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.

Application of Electrical Stimulation to Enhance Axon Regeneration Following Peripheral Nerve Injury.

Enhancing axon regeneration is a major focus of peripheral nerve injury research. Although peripheral axons possess a limited ability to regenerate, their functional recovery is very poor. Various activity-based therapies like exercise, optical stimulation, and electrical stimulation as well as pharmacologic treatments can enhance spontaneous axon regeneration. In this protocol, we use a custom-built cuff to electrically stimulate the whole sciatic nerve for an hour prior to transection and repair. We used a Thy-1-YFP-H mouse to visualize regenerating axon profiles. We compared the regeneration of axons from nerves that were electrically stimulated to nerves that were not stimulated (untreated). Electrically stimulated nerves had longer axon growth than the untreated nerves. We detail how variations of this method can be used to measure acute axon growth.

Conditioning electrical stimulation fails to enhance sympathetic axon regeneration.

Peripheral nerve injuries are common, and there is a critical need for the development of novel therapeutics to complement surgical repair. Conditioning electrical stimulation (CES) is a novel variation to the well-studied perioperative electrical stimulation, both of which have displayed success in enhancing the regeneration of motor and sensory axons in an injured peripheral nerve. CES is a clinically attractive alternative not only because of its ability to be performed at the bedside prior to a scheduled nerve repair surgery, but it has also been shown to be superior to perioperative electrical stimulation in the enhancement of motor and sensory regeneration. However, the effects of CES on sympathetic regeneration are unknown. Therefore, we tested the effects of two clinically relevant CES paradigms on sympathetic axon regeneration and distal target reinnervation. Because of the long history of evidence for the enhancement of motor and sensory axons in response to electrical stimulation, we hypothesize that CES will also enhance sympathetic axon regeneration. Our results indicate that the growth of sympathetic axons is acutely inhibited by CES; however, at a longer survival time point post-injury, there is no difference between sham CES and the CES groups. There has been evidence to suggest that the growth of sympathetic axons is inhibited by a conditioning lesion, and that sympathetic axons may respond to electrical stimulation by sprouting rather than elongation. Our data indicate that sympathetic axons may retain some regenerative ability after CES, but no enhancement is exhibited, which may be accounted for by the inability of the current clinically relevant electrical stimulation paradigm to recruit the small-caliber sympathetic axons into activity. Further studies will be needed to optimize electrical stimulation parameters in order to enhance the regeneration of all neuron types.

Angiogenesis is critical for the exercise-mediated enhancement of axon regeneration following peripheral nerve injury.

Enhancing axon regeneration is a major focus of nerve injury research, and the quality of the surgical nerve repair plays a large role in the aggregate success of nerve regeneration. Additionally, exercise is known to promote successful axon regeneration after surgical nerve repair. In this study, we asked how exercise-induced nerve regeneration is affected when a transected nerve is repaired with or without fibrin glue. Fibrin glue repaired nerves exhibited greater vasculature within the tissue bridge compared to nerves that were intrinsically repaired. Fibrin glue repaired nerves also exhibited more robust axon regeneration after exercise compared to nerves that were not repaired with fibrin glue. When angiogenesis of the tissue bridge was prevented, exercise was unable to enhance regeneration despite the presence of fibrin glue. These findings suggest that the biological properties of fibrin glue enhance angiogenesis within the repair site, and a vascularized bridge is required for enhanced axon elongation with exercise. The combination of fibrin glue repair and exercise resulted in notable differences in vascular growth, axon elongation, neuromuscular junction reinnervation, and functional recovery. Fibrin glue should be considered as an adjuvant for nerve repair to enhance the subsequent efficacy of activity- and physical therapy-based treatment interventions.

Neuronal androgen receptor is required for activity dependent enhancement of peripheral nerve regeneration.

Neuronal activity after nerve injury can enhance axon regeneration and the restoration of function. The mechanism for this enhancement relies in part on hormone receptors, and we previously demonstrated that systemic androgen receptor antagonism blocked the effect of exercise or electrical stimulation on enhancing axon regeneration after nerve injury in both sexes. Here, we tested the hypothesis that the site of this androgen receptor signaling is both neuronal and involves the classical, genomic signaling pathway. In vivo, dorsal root ganglion neurons successfully regenerate in response to activity-dependent neuronal activation, and conditional deletion of the DNA-binding domain of the androgen receptor in adults blocks this effect in males and females. Motoneurons in males and females also respond in this manner, but we also observed a sex difference. In vitro, cultured sensory dorsal root ganglion neurons respond to androgens via traditional androgen receptor signaling mechanisms leading to enhanced neurite growth and did not respond to a testosterone conjugate that is unable to cross the cell membrane. Given our previous observation of a requirement for activity-dependent androgen receptor signaling to promote regeneration in both sexes, we interpret our results to indicate that genomic neuronal androgen receptor signaling is required for activity-dependent axon regeneration in both sexes.

Asparagine Endopeptidase (δ Secretase), an Enzyme Implicated in Alzheimer's Disease Pathology, Is an Inhibitor of Axon Regeneration in Peripheral Nerves.

Asparagine endopeptidase (AEP) is a lysosomal protease implicated in the pathology of Alzheimer's disease (AD). It is known to cleave the axonal microtubule associated protein, Tau, and amyloid precursor protein (APP), both of which might impede axon regeneration following peripheral nerve injury (PNI). Active AEP, AEP-cleaved fragments of Tau (Tau N368), and APP (APP N585) were found in injured peripheral nerves. In AEP null mice, elongation of regenerating axons after sciatic nerve transection and repair was increased relative to wild-type (WT) controls. Compound muscle action potentials (M responses) were restored in reinnervated muscles twice as fast after injury in AEP knock-out (KO) mice as WT controls. Neurite elongation in cultures of adult dorsal root ganglion (DRG) neurons derived from AEP KO mice was increased significantly relative to cultures from WT controls. In AEP KO mice exposed to 1 h of 20-Hz electrical stimulation (ES) at the time of nerve injury, no further enhancement of axon regeneration was observed. These findings support inhibition of AEP as a therapeutic target to enhance axon regeneration after PNI.

Chimeric Peptide Species Contribute to Divergent Dipeptide Repeat Pathology in c9ALS/FTD and SCA36.

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.

Modified Anterior Tibial Artery Perforator-Pedicled Propeller Flap for Soft-Tissue Coverage of the Ankle and Heel.

BACKGROUND: The ankle and heel are challenging regions to reconstruct functionally. Here, we explored the feasibility and clinical outcomes of a modified anterior tibial artery perforator-pedicled propeller flap for the repair of soft-tissue defects of the ankle and heel. PATIENTS AND METHODS: Between January 2013 and December 2015, 12 patients with soft-tissue defects of the ankle and/or heel underwent reconstructive surgery that included our flap technique. The flaps measured 20 × 8 cm to 7 × 4 cm. A hand-held Doppler was used to identify a proper constant perforator in the distal ankle. In each case, the base of the flap was well preserved. The flap was transposed (180° rotation) to reach and cover the defect. RESULTS: The average follow-up time was 13 months (10-28 months). We observed good texture matches and contour in all of the flaps. All patients could walk and wear normal footwear. All but one flap survived completely without complications. Partial loss was observed in one patient, and the necrotic region was healed with secondary intention. CONCLUSION: Our modified anterior tibial artery free-style perforator-pedicled propeller flap provides a novel option for functional ankle and heel reconstruction. LEVEL OF EVIDENCE: Level IV.

Optical Stimulation and Electrophysiological Analysis of Regenerating Peripheral Axons.

Although axons in the peripheral nervous system can regenerate, functional recovery after nerve injuries is poor. Activity-based therapies, such as exercise and electrical stimulation, enhance the regeneration of cut peripheral axons. Despite their effectiveness, clinical application of these experimental techniques has been limited. At least part of the basis for this translational barrier has been a lack of information as to the precise mechanism of activity-based therapies on peripheral axon regeneration. To evaluate the requirements for neuron-type specific activation to promote regeneration using these therapies, in the current protocol, we employed optogenetics. Utilizing the advantages of transgenic mouse lines we targeted opsin expression to different neuron types. Using fiber optics we activated those neurons with high temporal specificity as a model of activity-based intervention after nerve injury and to measure functional recovery achieved after such a treatment.

Effects of Repeated 20-Hz Electrical Stimulation on Functional Recovery Following Peripheral Nerve Injury.

One hour of 20-Hz continuous electrical stimulation (ES) applied at the time of injury promotes the regeneration of axons in cut peripheral nerves. A more robust enhancement of peripheral axon regeneration is achieved by 2 weeks of daily treadmill exercise. We investigated whether repeated applications of brief ES (mES) would be more effective in promoting regeneration than a single application. Sciatic nerves of C57B6 mice were cut and repaired by end-to-end anastomosis. At that time and every third day for 2 weeks, the repaired nerve was stimulated for 1 hour at 20 Hz. In controls, injured mice were either untreated or treated with ES only once. Direct muscle responses recorded from reinnervated muscles in awake animals were observed earlier both in mice treated with ES and mES than untreated controls. Their amplitudes increased progressively over the post transection study period, but the rate of this progression was increased significantly only in animals treated once with ES. Monosynaptic H reflexes recovered to pretransection levels in both untreated and singly treated mice but in the animals treated repeatedly, they were maintained at more than twice that of the same reflexes recorded prior to injury. In anatomical analyses, both excitatory and inhibitory synaptic contacts with the cell bodies of injured motoneurons, including those expressing the vesicular glutamate transporter 1 (VGLUT1), were sustained in mice treated repeatedly but not in singly treated or untreated mice. Repeated ES does not enhance the rate of restoration of functional muscle reinnervation and results in the retention of exaggerated reflexes.

Chemogenetic Enhancement of Axon Regeneration Following Peripheral Nerve Injury in the SLICK-A Mouse.

The effects of chemogenetics on axon regeneration following peripheral nerve transection and repair were studied in mice expressing a Cre-dependent excitatory designer receptor exclusively activated by designer drugs (DREADD) and Cre-recombinase/yellow fluorescent protein (YFP) in a subset of motor and sensory neurons and cortical motoneurons (SLICK-A). Sciatic nerves were cut and repaired and mice were treated either once, at the time of injury, or five days per week for two weeks with clozapine N-oxide (CNO) (1 mg/kg, i.p.), or were untreated controls. Two weeks after injury, the lengths of YFP+ axon profiles were measured in nerves harvested from euthanized animals. Compared to untreated controls, regenerating axon lengths were not significantly longer in mice treated only once with CNO, but they were more than three times longer in mice receiving CNO repeatedly. Based on results of retrograde labeling experiments, axons of more sensory and motor neurons had regenerated successfully in mice receiving multiple CNO treatments than animals receiving only one treatment or no treatments. The increase in numbers of labeled sensory, but not motor neurons could be accounted for by increases in the proportion of retrogradely labeled neurons also expressing the DREADD. Chemogenetic increases in neuronal excitability represent a potent and innovative treatment to promote peripheral nerve regeneration.

Optogenetically enhanced axon regeneration: motor versus sensory neuron-specific stimulation.

Brief neuronal activation in injured peripheral nerves is both necessary and sufficient to enhance motor axon regeneration, and this effect is specific to the activated motoneurons. It is less clear whether sensory neurons respond in a similar manner to neuronal activation following peripheral axotomy. Further, it is unknown to what extent enhancement of axon regeneration with increased neuronal activity relies on a reflexive interaction within the spinal circuitry. We used mouse genetics and optical tools to evaluate the precision and selectivity of system-specific neuronal activation to enhance axon regeneration in a mixed nerve. We evaluated sensory and motor axon regeneration in two different mouse models expressing the light-sensitive cation channel, channelrhodopsin (ChR2). We selectively activated either sensory or motor axons using light stimulation combined with transection and repair of the sciatic nerve. Regardless of genotype, the number of ChR2-positive neurons whose axons had regenerated successfully was greater following system-specific optical treatment, with no effect on the number of ChR2-negative neurons (whether motor or sensory neurons). We conclude that acute system-specific neuronal activation is sufficient to enhance both motor and sensory axon regeneration. This regeneration-enhancing effect is likely cell autonomous.

Selective Requirement for Maintenance of Synaptic Contacts onto Motoneurons by Target-Derived trkB Receptors.

Synaptic contacts onto motoneurons were studied in mice in which the gene for the trkB neurotrophin receptor was knocked out selectively in a subset of spinal motoneurons. The extent of contacts by structures immunoreactive for either of two different vesicular glutamate transporters (VGLUT1 and VGLUT2), the vesicular GABA transporter, or glutamic acid decarboxylase 67 (GAD67) with the somata of motoneurons, was studied in wild type and trkB knockout cells in tamoxifen treated male and female SLICK-trkB(-/-) mice. Selective knockout of the trkB gene resulted in a marked reduction in contacts made by VGLUT2- and GAD67-immunoreactive structures in both sexes and a significant reduction in contacts containing only glycine in male mice. No reduction was found for glycinergic contacts in female mice or for VGLUT1 immunoreactive contacts in either sex. Signaling through postsynaptic trkB receptors is considered to be an essential part of a cellular mechanism for maintaining the contacts of some, but not all, synaptic contacts onto motoneurons.

Optically-Induced Neuronal Activity Is Sufficient to Promote Functional Motor Axon Regeneration In Vivo.

Peripheral nerve injuries are common, and functional recovery is very poor. Beyond surgical repair of the nerve, there are currently no treatment options for these patients. In experimental models of nerve injury, interventions (such as exercise and electrical stimulation) that increase neuronal activity of the injured neurons effectively enhance axon regeneration. Here, we utilized optogenetics to determine whether increased activity alone is sufficient to promote motor axon regeneration. In thy-1-ChR2/YFP transgenic mice in which a subset of motoneurons express the light-sensitive cation channel, channelrhodopsin (ChR2), we activated axons in the sciatic nerve using blue light immediately prior to transection and surgical repair of the sciatic nerve. At four weeks post-injury, direct muscle EMG responses evoked with both optical and electrical stimuli as well as the ratio of these optical/electrical evoked EMG responses were significantly greater in mice that received optical treatment. Thus, significantly more ChR2+ axons successfully re-innervated the gastrocnemius muscle in mice that received optical treatment. Sections of the gastrocnemius muscles were reacted with antibodies to Synaptic Vesicle Protein 2 (SV2) to quantify the number of re-occupied motor endplates. The number of SV2+ endplates was greater in mice that received optical treatment. The number of retrogradely-labeled motoneurons following intramuscular injection of cholera toxin subunit B (conjugated to Alexa Fluor 555) was greater in mice that received optical treatment. Thus, the acute (1 hour), one-time optical treatment resulted in robust, long-lasting effects compared to untreated animals as well as untreated axons (ChR2-). We conclude that neuronal activation is sufficient to promote motor axon regeneration, and this regenerative effect is specific to the activated neurons.

Exercise, neurotrophins, and axon regeneration in the PNS.

Electrical stimulation and exercise are treatments to enhance recovery from peripheral nerve injuries. Brain-derived neurotrophic factor and androgen receptor signaling are requirements for the effectiveness of these treatments. Increased neuronal activity is adequate to promote regeneration in injured nerves, but the dosing of activity and its relationship to neurotrophins and sex steroid hormones is less clear. Translation of these therapies will require principles associated with their cellular mechanisms.

The effects of exercise on synaptic stripping require androgen receptor signaling.

Following peripheral nerve injury, synapses are withdrawn from axotomized motoneurons. Moderate daily treadmill exercise, which promotes axon regeneration of cut peripheral nerves, also influences this synaptic stripping. Different exercise protocols are required to promote axon regeneration in male and female animals, but the sex requirements for an effect of exercise on synaptic stripping are unknown. In male and female C57BL/6 mice, the sciatic nerve was transected in the mid-thigh. Mice were then exercised five days per week for two weeks, beginning on the third post-transection day. Half of the exercised mice were trained by walking slowly (10 M/min) on a level treadmill for one hour per day (continuous training). Other mice were interval trained; four short (two min) sprints at 20 M/min separated by five minute rest periods. A third group was untrained. The extent of synaptic contacts made by structures immunoreactive to vesicular glutamate transporter 1 and glutamic acid decarboxylase 67 onto axotomized motoneurons was studied in confocal images of retrogradely labeled cells. Both types of presumed synaptic contacts were reduced markedly in unexercised mice following nerve transection, relative to intact mice. No significant reduction was found in continuous trained males or interval trained females. Reductions in these contacts in interval trained males and continuous trained females were identical to that observed in untrained mice. Treatments with the anti-androgen, flutamide, blocked the effect of sex-appropriate exercise on synaptic contacts in both males and females. Moderate daily exercise has a potent effect on synaptic inputs to axotomized motoneurons. Successful effects of exercise have different requirements in males and females, but require androgen receptor signaling in both sexes.

Novel multi-system functional gains via task specific training in spinal cord injured male rats.

Locomotor training (LT) after spinal cord injury (SCI) is a rehabilitative therapy used to enhance locomotor recovery. There is evidence, primarily anecdotal, also associating LT with improvements in bladder function and reduction in some types of SCI-related pain. In the present study, we determined if a step training paradigm could improve outcome measures of locomotion, bladder function, and pain/allodynia. After a T10 contusive SCI trained animals (adult male Wistar rats), trained animals began quadrupedal step training beginning 2 weeks post-SCI for 1 h/day. End of study experiments (3 months of training) revealed significant changes in limb kinematics, gait, and hindlimb flexor-extensor bursting patterns relative to non-trained controls. Importantly, micturition function, evaluated with terminal transvesical cystometry, was significantly improved in the step trained group (increased voiding efficiency, intercontraction interval, and contraction amplitude). Because both SCI and LT affect neurotrophin signaling, and neurotrophins are involved with post-SCI plasticity in micturition pathways, we measured bladder neurotrophin mRNA. Training regulated the expression of nerve growth factor (NGF) but not BDNF or NT3. Bladder NGF mRNA levels were inversely related to bladder function in the trained group. Monitoring of overground locomotion and neuropathic pain throughout the study revealed significant improvements, beginning after 3 weeks of training, which in both cases remained consistent for the study duration. These novel findings, improving non-locomotor in addition to locomotor functions, demonstrate that step training post-SCI could contribute to multiple quality of life gains, targeting patient-centered high priority deficits.

Persistent polyuria in a rat spinal contusion model.

Polyuria contributes to bladder overdistention, which confounds both lower and upper urinary tract management in individuals having a spinal cord injury (SCI). Bladder overdistention post-SCI is one of the most common triggers for autonomic dysreflexia, a potentially life-threatening condition. Post-SCI polyuria is thought to result from loss of vascular tone in the lower extremities, leading to edema and subsequent excess fluid, resulting in polyuria. Mild SCIs that have near complete recovery would therefore be expected to have little to no polyuria, while severe injuries resulting in flaccid limbs and lower extremity edema would be expected to exhibit severe polyuria. Since interventions that may decrease lower extremity edema are recommended to lessen the severity of polyuria, step training (which promotes vascular circulation) was evaluated as a therapy to reduce post-SCI polyuria. In the present study, polyuria was evaluated in mild, moderate, and severe contusive SCI in adult male rats. The animals were housed in metabolic cages for 24-hour periods pre- and post-SCI (to 6 weeks). Urine, feces, food, water, and body weights were collected. Other assessments included residual expressed urine volumes, locomotor scoring, in-cage activity, and lesion histology. SCI produced an immediate increase in 24-hour urine collection, as early as 3 days post-SCI. Approximately 2.6-fold increases in urine collection occurred from weeks 1-6 post-SCI for all injury severities. Even with substantial gains in locomotor and bladder function following a mild SCI, polyuria remained severe. Step training (30 min/day, 6 days/week) did not alleviate polyuria in the moderate SCI contusion group. These results indicate that (1) mild injuries retaining weight-bearing locomotion that should have mild, if any, edema/loss of vascular tone still exhibit severe polyuria, and (2) step training was unable to reduce post-SCI polyuria. Taken together, these results indicate that the current mechanistic hypothesis of post-SCI polyuria may be incomplete.