Human motor endplate remodeling after traumatic nerve injury

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  • 1 Peripheral Nerve Research Lab, Department of Orthopaedic Surgery, University of California, Irvine;
  • 2 Department of Orthopaedic Surgery, University of California, San Diego;
  • 3 Congress Medical Foundation, Pasadena; and
  • 4 Reeve-Irvine Research Center, University of California, Irvine, California
Open access

OBJECTIVE

Current management of traumatic peripheral nerve injuries is variable with operative decisions based on assumptions that irreversible degeneration of the human motor endplate (MEP) follows prolonged denervation and precludes reinnervation. However, the mechanism and time course of MEP changes after human peripheral nerve injury have not been investigated. Consequently, there are no objective measures by which to determine the probability of spontaneous recovery and the optimal timing of surgical intervention. To improve guidance for such decisions, the aim of this study was to characterize morphological changes at the human MEP following traumatic nerve injury.

METHODS

A prospective cohort (here analyzed retrospectively) of 18 patients with traumatic brachial plexus and axillary nerve injuries underwent biopsy of denervated muscles from the upper extremity from 3 days to 6 years after injury. Muscle specimens were processed for H & E staining and immunohistochemistry, with visualization via confocal and two-photon excitation microscopy.

RESULTS

Immunohistochemical analysis demonstrated varying degrees of fragmentation and acetylcholine receptor dispersion in denervated muscles. Comparison of denervated muscles at different times postinjury revealed progressively increasing degeneration. Linear regression analysis of 3D reconstructions revealed significant linear decreases in MEP volume (R = −0.92, R2 = 0.85, p = 0.001) and surface area (R = −0.75, R2 = 0.56, p = 0.032) as deltoid muscle denervation time increased. Surprisingly, innervated and structurally intact MEPs persisted in denervated muscle specimens from multiple patients 6 or more months after nerve injury, including 2 patients who had presented > 3 years after nerve injury.

CONCLUSIONS

This study details novel and critically important data about the morphology and temporal sequence of events involved in human MEP degradation after traumatic nerve injuries. Surprisingly, human MEPs not only persisted, but also retained their structures beyond the assumed 6-month window for therapeutic surgical intervention based on previous clinical studies. Preoperative muscle biopsy in patients being considered for nerve transfer may be a useful prognostic tool to determine MEP viability in denervated muscle, with surviving MEPs also being targets for adjuvant therapy.

ABBREVIATIONS AChR = acetylcholine receptor; BPI = brachial plexus injury; EMG = electromyography; MEP = motor endplate; NMJ = neuromuscular junction.

OBJECTIVE

Current management of traumatic peripheral nerve injuries is variable with operative decisions based on assumptions that irreversible degeneration of the human motor endplate (MEP) follows prolonged denervation and precludes reinnervation. However, the mechanism and time course of MEP changes after human peripheral nerve injury have not been investigated. Consequently, there are no objective measures by which to determine the probability of spontaneous recovery and the optimal timing of surgical intervention. To improve guidance for such decisions, the aim of this study was to characterize morphological changes at the human MEP following traumatic nerve injury.

METHODS

A prospective cohort (here analyzed retrospectively) of 18 patients with traumatic brachial plexus and axillary nerve injuries underwent biopsy of denervated muscles from the upper extremity from 3 days to 6 years after injury. Muscle specimens were processed for H & E staining and immunohistochemistry, with visualization via confocal and two-photon excitation microscopy.

RESULTS

Immunohistochemical analysis demonstrated varying degrees of fragmentation and acetylcholine receptor dispersion in denervated muscles. Comparison of denervated muscles at different times postinjury revealed progressively increasing degeneration. Linear regression analysis of 3D reconstructions revealed significant linear decreases in MEP volume (R = −0.92, R2 = 0.85, p = 0.001) and surface area (R = −0.75, R2 = 0.56, p = 0.032) as deltoid muscle denervation time increased. Surprisingly, innervated and structurally intact MEPs persisted in denervated muscle specimens from multiple patients 6 or more months after nerve injury, including 2 patients who had presented > 3 years after nerve injury.

CONCLUSIONS

This study details novel and critically important data about the morphology and temporal sequence of events involved in human MEP degradation after traumatic nerve injuries. Surprisingly, human MEPs not only persisted, but also retained their structures beyond the assumed 6-month window for therapeutic surgical intervention based on previous clinical studies. Preoperative muscle biopsy in patients being considered for nerve transfer may be a useful prognostic tool to determine MEP viability in denervated muscle, with surviving MEPs also being targets for adjuvant therapy.

ABBREVIATIONS AChR = acetylcholine receptor; BPI = brachial plexus injury; EMG = electromyography; MEP = motor endplate; NMJ = neuromuscular junction.

In Brief

The study presents the first-ever description of the temporal profile of human motor endplate (MEP) degeneration following peripheral nerve injury (PNI) with the findings that MEP degeneration differs in time course between humans and mice or rodents and that denervated MEPs may persist in humans for years. The results highlight the importance of species-specific findings and may serve to objectively answer critical questions regarding the optimal timing of both surgical intervention and adjuvant treatments for PNIs.

Patients with traumatic brachial plexus injuries (BPIs) and injuries to individual peripheral nerves have particularly poor outcomes with limited functional recovery, even after optimal surgical management. As improvements in recovery have plateaued with surgical manipulations alone, cellular and molecular therapeutic regimens are required to further improve outcome. Yet, the potential targets and appropriate time to intervene can only be determined if there is a true understanding of the process of nerve and muscular degeneration1 secondary to a traumatic nerve injury. While animal models have shed light on molecular changes to the muscle and motor endplate (MEP) postinjury, the time course of degeneration in animal models is unlikely to be the same as in the human condition and thus cannot provide precise information that would help inform surgical intervention and the timing of adjuvant therapy.

Clinical studies in this area have focused on improving surgical treatment including primary repair; reconstruction using autograft, allograft, or nerve conduits; and, more recently, nerve transfers. However, these efforts only partially restore function to the affected limb, may result in donor site morbidity, and yield unpredictable outcomes.2 One reason for unpredictable results may be variable postsynaptic changes at the neuromuscular junction (NMJ) resulting in irreversible MEP degradation. The NMJ is the interface between the peripheral nervous system and the muscles it innervates, consisting of a presynaptic nerve terminal, an MEP, and perisynaptic Schwann cells. Mature MEPs are characterized by a pretzel-like form with multiple perforations, while immature plaque endplates are defined by their smaller size and lack of perforations.3,4 Following axonotmesis or neurotmesis, Wallerian degeneration of the original nerve and subsequent axonal regeneration can reconstitute the presynaptic nerve terminal under appropriate conditions.5 However, even if axonal regeneration is successful, degradation of the MEP can impede reinnervation and signal transduction across the NMJ, effectively resulting in permanent denervation of the target muscle.

Animal studies have demonstrated that the architectural arrangement of acetylcholine receptors (AChRs) at the postsynaptic MEP is critical for effective signal transduction at the NMJ. Long-term denervation in animals results in declustering and dispersion of AChRs, leading to disassembly of the MEPs.6–8 This phenomenon would effectively limit the therapeutic window for operative intervention in patients with axonotmesis or neurotmesis, with resulting atrophy of the target muscles. However, despite advances in understanding MEP plasticity in animals, temporal and spatial patterns of MEP degradation in humans are unknown.

The current understanding of neurological injury and regenerative outcomes in humans is based solely on clinical observations. Outcomes have been found to depend on a number of factors, including patient age, level of injury, gap size (in the case of transection), patient comorbidities, smoking status, associated injuries, and time to surgery.9–11 Of these, the only factor that can be modified by the surgeon is the time to surgery. Historically, studies have suggested that even in distal lesions, surgical interventions more than 6 months after injury rarely result in meaningful recovery.10,12,13 The reasons for this are not known but could reflect the fact that postsynaptic MEP degradation is approximately complete and irreversible by this time point. However, no studies have histologically examined or described the temporal profile of human MEP degradation after acute nerve injury to test this idea. While animal studies and clinical observations have historically served to guide surgical decision-making, appropriate timing of surgical intervention in humans can only be conclusively determined through a thorough understanding of the mechanisms underlying MEP degeneration following acute nerve injury in humans. It is reasonable to assume that human MEP degradation would occur similarly to murine or rodent MEP degradation but may have a different time course. The purpose of this study was to histologically define the morphology and temporal profile of MEP degradation in humans following nerve injury and provide objective evidence to aid surgeons in determining the appropriate timing of operative intervention for these injuries.

Methods

IRB approval along with informed patient consent was obtained to perform biopsies from denervated muscles in patients undergoing surgery for nerve injuries ranging from complete preganglionic C5–T1 BPI to less severe traumatic injuries such as isolated axillary nerve transections. Prior to performing any surgical intervention, electromyography (EMG) was performed by a board-certified neuromuscular-trained neurologist in all patients to confirm the absence of nerve action potentials, along with the presence of fibrillation potentials and positive sharp waves, indicating denervation. Muscle biopsies were obtained from a prospective cohort of patients beginning in 2015 and are here analyzed retrospectively. The timing of the muscle biopsy was based on the time of presentation to the operating surgeon as well as the clinical decision-making to provide the patient with the best therapeutic option currently available. Thus, muscle samples from multiple time points after injury were analyzed, as were control specimens from innervated muscles of healthy donors (obtained during routine standard-of-care procedures), to create a temporal sequence of events for human MEP degradation following traumatic nerve injury. For the one patient in this series who had an innervated free gracilis for a late BPI injury, we were able to successfully biopsy a positive control along with the denervated muscle from the same patient.

Specimens from the operating room were cryoprotected in Tissue-Tek optimal cutting temperature (OCT) mounting medium and snap-frozen using liquid nitrogen. Twenty-micrometer cross-sections were generated at −20°C using a microtome. Sections were then stained with H & E to evaluate the overall tissue composition and structure. Images were captured using an inverted microscope (IX71, Olympus).

A portion of the harvested specimens from the operating room was immediately snap-frozen, processed for immunohistochemistry, and visualized initially with confocal and subsequently with two-photon microscopy. Following overnight fixation in 4% paraformaldehyde, specimens were incubated in recombinant mouse anti–human synaptophysin (1:250, Dako) and purified mouse anti–human neurofilament (1:300, Covance) to label presynaptic vesicles and axons, respectively. After rinsing, specimens were then incubated in secondary antibodies conjugated to donkey anti–mouse Alexa Fluor 488 (1:300, Thermo Fisher Scientific) and α-bungarotoxin, Alexa Fluor 594 conjugate (1:1000, Thermo Fisher Scientific), to directly label MEPs.

Unsectioned tissue sample mounts of approximately 1-mm thickness were visualized via two-photon excitation microscopy given its primary advantage of superior optical sectioning in 3D imaging of thick specimens. The deeper penetration of two-photon excitation compared to standard confocal imaging allows for assessment of the spatial arrangement and morphometric properties of MEPs. Two-photon excitation was achieved using an 810-nm laser to excite both fluorophores simultaneously. Images were acquired with a custom microscope system by Intelligent Imaging Innovations (3i) using a 20×/0.8 water immersion objective lens.

Images obtained via two-photon excitation microscopy were used to create 3D reconstructions with Volocity imaging software (PerkinElmer). MEP surface area and volume were quantified using ImageJ (National Institutes of Health) with the 3D Object Counter plug-in using the optical fractionator method.14

Linear regression analysis was used to investigate any linear relationship between denervated muscle time in months and MEP volume or surface area. Analysis was performed using GraphPad Prism 8.0 (GraphPad Software Inc.). Data are reported as the mean ± standard error of the mean. An α value of 0.05 was set as the level of significance.

Results

All muscle biopsies were taken from 18 patients (17 males and 1 female, age range 22–61 years) with a definitive, clearly identifiable date of injury and were obtained during a clinically indicated standard-of-care surgical intervention. Thus, we obtained muscle biopsies from the same surgical incision used for these procedures, which ranged from a partial radial nerve to axillary nerve transfer to an Oberlin transfer. The time from injury to muscle biopsy ranged from 3 days to 6 years postinjury (Table 1). After analysis of the 15 deltoids, 3 biceps, and 2 first dorsal interossei, we were unable to detect any muscle-specific pattern of degeneration; rather, there appeared to be a time-dependent pattern of degeneration. As shown in Fig. 1A, gross atrophy of denervated deltoid muscle was clinically apparent. Biopsy of these muscles showed marked histological changes, including shrinkage of muscle fibers and perifascicular fat accumulation (Fig. 1B).

TABLE 1.

Summary of patient characteristics

Case No.Age (yrs)SexInjury Duration (mos)*Type of InjurySurgical TreatmentBiopsied Muscle
154M4Pan plexus BPINerve transfers ×4First dorsal interosseous
227M6ANIPartial radial to axillary nerveDeltoid
359M36ANIReverse total shoulder arthroplastyDeltoid
444M5BPIFree gracilisBiceps, deltoid, & gracilis
526M1GSW w/ BPIORIF humerusDeltoid
652M7ANIPartial radial to axillary nerveDeltoid
753M3BPIRemoval of hardwareDeltoid
835M3ANIPartial radial to axillary nerveDeltoid
924M9BPIPartial radial to axillary nerveDeltoid
1024M8GSW w/ BPINerve transfer for radial nerveDeltoid
1124M10Acromioclavicular separation w/ BPIAcromioclavicular reconstruction w/ allograftDeltoid
1222M72ANIPartial radial to axillary nerveDeltoid
1329F7Ulnar nerve injuryDegloving injury w/ segmental nerve defectFirst dorsal interosseous
1444M6BPINerve transfers ×2Biceps & deltoid
1528M6Proximal humerus osteomyelitis w/ ANIRemoval of hardwareDeltoid
1629M3GSW w/ BPIORIF clavicle fractureDeltoid
1727M5BPINerve transferBiceps
1861M4ANIPartial radial to axillary nerveDeltoid

ANI = axillary nerve injury; GSW = gunshot wound; ORIF = open reduction internal fixation.

Duration of patient’s nerve injury at the time of muscle biopsy procurement.

Expressed in days.

FIG. 1.
FIG. 1.

A: Clinical image showing prominent deltoid atrophy in a patient with traumatic axillary nerve injury. B: H & E staining of cross-sectional deltoid muscle fibers from muscle biopsy of patient. Bar = 100 µm. Figure is available in color online only.

Immunohistochemical analysis of denervated first dorsal interosseous, biceps, and deltoid muscle samples for MEP markers revealed distinct differences from innervated muscles of control specimens (Fig. 2), including the relative absence of normal-appearing MEPs, fragmentation and dispersion of AChRs, and an apparent shift toward plaque-like endplate morphology. Endplate morphology has been characterized as a spectrum ranging from mature pretzel-shaped endplates to immature plaque endplates, according to their distinct topographic features at the postsynaptic membrane.3 This morphometric dichotomy, as the MEP transitions from plaque to pretzel, is a hallmark of NMJ development.15,16 Interestingly, denervation seems to cause the mature pretzel morphology to regress back to the plaque morphology of the early embryonic developmental state.

FIG. 2.
FIG. 2.

Confocal images of human MEPs. A: Innervated deltoid. B: Four-month denervated first dorsal interosseous. C: Five-month denervated biceps. D: One-year denervated biceps. Red indicates α-bungarotoxin; green, neurofilament and synaptophysin; blue, DAPI. Bar = 50 μm. Figure is available in color online only.

Morphological comparison of denervated first dorsal interosseous, biceps, and deltoid muscles revealed temporal progression of degeneration. MEPs from recently denervated muscles demonstrated well-preserved circular morphology, with AChRs arranged in characteristic folding patterns (Figs. 2B, 2C, 3C, and 3D). By 1 year, MEPs exhibited greater fragmentation (Figs. 2D and 4). Moreover, synaptic gutters started to fade, and asymmetry in AChR distribution was noted (Figs. 2D and 4). This gradient of MEP degeneration needs to be rigorously characterized with future studies. Interestingly, even after 1 year of denervation, morphologically normal MEPs persisted. Although images using two-photon microscopy revealed a decrease in MEP surface area and volume, as seen in 3D reconstruction, as well as a loss of mature AChR morphology and a trend toward plaque endplate morphology, MEPs from more prolonged denervated time points did not demonstrate complete degeneration and disintegration (Fig. 5I and J).

FIG. 3.
FIG. 3.

Two-photon microscopy of human MEPs. A and B: Innervated deltoid muscle. C and D: Biceps muscle 4 months after denervation due to traumatic peripheral nerve injury. Red indicates α-bungarotoxin; green, neurofilament and synaptophysin. Bar = 50 μm. Figure is available in color online only.

FIG. 4.
FIG. 4.

Confocal images of human MEPs from biceps muscle 1 year after traumatic BPI. A: MEPs at high magnification. B: MEPs at lower magnification. Red indicates α-bungarotoxin; blue, DAPI; green, neurofilament and synaptophysin. Bar = 10 μm. Figure is available in color online only.

FIG. 5.
FIG. 5.

A–E: H & E staining of cross-sectional deltoid muscle fibers. Bar = 100 µm. F–J: Two-photon excitation microscopy of human MEPs. Red indicates α-bungarotoxin (BTX); green, neurofilament (NF) and synaptophysin (Syn). Bar = 50 µm. Figure is available in color online only.

Overall, comparison of denervated muscles revealed temporal degeneration. MEPs from acutely denervated muscles demonstrated well-preserved circular morphology with definite AChRs arranged in distinct folding patterns, while MEPs from more chronically denervated muscles revealed a trend toward plaque endplate morphology. Remarkably, innervated, morphologically preserved MEPs persisted in muscles that had been denervated for more than 3 years (Fig. 5I and J).

Morphometric quantification of MEPs from denervated deltoids compared to normal deltoid (Fig. 6) revealed a decrease in both MEP volume and surface area at the 0–10 months postdenervation time points, with an increase in MEP volume and surface area close to normal at the 36- and 72-month postdenervation time points. Significant negative correlations were observed between MEP volume and denervated time from 0 to 10 months (R = −0.92, R2 = 0.85, p = 0.001) as well as between MEP surface area and denervated time from 0 to 10 months (R = −0.75, R2 = 0.56, p = 0.032).

FIG. 6.
FIG. 6.

Regression analysis of human MEP morphometry from denervated human deltoids. MEP volume (A) and MEP surface area (B). Data are presented as the mean ± standard error of the mean.

Discussion

In this study we describe a novel method for assessing the functional potential of denervated human muscle tissue as a first step toward characterizing the temporal profile of human MEP degeneration after traumatic peripheral nerve injury. Although systematic sampling was not possible because the time postinjury was determined by the timing of standard-of-care surgeries for traumatic axillary nerve injuries and BPIs, the combination of data from multiple specimens herein provides critically important data about the timing of events involved in human MEP degradation after traumatic nerve injury. The key finding is that MEPs in denervated human muscles persist and retain their architecture for much longer than has been reported in murine and rodent studies. Indeed, some MEPs persist for years in muscles that are denervated based on all clinical criteria. This is of major importance clinically because MEPs persist long after the 6-month period that is considered by most clinicians to be the end of the window of opportunity for meaningful functional recovery. These findings are surprising because the consensus view based on data from animal studies is that AChRs would have dispersed and MEPs would have disappeared by the end of the critical 6-month window after injury.

Implications for Timing of Reparative Surgery Following Peripheral Nerve Injury

As adult human axons regenerate at a peak rate of approximately 1–3 mm per day,17 it often requires months before clinical signs of regeneration are apparent. However, late surgical intervention risks irreversible degradation of the target end organ, thus missing the critical window during which functional recovery is achievable.18 Degeneration of denervated MEPs is thought to be an important factor that limits this functional reinnervation of muscle. If so, an important factor in decision-making regarding surgical interventions to improve function is understanding the actual timing of human MEP degeneration. Cellular and molecular pathways at the NMJ offer potential targets for therapeutic intervention to improve outcomes for patients who have suffered traumatic nerve injuries, but to deploy therapeutic interventions, it is critical to first understand the unique aspects of human MEP degeneration in terms of both process and timing. Our results provide an initial glimpse into this important question. Most importantly, our results suggest that the window of opportunity for surgical intervention may extend far beyond the 6-month period that dominates in clinical decision-making. Indeed, our results suggest the provocative idea that it is not time after injury per se, but rather whether MEPs persist in human denervated muscles that would be receptive to reinnervation.

Our results also provide a framework in which to consider whether adjuvant therapies that have shown efficacy in mice and rodents warrant clinical trials in humans. Whereas murine and rodent models show significant functional deficits when reinnervation occurs beyond 2 months following injury,19 clinical observations suggest that this does not translate to optimal timing of surgical intervention in humans.13 One suggested surgical solution for the treatment of axillary nerve injuries is a partial radial nerve to axillary nerve transfer.20 However, this procedure has variable results and is not widely accepted currently. If human MEPs persist and retain their structures even after the previously postulated 6-month therapeutic window for operative intervention, then a partial radial nerve to axillary nerve transfer may indeed be indicated. The decision to undertake a nerve transfer is currently made clinically, with little guidance from objective data. Our results suggest that a preoperative muscle biopsy may offer additional data to aid in the decision-making process by providing direct visualization of the MEP, including its innervation status and morphometric properties.

Molecular Pathways Underlying MEP Degradation

Cellular and molecular pathways at the NMJ offer a novel target for therapeutic intervention to improve outcomes for patients who have suffered traumatic nerve injuries. Prior animal studies have demonstrated the therapeutic potential of targeting signaling molecules and enzymes such as agrin and matrix metalloproteinase 3 (MMP-3), which are involved in the maintenance of normal MEP architecture.19,21,22 However, no studies have explored the use of these therapeutic targets in humans. One of the greatest challenges facing translational research in this area lies in the species-specific differences between human and murine/rodent MEPs. Recent studies have demonstrated that human MEPs are morphologically distinct and molecularly divergent from murine/rodent MEPs.23 Furthermore, contrary to murine/rodent findings that have suggested that MEP degeneration increases with age,24,25 human MEPs have been shown to remain stable across the adult lifespan.23 Murine/rodent models have shown that the release of agrin by motor neurons is critical to the aggregation of AChRs at the MEP and that damage to the NMJ results in molecular changes to the MEP, including the dispersion of AChRs.7,26 Although studies in mice and rodents have provided crucial information, our current results suggest that information from murine/rodent studies may be of limited use in predicting the timing of postdenervation sequelae and possible treatment windows for future therapeutic interventions targeting the molecular machinery involved in MEP degradation.

We have previously demonstrated that long-term denervation in murine/rodent models is characterized by alterations in the MEP morphology from a mature pretzel appearance (perforated and containing membranous infoldings) to an immature plaque (diminished size and increased density), as well as an intermediate morphology in between the two.19 This switch to a plaque-like morphology is directly correlated with the critical time window beyond which functional recovery by reinnervation is severely limited because of degeneration of the MEP. An understanding of this degradation process is critical and applies not only to the treatment of traumatic peripheral nerve injuries, but also to progressive neurodegenerative diseases. For example, denervation without motor neuron loss has also been implicated in the early stages of amyotrophic lateral sclerosis in murine/rodent models as well as in humans.27

Unexpected Persistence of MEPs in Denervated Human Muscle

The unexpected persistence of human MEPs after prolonged denervation may have several explanations. One possibility is that these persistent MEPs arise from newly formed MEPs associated with new regenerating muscle fibers formed after denervation.28 Against this view, however, is the clinical evidence that indicates a persistent loss of motor input. Another possibility is reinnervation by adrenergic sympathetic axons from blood vessels in the muscle, which would restore input but not motor function. Recent findings also suggest a likely role of this sympathetic innervation at the MEP in the homeostatic regulation of AChR stability and maintenance, which may explain the persistence of human MEPs despite long-term motor denervation. For instance, sympathetic ablation experiments via surgical or chemical sympathectomy29 have implicated sympathetic-dependent regulation of the levels of postsynaptic membrane AChRs through AChR recycling and degradation via sympathetic control of two main pathways: PKA/cAMP30–32 and Gαi2-Hdac4-Myogenin-MuRF1.33–35 Moreover, studies of chemical sympathectomy of mouse tibialis anterior muscles resulted in significant electrophysiological and morphological deficits of the MEPs, but both phenotypes were rescued when treated with a sympathomimetic drug, suggesting a critical role of sympathetic innervation in the homeostatic maintenance of MEPs.29 While the identity of the axons at some surviving MEPs from functionally denervated muscles was unknown in this study, other possible sources besides adrenergic sympathetic axons from blood vessels in the muscle could be motor axons that have sprouted from nearby innervated muscles,36,37 cholinergic sympathetic axons from cutaneous sweat glands, or sensory axons.38 It is the goal of our ongoing and future studies to determine the identity of these axons.

Furthermore, the evaluation of MEP persistence may provide additional context for imaging modalities and EMG studies. Magnetic resonance imaging and ultrasound can be useful for identifying damaged nerves or muscle preoperatively, but they cannot predict regeneration potential,39–41which depends on the viability of the MEP within targeted muscle fibers.42 Clinicians also routinely use EMG studies to gather objective data about the status of muscle innervation, with fibrillation potentials and positive sharp waves as the hallmarks of a denervated muscle. It is well known from the poliomyelitis literature that these fibrillation potentials and positive sharp waves can persist for up to 30 years even in muscles that are end stage.43 This EMG finding does not reflect that the muscle is “viable” for reinnervation, but rather implies that the muscle membranes (sarcolemma) are unstable and prone to spontaneous depolarization.44 Currently, there is no EMG finding from a denervated muscle that positively correlates with receptivity for neural regeneration. And as reinnervation takes longer than 3 months, the absence of motor unit potential (MUP) does not suggest a neurotmesis or fourth- or fifth-degree Sunderland injury.45 Rather, recovery potential is related to compound muscle action potential (CMAP) amplitude, as it implies that there is continuity of the nerve, while the lack of volitional units implies that there is no nerve continuity. From these findings, it is clear that EMG has room for operator-dependent interpretation, and additional methods for objectively describing the innervation status of the muscle are required.

Our study herein is a critical first step toward defining the nature and time course of human MEP degeneration after traumatic nerve injury and provides a framework and quantifiable measures for future studies. By analyzing the morphometric properties of an even larger sample size of human MEPs at multiple time points after a distinct, identifiable injury from multiple academic centers, we will be able to define the actual sequence of structural changes postinjury to characterize human MEP degeneration. These ongoing multicenter studies could thereby provide objective data to guide decisions about the timing of surgical interventions and allow the formulation of testable hypotheses about the mechanisms that preserve denervated human MEPs. It is our targeted goal to determine preoperatively the currently unaddressed question of what density of viable MEPs is needed for a reinnervated muscle to achieve a good functional outcome. Further work is also necessary to better understand how and why presynaptic neuronal elements are actually present at these retained MEPs after chronic denervation injuries. As human traumatic nerve injuries are variable in both nature and severity, it would be prudent to conduct parallel studies in nonhuman primates with precise surgically created injuries to validate the human findings to ensure that there is no evidence of axonal sprouting from residual intact, though nonfunctional, nerve fibers. In addition, surrogate molecular biomarkers of reinnervation competency on preoperative muscle biopsy with successful nerve transfers could be identified to develop objective, predictive tools in surgical decision-making. The goal is to establish new evidence-based prognostic and diagnostic criteria to transform the surgical care and management of traumatic nerve injuries toward data-driven clinical decision-making. With the tremendous variability in clinical nerve surgery decision-making, additional efforts must be made toward creating objective evidence-based prognostic and diagnostic guides that can be widely disseminated.

Conclusions

This study highlights the implications and clinical relevance of species-specific findings in improving upper-extremity muscle function in human beings with denervation injuries and provides invaluable data regarding the persistence of MEPs beyond the anticipated 6-month clinical window for therapeutic intervention in select patients. These novel data from muscle biopsies may aid surgeons in determining the optimal timing of surgical intervention in the future and serve as an important first step toward translational research on adjuvant treatments for preventing or reversing muscle atrophy after nerve injury as well as establishing objective evidence-based prognostic and diagnostic criteria to guide decisions about the timing of surgical interventions.

Acknowledgments

This work was supported by intramural funding from the Department of Orthopaedic Surgery, University of California, Irvine.

Disclosures

Dr. Steward is a cofounder and has economic interests in the company Axonis, which holds a license on patents relating to PTEN deletion and axon regeneration. Dr. Ward is the Deputy Editor (compensated) of the journal Physical Therapy.

Author Contributions

Conception and design: Gupta, Palispis, Shah, Ward, Lee, Steward. Acquisition of data: Gupta, Chan, Uong, Palispis, Wright. Analysis and interpretation of data: all authors. Drafting the article: Gupta, Chan, Uong, Wright, Ward, Lee, Steward. Critically revising the article: Gupta, Uong, Shah, Steward. Reviewed submitted version of manuscript: Gupta, Uong, Shah, Ward, Lee, Steward. Approved the final version of the manuscript on behalf of all authors: Gupta. Statistical analysis: Gupta, Chan, Uong. Study supervision: Gupta.

Supplemental Information

Previous Presentations

Portions of this work were presented at the 2017 American Association of Neurological Surgeons Annual Scientific Meeting held in Los Angeles, California, on April 22–26, 2017, and the 2019 American Shoulder and Elbow Surgeons Annual Meeting held in New York, New York, on October 17–19, 2019.

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    Sunderland S. Rate of regeneration in human peripheral nerves; analysis of the interval between injury and onset of recovery. Arch Neurol Psychiatry. 1947;58(3):251295.

    • Search Google Scholar
    • Export Citation
  • 18

    Palispis WA, Gupta R. Surgical repair in humans after traumatic nerve injury provides limited functional neural regeneration in adults. Exp Neurol. 2017;290:106114.

    • Search Google Scholar
    • Export Citation
  • 19

    Chao T, Frump D, Lin M, Matrix metalloproteinase 3 deletion preserves denervated motor endplates after traumatic nerve injury. Ann Neurol. 2013;73(2):210223.

    • Search Google Scholar
    • Export Citation
  • 20

    Desai MJ, Daly CA, Seiler JG III, Radial to axillary nerve transfers: a combined case series. J Hand Surg Am. 2016;41(12):11281134.

  • 21

    Bezakova G, Rabben I, Sefland I, Neural agrin controls acetylcholine receptor stability in skeletal muscle fibers. Proc Natl Acad Sci U S A. 2001;98(17):99249929.

    • Search Google Scholar
    • Export Citation
  • 22

    Bezakova G, Helm JP, Francolini M, Lømo T. Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo. J Cell Biol. 2001;153(7):14411452.

    • Search Google Scholar
    • Export Citation
  • 23

    Jones RA, Harrison C, Eaton SL, Cellular and molecular anatomy of the human neuromuscular junction. Cell Rep. 2017;21(9):23482356.

  • 24

    Anis NA, Robbins N. General and strain-specific age changes at mouse limb neuromuscular junctions. Neurobiol Aging. 1987;8(4):309318.

    • Search Google Scholar
    • Export Citation
  • 25

    Valdez G, Tapia JC, Kang H, Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proc Natl Acad Sci U S A. 2010;107(33):1486314868.

    • Search Google Scholar
    • Export Citation
  • 26

    Reist NE, Werle MJ, McMahan UJ. Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions. Neuron. 1992;8(5):865868.

    • Search Google Scholar
    • Export Citation
  • 27

    Fischer LR, Culver DG, Tennant P, Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp Neurol. 2004;185(2):232240.

    • Search Google Scholar
    • Export Citation
  • 28

    Rodrigues A de C, Schmalbruch H. Satellite cells and myonuclei in long-term denervated rat muscles. Anat Rec. 1995;243(4):430437.

  • 29

    Khan MM, Lustrino D, Silveira WA, Sympathetic innervation controls homeostasis of neuromuscular junctions in health and disease. Proc Natl Acad Sci U S A. 2016;113(3):746750.

    • Search Google Scholar
    • Export Citation
  • 30

    Choi KR, Berrera M, Reischl M, Rapsyn mediates subsynaptic anchoring of PKA type I and stabilisation of acetylcholine receptor in vivo. J Cell Sci. 2012;125(Pt 3):714723.

    • Search Google Scholar
    • Export Citation
  • 31

    Röder IV, Strack S, Reischl M, Participation of myosin Va and Pka type I in the regeneration of neuromuscular junctions. PLoS One. 2012;7(7):e40860.

    • Search Google Scholar
    • Export Citation
  • 32

    Martinez-Pena y Valenzuela I, Pires-Oliveira M, Akaaboune M. PKC and PKA regulate AChR dynamics at the neuromuscular junction of living mice. PLoS One. 2013;8(11):e81311.

    • Search Google Scholar
    • Export Citation
  • 33

    Khan MM, Strack S, Wild F, Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors. Autophagy. 2014;10(1):123136.

    • Search Google Scholar
    • Export Citation
  • 34

    Wild F, Khan MM, Straka T, Rudolf R. Progress of endocytic CHRN to autophagic degradation is regulated by RAB5-GTPase and T145 phosphorylation of SH3GLB1 at mouse neuromuscular junctions in vivo. Autophagy. 2016;12(12):23002310.

    • Search Google Scholar
    • Export Citation
  • 35

    Rudolf R, Bogomolovas J, Strack S, Regulation of nicotinic acetylcholine receptor turnover by MuRF1 connects muscle activity to endo/lysosomal and atrophy pathways. Age (Dordr). 2013;35(5):16631674.

    • Search Google Scholar
    • Export Citation
  • 36

    English AW. Cytokines, growth factors and sprouting at the neuromuscular junction. J Neurocytol. 2003;32(5-8):943960.

  • 37

    Koob JW, Moradzadeh A, Tong A, Induction of regional collateral sprouting following muscle denervation. Laryngoscope. 2007;117(10):17351740.

    • Search Google Scholar
    • Export Citation
  • 38

    Liu H, Wang L, Li H, Formation of neuromuscular junction-like structure between primary sensory terminals and skeletal muscle cells in vitro. Anat Rec (Hoboken). 2011;294(3):472478.

    • Search Google Scholar
    • Export Citation
  • 39

    Gupta R, Villablanca PJ, Jones NF. Evaluation of an acute nerve compression injury with magnetic resonance neurography. J Hand Surg Am. 2001;26(6):10931099.

    • Search Google Scholar
    • Export Citation
  • 40

    Nwawka OK, Casaletto E, Wolfe SW, Feinberg JH. Ultrasound imaging of brachial plexus trauma in gunshot injury. Muscle Nerve. 2019;59(6):707711.

    • Search Google Scholar
    • Export Citation
  • 41

    Yoshikawa T, Hayashi N, Yamamoto S, Brachial plexus injury: clinical manifestations, conventional imaging findings, and the latest imaging techniques. Radiographics. 2006;26(suppl 1):S133S143.

    • Search Google Scholar
    • Export Citation
  • 42

    Feinberg JH, Radecki J, Wolfe SW, Brachial plexopathy/nerve root avulsion in a football player: the role of electrodiagnostics. HSS J. 2008;4(1):8795.

    • Search Google Scholar
    • Export Citation
  • 43

    Trojan DA, Gendron D, Cashman NR. Electrophysiology and electrodiagnosis of the post-polio motor unit. Orthopedics. 1991;14(12):13531361.

    • Search Google Scholar
    • Export Citation
  • 44

    Schiffer D, Palmucci L, Bertolotto A, Monga G. Mitochondrial abnormalities of late motor neuron degeneration following poliomyelitis and other neurogenic muscular atrophies. J Neurol. 1979;221(3):193201.

    • Search Google Scholar
    • Export Citation
  • 45

    Robinson LR. Role of neurophysiologic evaluation in diagnosis. J Am Acad Orthop Surg. 2000;8(3):190199.

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

Correspondence Ranjan Gupta: University of California, Irvine, CA. ranjang@uci.edu.

INCLUDE WHEN CITING Published online September 18, 2020; DOI: 10.3171/2020.8.JNS201461.

Disclosures Dr. Steward is a cofounder and has economic interests in the company Axonis, which holds a license on patents relating to PTEN deletion and axon regeneration. Dr. Ward is the Deputy Editor (compensated) of the journal Physical Therapy.

  • View in gallery

    A: Clinical image showing prominent deltoid atrophy in a patient with traumatic axillary nerve injury. B: H & E staining of cross-sectional deltoid muscle fibers from muscle biopsy of patient. Bar = 100 µm. Figure is available in color online only.

  • View in gallery

    Confocal images of human MEPs. A: Innervated deltoid. B: Four-month denervated first dorsal interosseous. C: Five-month denervated biceps. D: One-year denervated biceps. Red indicates α-bungarotoxin; green, neurofilament and synaptophysin; blue, DAPI. Bar = 50 μm. Figure is available in color online only.

  • View in gallery

    Two-photon microscopy of human MEPs. A and B: Innervated deltoid muscle. C and D: Biceps muscle 4 months after denervation due to traumatic peripheral nerve injury. Red indicates α-bungarotoxin; green, neurofilament and synaptophysin. Bar = 50 μm. Figure is available in color online only.

  • View in gallery

    Confocal images of human MEPs from biceps muscle 1 year after traumatic BPI. A: MEPs at high magnification. B: MEPs at lower magnification. Red indicates α-bungarotoxin; blue, DAPI; green, neurofilament and synaptophysin. Bar = 10 μm. Figure is available in color online only.

  • View in gallery

    A–E: H & E staining of cross-sectional deltoid muscle fibers. Bar = 100 µm. F–J: Two-photon excitation microscopy of human MEPs. Red indicates α-bungarotoxin (BTX); green, neurofilament (NF) and synaptophysin (Syn). Bar = 50 µm. Figure is available in color online only.

  • View in gallery

    Regression analysis of human MEP morphometry from denervated human deltoids. MEP volume (A) and MEP surface area (B). Data are presented as the mean ± standard error of the mean.

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    Chan JP, Clune J, Shah SB, Examination of the human motor endplate after brachial plexus injury with two-photon microscopy. Muscle Nerve. 2020;61(3):390395.

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    Steinbach JH. Developmental changes in acetylcholine receptor aggregates at rat skeletal neuromuscular junctions. Dev Biol. 1981;84(2):267276.

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    • Export Citation
  • 17

    Sunderland S. Rate of regeneration in human peripheral nerves; analysis of the interval between injury and onset of recovery. Arch Neurol Psychiatry. 1947;58(3):251295.

    • Search Google Scholar
    • Export Citation
  • 18

    Palispis WA, Gupta R. Surgical repair in humans after traumatic nerve injury provides limited functional neural regeneration in adults. Exp Neurol. 2017;290:106114.

    • Search Google Scholar
    • Export Citation
  • 19

    Chao T, Frump D, Lin M, Matrix metalloproteinase 3 deletion preserves denervated motor endplates after traumatic nerve injury. Ann Neurol. 2013;73(2):210223.

    • Search Google Scholar
    • Export Citation
  • 20

    Desai MJ, Daly CA, Seiler JG III, Radial to axillary nerve transfers: a combined case series. J Hand Surg Am. 2016;41(12):11281134.

  • 21

    Bezakova G, Rabben I, Sefland I, Neural agrin controls acetylcholine receptor stability in skeletal muscle fibers. Proc Natl Acad Sci U S A. 2001;98(17):99249929.

    • Search Google Scholar
    • Export Citation
  • 22

    Bezakova G, Helm JP, Francolini M, Lømo T. Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo. J Cell Biol. 2001;153(7):14411452.

    • Search Google Scholar
    • Export Citation
  • 23

    Jones RA, Harrison C, Eaton SL, Cellular and molecular anatomy of the human neuromuscular junction. Cell Rep. 2017;21(9):23482356.

  • 24

    Anis NA, Robbins N. General and strain-specific age changes at mouse limb neuromuscular junctions. Neurobiol Aging. 1987;8(4):309318.

    • Search Google Scholar
    • Export Citation
  • 25

    Valdez G, Tapia JC, Kang H, Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proc Natl Acad Sci U S A. 2010;107(33):1486314868.

    • Search Google Scholar
    • Export Citation
  • 26

    Reist NE, Werle MJ, McMahan UJ. Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions. Neuron. 1992;8(5):865868.

    • Search Google Scholar
    • Export Citation
  • 27

    Fischer LR, Culver DG, Tennant P, Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp Neurol. 2004;185(2):232240.

    • Search Google Scholar
    • Export Citation
  • 28

    Rodrigues A de C, Schmalbruch H. Satellite cells and myonuclei in long-term denervated rat muscles. Anat Rec. 1995;243(4):430437.

  • 29

    Khan MM, Lustrino D, Silveira WA, Sympathetic innervation controls homeostasis of neuromuscular junctions in health and disease. Proc Natl Acad Sci U S A. 2016;113(3):746750.

    • Search Google Scholar
    • Export Citation
  • 30

    Choi KR, Berrera M, Reischl M, Rapsyn mediates subsynaptic anchoring of PKA type I and stabilisation of acetylcholine receptor in vivo. J Cell Sci. 2012;125(Pt 3):714723.

    • Search Google Scholar
    • Export Citation
  • 31

    Röder IV, Strack S, Reischl M, Participation of myosin Va and Pka type I in the regeneration of neuromuscular junctions. PLoS One. 2012;7(7):e40860.

    • Search Google Scholar
    • Export Citation
  • 32

    Martinez-Pena y Valenzuela I, Pires-Oliveira M, Akaaboune M. PKC and PKA regulate AChR dynamics at the neuromuscular junction of living mice. PLoS One. 2013;8(11):e81311.

    • Search Google Scholar
    • Export Citation
  • 33

    Khan MM, Strack S, Wild F, Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors. Autophagy. 2014;10(1):123136.

    • Search Google Scholar
    • Export Citation
  • 34

    Wild F, Khan MM, Straka T, Rudolf R. Progress of endocytic CHRN to autophagic degradation is regulated by RAB5-GTPase and T145 phosphorylation of SH3GLB1 at mouse neuromuscular junctions in vivo. Autophagy. 2016;12(12):23002310.

    • Search Google Scholar
    • Export Citation
  • 35

    Rudolf R, Bogomolovas J, Strack S, Regulation of nicotinic acetylcholine receptor turnover by MuRF1 connects muscle activity to endo/lysosomal and atrophy pathways. Age (Dordr). 2013;35(5):16631674.

    • Search Google Scholar
    • Export Citation
  • 36

    English AW. Cytokines, growth factors and sprouting at the neuromuscular junction. J Neurocytol. 2003;32(5-8):943960.

  • 37

    Koob JW, Moradzadeh A, Tong A, Induction of regional collateral sprouting following muscle denervation. Laryngoscope. 2007;117(10):17351740.

    • Search Google Scholar
    • Export Citation
  • 38

    Liu H, Wang L, Li H, Formation of neuromuscular junction-like structure between primary sensory terminals and skeletal muscle cells in vitro. Anat Rec (Hoboken). 2011;294(3):472478.

    • Search Google Scholar
    • Export Citation
  • 39

    Gupta R, Villablanca PJ, Jones NF. Evaluation of an acute nerve compression injury with magnetic resonance neurography. J Hand Surg Am. 2001;26(6):10931099.

    • Search Google Scholar
    • Export Citation
  • 40

    Nwawka OK, Casaletto E, Wolfe SW, Feinberg JH. Ultrasound imaging of brachial plexus trauma in gunshot injury. Muscle Nerve. 2019;59(6):707711.

    • Search Google Scholar
    • Export Citation
  • 41

    Yoshikawa T, Hayashi N, Yamamoto S, Brachial plexus injury: clinical manifestations, conventional imaging findings, and the latest imaging techniques. Radiographics. 2006;26(suppl 1):S133S143.

    • Search Google Scholar
    • Export Citation
  • 42

    Feinberg JH, Radecki J, Wolfe SW, Brachial plexopathy/nerve root avulsion in a football player: the role of electrodiagnostics. HSS J. 2008;4(1):8795.

    • Search Google Scholar
    • Export Citation
  • 43

    Trojan DA, Gendron D, Cashman NR. Electrophysiology and electrodiagnosis of the post-polio motor unit. Orthopedics. 1991;14(12):13531361.

    • Search Google Scholar
    • Export Citation
  • 44

    Schiffer D, Palmucci L, Bertolotto A, Monga G. Mitochondrial abnormalities of late motor neuron degeneration following poliomyelitis and other neurogenic muscular atrophies. J Neurol. 1979;221(3):193201.

    • Search Google Scholar
    • Export Citation
  • 45

    Robinson LR. Role of neurophysiologic evaluation in diagnosis. J Am Acad Orthop Surg. 2000;8(3):190199.

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