparison, the stimulus intensity of ES in mouse models of PNI typically falls in the range of 3-4 V. 53 Therefore, in order for the ES paradigm to recruit sympathetic neurons into activity, the stimulus intensity may not be in the realm of comfort for patients.
ES in itself is also non-specific in nature and can activate other cells around the axons that can influence axonal growth.Nerve growth factor (NGF), which binds to the tropomyosin receptor kinase A (TrkA) receptor, heavily influences sympathetic outgrowth, particularly sprouting.NGF is released by Schwann cells in response to ES, which may then increase sympathetic sprouting, 54 a result that may hinder elongation and eventual functional reinnervation of distal targets.The presence of axonal sprouts, when coupled with elongation and the formation of new synapses, are critical for the reinnervation of distal targets after PNI; however, axonal sprouting alone without proper elongation may result in off-target effects and incomplete distal target reinnervation.
6][57] This conditioning lesion is believed to prime the neurons for regeneration; however, the regeneration of sympathetic axons in the sciatic nerve were shown to be inhibited by this technique. 57Thus, different axon types in the peripheral nerves may respond to activity-dependent interventions differently.
The inhibitory effect of the conditioning lesion on sympathetic outgrowth is inconclusive given that Navarro's group found that a conditioning lesion at the same site as a test lesion accelerated the growth of the sympathetic axons and shortened the time until most sweat glands became functionally reinnervated. 58dditionally, some studies have found that the superior cervical ganglia do respond positively to neuronal stimulation interventions such as conditioning lesions. 59ome of these incongruous findings may be attributable to the differing molecular profiles and heterogenous NGF requirements for target organ innervation of the sympathetic chain ganglia at different spinal levels. 60,61Therefore, further investigations are necessary to characterize how activity-based therapeutics such as ES affect different peripheral nerves.
Although several studies have investigated the impact of the conditioning lesion on sympathetic regeneration, the effects of ES on sympathetic regener-ation and functional recovery over time after PNI are still unknown.Preliminary data suggest that ES at the very least is not improving the rate of sweat gland reinnervation and does not enhance sympathetic axon regeneration shortly after injury (Tian, unpublished).Additionally, it appears that activity-based therapies may also be detrimental to sympathetic regeneration overall, with specific stimulation of sympathetic neurons using bioluminescent optogenetics resulting in a decrease in the number of retrogradely-traced sympathetic neurons after sciatic nerve injury. 917][68] Thus, a need exists for treatments of PNI that promote the regeneration of all axons while avoiding potential adverse effects.

Sympathetic Sprouting and Pain After Peripheral Nerve Injury
After PNI, patients may experience various degrees of neuropathic pain.0][71][72][73] Sympathetic sprouting may include the formation of ''baskets'' around individual somas, particularly large-diameter neurons, and have also been observed in the DRGs of patients with neuropathic pain. 74,75In rats, a sympathectomy significantly reduces pain after spinal nerve ligation. 70Additionally, injury-induced spontaneous activity of primary sensory neurons at the level of the DRG have been implicated to play a critical role in the development of sympathetically maintained pain. 769][80] Notably, the small unmyelinated DRG neurons typically do not exhibit these discharge patterns, more often exhibiting an irregular, low-frequency pattern instead. 80,81hese findings in the pain literature may prove concerning for the use of ES, especially because evidence supports that early spontaneous activity is a trigger for persistent neuropathic pain after PNI, 76 although this observation has not been systematically recorded in clinical populations.In many rodent models of PNI with ES, stimulation is applied at the time of injury, triggering a synchronized firing of mostly largercaliber neurons.Synchronized cluster firing, defined as ''more than three adjacently located DRG neurons that exhibit calcium transients within several continuous imaging frames,'' is a characteristic found in DRG neu-rons that leads to spontaneous pain behavior. 82Cluster firing activity was found to be triggered by the sympathetic activity of fibers that had sprouted into DRGs after injury and is mediated by norepinephrine.
Further, ES has been shown to induce NGF release from Schwann cells. 54TrkA, which are high-affinity-NGF receptors, are found on nociceptors and sympathetic efferent fibers, 83 and signaling through the NGF/TrkA pathway has been a target for treatment of pain. 84Transdermal ES after NGF administration to the hindlimbs of pigs resulted in a hyperalgesia, which was increased with higher-frequency stimulation FIG. 3. Post-ganglionic sympathetic neurons sprouting into the dorsal root ganglia (DRG) in response to peripheral nerve injury (PNI).The cell bodies of pre-ganglionic sympathetic neurons (green) are in the lateral horns of spinal cord levels T1-L2.Their projections travel along the ventral roots then through the white rami to innervate post-ganglionic sympathetic neurons (magenta) in the sympathetic chain.The preganglionic neurons (green) can traverse several levels of the sympathetic chain before synapsing on postganglionic neurons.Most post-ganglionic axons exit the sympathetic ganglia (SG) through the gray rami and into the ventral and dorsal rami of the spinal nerves to innervate distal targets.Some will send projections to their adjacent DRG (sensory neurons in blue) to reach blood vessels and the surface of the DRG.These projections may sprout and contribute to neuropathic pain after PNI, such as that to the sciatic nerve, which in a mouse contains contributions mainly from the L3, L4, and L5 spinal nerve roots. 89,90For simplicity, only one neuron at each potential synapse is represented.Figure adapted from Xie and colleagues 2016. 73 ‡20 Hz) and was peaked at 3 weeks post-NGF administration. 85Pre-clinical evaluation of NGF and TrkA antagonism consistently prevented hyperalgesia and allodynia, 86,87 but anti-NGF monoclonal antibodies in humans have shown mixed efficacy and lack longterm follow-up studies. 88

Conclusion
Despite decades of research in the viability of ES, the effects of this clinically relevant adjunctive treatment option on sympathetic regeneration remains unknown.Sympathetic axons comprise of nearly one fourth of axons in the sciatic nerve and account for many major bodily functions.Further, with the high likelihood that the current clinical paradigm is unable to recruit the small-diameter sympathetic fibers into activity, the effects of ES may be more dependent on factors released by surrounding cells.Therefore, optimization of the stimulation paradigm needs to be explored alongside the effects of activity-dependent therapeutics on the regeneration of different neuron types.And, finally, with the relationship between neuropathic pain and sympathetic sprouting, which also appears to be activity-dependent and related to the injury-induced spontaneous activity of larger cells, the potential adverse effects of ES need to be further examined.With this intervention gaining more traction in the clinical realm, it becomes imperative that the effects of ES on peripheral nerve regeneration must be studied in sympathetic neurons in peripheral nerves.

FIG. 2 .
FIG. 2. Anatomy schematic of the sciatic nerve.Cross-section diagram of the rat sciatic nerve.Unmyelinated sensory and post-ganglionic sympathetic neurons are clustered in Remak bundles.Figure adapted from Schmalbruch, 1986.48