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2021 BY T HE J OURNAL OF B ONE AND J OINT S URGERY, I NCORPORATED

Brief Electrical Stimulation Accelerates Axon

Regeneration and Promotes Recovery Following
Nerve Transection and Repair in Mice
Junichi Sayanagi, MD, Jesús A. Acevedo-Cintrón, BS, Deng Pan, BS, Lauren Schellhardt, BA, Daniel A. Hunter,
Alison K. Snyder-Warwick, MD, Susan E. Mackinnon, MD, and Matthew D. Wood, PhD
Downloaded from http://journals.lww.com/jbjsjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 04/10/2022

Investigation performed at the Division of Plastic and Reconstructive Surgery, Department of Surgery, Washington University School of Medicine,
St. Louis, Missouri

Background: Clinical outcomes following nerve injury repair can be inadequate. Pulsed-current electrical stimulation
(ES) is a therapeutic method that facilitates functional recovery by accelerating axon regeneration. However, current
clinical ES protocols involve the application of ES for 60 minutes during surgery, which can increase operative complexity
and time. Shorter ES protocols could be a strategy to facilitate broader clinical adoption. The purpose of the present study
was to determine if a 10-minute ES protocol could improve outcomes.
Methods: C57BL/6J mice were randomized to 3 groups: no ES, 10 minutes of ES, and 60 minutes of ES. In all groups, the
sciatic nerve was transected and repaired, and, in the latter 2 groups, ES was applied after repair. Postoperatively, changes to
gene expression from dorsal root ganglia were measured after 24 hours. The number of motoneurons regenerating axons was
determined by retrograde labeling at 7 days. Histomorphological analyses of the nerve were performed at 14 days. Function
was evaluated serially with use of behavioral tests up to 56 days postoperatively, and relative muscle weight was evaluated.
Results: Compared with the no-ES group, both ES groups demonstrated increased regeneration-associated gene
expression within dorsal root ganglia. The 10-minute and 60-minute ES groups demonstrated accelerated axon regen-
eration compared with the no-ES group based on increased numbers of labeled motoneurons regenerating axons (mean
difference, 202.0 [95% confidence interval (CI), 17.5 to 386.5] and 219.4 [95% CI, 34.9 to 403.9], respectively) and
myelinated axon counts (mean difference, 559.3 [95% CI, 241.1 to 877.5] and 339.4 [95% CI, 21.2 to 657.6],
respectively). The 10-minute and 60-minute ES groups had improved behavioral recovery, including on grid-walking
analysis, compared with the no-ES group (mean difference, 11.9% [95% CI, 3.8% to 20.0%] and 10.9% [95% CI, 2.9% to
19.0%], respectively). There was no difference between the ES groups in measured outcomes.
Conclusions: A 10-minute ES protocol accelerated axon regeneration and facilitated functional recovery.
Clinical Relevance: The brief (10-minute) ES protocol provided similar benefits to the 60-minute protocol in an acute
sciatic nerve transection/repair mice model and merits further studies.

P
eripheral nerve injuries are common, affecting 2.6% of
patients with upper-extremity trauma and 1.2% of those
with lower-extremity trauma1. Despite repair of nerve
injuries, clinical outcomes can be inadequate2-5. Slow axonal
growth can be a cause of inadequate recovery, and, for severe
axonotmetic (Sunderland III) or neurotmetic (Sunderland IV
and V) nerve injuries, axon outgrowth across a repair site
occurs at different rates in a staggered fashion, further delaying
regeneration and recovery6. Thus, strategies for accelerating
axonal regeneration would improve surgical management.
Therapeutic electrical stimulation (ES) protocols, with
ES being applied intraoperatively during nerve surgery, are a
promising strategy. Animal data have demonstrated that the
application of pulsed-current ES for 60 minutes accelerates the
number of motor and sensory axons regenerating and reaching
their appropriate end-organ targets6-10. ES achieves these effects
by augmenting early expression of regeneration-associated genes
(RAGs) within neurons, including brain-derived neurotrophic
factors (BDNFs)7,11-13. From these promising data, clinical trials
involving the use of a 60-minute ES protocol have demonstrated

Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJS/G615).

J Bone Joint Surg Am. 2020;103:e80(1-9) d http://dx.doi.org/10.2106/JBJS.20.01965

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Fig. 1
Schematic illustrating the experimental design and the allocation of animals for the various outcome measures, with end points indicated for each metric.

improved outcomes after nerve surgery from as early as 201014,
yet only 3 additional trials have been performed since 201015-17.
A factor that may limit translation of ES is the current
utilization of a 60-minute stimulation period. Care providers
will find a lengthy ES protocol during surgery to be an obstacle as
it can increase operative time, which negatively affects costs and
complication risks18-21, or necessitate more complex recovery
procedures to implement ES protocols17. Shorter ES protocols,
therefore, could increase translation.
In the present study, we assessed whether a shorter ES
protocol could promote improved nerve regeneration and re-
covery in the context of a repaired traumatic nerve injury. We
hypothesized that a shorter ES protocol could improve nerve
regeneration and functional recovery and tested our hypothesis
with use of a mouse sciatic nerve transection and repair model.
ticular nerve injury and repair model was unlikely to show dif-
ferences between groups regarding histological findings at late end
points, axon regeneration was measured at early end points as it
could detect differences in rates of regeneration25,26. Functional
recovery alone was measured to a longer end point as this longer
end point would still capture whether accelerated axon regener-
ation led to differences in recovery.
Surgical Procedures
Mice were anesthetized with a cocktail of ketamine (100 mg/kg)
(Fort Dodge Animal Health) and dexmedetomidine (0.5 mg/kg)
(Pfizer Animal Health). The right sciatic nerve was exposed under
sterile conditions, transected 2 mm proximal to the sciatic tri-
furcation, and repaired immediately with 11-0 nylon epineural

Materials and Methods

Experimental Design

A ll experiments were approved by the Washington University

in St. Louis Institutional Animal Care and Use Committee
and performed in compliance with the National Institutes of
Health guidelines. Ninety-nine male C57BL/6J mice (age, 7 weeks;
weight, 20 to 25 g) (Jackson Laboratories) were used to evaluate
the effects of ES on nerve regeneration (Fig. 1), 20 mice were used
to determine an evaluation end point for axon regeneration, and 5
mice were used as controls for gene analysis. For the primary
experiments, mice were randomized to 3 groups: (1) no ES, (2) 10
minutes of ES, and (3) 60 minutes of ES. Ten minutes was chosen
as the lower limit for ES based on previous studies22-24. Mice were
monitored daily in a temperature-controlled central animal
facility with free access to chow and water.
Previous studies assessing the effects of electrical stimula-
tion on nerve regeneration after repair showed that the primary
effect was to accelerate axon regeneration, which led to functional
recovery7,11. Therefore, we designed the study with specific end Fig. 2
points representing early and still-ongoing axon regeneration in Photograph made during the experimental procedure. The sciatic nerve
order to determine whether the application of ES accelerated axon was transected and repaired 2 mm proximal to the sciatic trifurcation. ES
regeneration compared with that after repair alone. Gene analysis was applied after repair for 10 or 60 minutes at a location 2 mm proximal to
was performed on dorsal root ganglia at 24 hours, as neuron the repair site, with a hook electrode being used to secure the nerve and a
bodies are affected by ES within the first 48 hours. As this par- return electrode placed proximal to the injury in the fascia.
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sutures (Sharpoint). For applicable groups, ES was performed as
described previously27. A stainless-steel wire electrode (Compo-
nent Supply Company) was hooked around the sciatic nerve
2 mm proximal to the repair site. A return electrode was placed
into subcutaneous tissue. The electrodes were connected to the ES
device (Checkpoint Stimulator/Locator; Checkpoint Surgical). ES
was delivered for 10 or 60 minutes (16 Hz, 0.5 mA) (Fig. 2). After
ES, the electrodes were removed and the fascia and skin were
closed with 6-0 nylon sutures (Ethicon). Postoperative pain was
managed with buprenorphine SR (sustained-release) (0.05 mg/
kg) (ZooPharm). At terminal end points, mice were killed with
administration of sodium pentobarbital (>200 mg/kg).
Gene Analysis (qRT-PCR) of Dorsal Root Ganglia
Gene expression from dorsal root ganglia was assessed 24 hours
postoperatively; this end point was chosen on the basis of previ-
ous studies11. Total RNA was prepared from combined L3 and L4
dorsal root ganglia explants from each animal. RNA was extract-
ed, measured, and reverse-transcribed to cDNA as described
previously28. Quantitative real-time polymerase chain reaction
(qRT-PCR) was performed with use of a StepOnePlus thermo-
cycler (Applied Biosystems) using Taqman Master Mix (Applied
Biosystems) reagents with the following primer pairs: Bdnf
(Mm04230607_s1), Atf3 (Mm00476033_m1), Il6 (Mm00446190_
m1), Csf1 (Mm00432684_m1), Ccl2 (Mm00441242_m1), and
Actb (Mm02619580_g1) (Thermo Fisher Scientific). Genetic
expression levels were normalized to Actb. Relative gene expression
changes were calculated from gene expression levels obtained from
dorsal root ganglia of uninjured mice.
Retrograde Labeling of Motoneurons
Retrograde labeling of motoneurons regenerating their axons was
performed on anesthetized mice in which the sciatic nerve was re-
explored and transected 3 mm distal to the original repair site. The
proximal cut end was placed in a well of petroleum jelly (Vaseline;
Unilever) filled with 4% Fluoro-Gold (Sigma-Aldrich). After 45
minutes, the well and Fluoro-Gold were removed, and the wounds
were closed. One week later, spinal cords were harvested and fixed
in 4% paraformaldehyde in phosphate-buffered saline solution
(PBS) overnight, followed by immersion in 30% sucrose in PBS for
24 hours. Tissues were frozen in OCT Compound (VWR) and cut
into 30-mm longitudinal sections on glass slides. The total number
of Fluoro-Gold-labeled cell bodies on every section was counted
with use of an Olympus IX81 microscope (Olympus).
Histomorphological Analysis
Histomorphological analyses of the sciatic nerve 3 mm distal to the
repair site were performed 14 days postoperatively, as described
previously29,30. Briefly, harvested sciatic nerves were fixed in 3%
glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated,

Fig. 3
Bar graphs illustrating that ES elevates regeneration-associated gene expression from dorsal root ganglia. Expression levels of Bdnf, Atf3, IL-6, Csf1, and Ccl2 were
measured from pooled L3-L4 dorsal root ganglia harvested at 24 hours postoperatively. Uninjured nerve dorsal root ganglia were used for comparison. All data are
expressed as the mean and the standard deviation (n = 8 for the experimental groups and n = 5 for the control group [Normal]). *P < 0.05. **P < 0.01. ***P < 0.001.
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Fig. 4
Figs. 4-A through 4-E Illustrations depicting how ES improves motor axon regeneration. Fig. 4-A Schematic of retrograde labeling, with the Fluoro-Gold label
being applied 3 mm distal to the nerve repair site. Fig. 4-B Fluorescence photomicrograph showing representative longitudinal sections of spinal cords with Fluoro-
Gold-labeled motoneurons (yellow) for the no-ES group. Scale bars = 200 mm. Fig. 4-C Line graph showing the increase in the number of labeled motoneurons over
time in the no-ES group. Uninjured mice were used as controls (Normal group), represented as the dotted line. The data are expressed as the mean and the
standard deviation. ***P < 0.001 compared with Day 7. Fig. 4-D Fluorescence photomicrographs showing longitudinal sections of spinal cords with Fluoro-Gold-
labeled motoneurons (yellow) for all experimental groups at 7 days postoperatively. Scale bars = 200 mm. Fig. 4-E Line graph showing the number of motoneurons
at Day 7 in the no-ES group and both ES groups. The data are expressed as the mean and the standard deviation. N = 5 per group. *P < 0.05.

embedded in epoxy resin, and sectioned on an ultramicrotome to Grid-Walking Analysis

yield 1-mm cross-sections. Slides were counterstained with 1%
toluidine blue and analyzed at 1,000· with a Laborlux S micro-
scope (Leitz). Slides were assessed with use of custom histomor-
phometry software (Clemex Vision Professional; Clemex
Technologies). Histological measures were calculated with use of
>75% of the cross-sectional area of the nerve.
A grid-walking test was performed twice per week from pre-
operatively (Day 0) to 56 days postoperatively28,31. Mice were
acclimated to an elevated wire mesh with a grid size measuring
2.5 · 2.5 cm for 5 minutes before recording of the total number
of steps of the ipsilateral hindlimb for 3 minutes. The number
of total steps and slipped steps missing the mesh and going
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Fig. 5
Histomorphological sections of the sciatic nerve, made 3 mm distal to the repair site at 14 days postoperatively, illustrating that ES improved myelinated
axon regeneration. Toluidine blue staining of cross-sectional slices showed myelinated axons in all groups. Scale bars = 20 mm.

through the grid were counted, and the proportion of foot
faults was reported.
Mechanical and Cold Sensitivity Analyses
Mechanical and cold sensitivity analyses were performed at a 56-
day end point. Mice were acclimated to an elevated meshed metal
grid for 1 hour before measuring paw-withdrawal thresholds
with use of von Frey filaments (0.02 to 2.56 g) (TouchTest; North
Coast Medical), where diminished sensation was indicated by an
ipsilateral:contralateral ratio of <126,32. Cold sensitivity was as-
sessed by means of acetone-evoked evaporative cooling27. A drop
of acetone was released onto the plantar surface of the hindlimb,
and the time of response was measured.
required to detect significant differences. Therefore, the group
size for each experiment was at least 5 mice.
For comparisons of retrograde labeling, histomorphometry,
sensitivity tests, and muscle wet weight, 1-way analysis of variance
(ANOVA) followed by Tukey-Kramer post hoc testing was used. In
the grid-walking analysis, following 2-way repeated-measures
ANOVA, Tukey-Kramer post hoc testing was used for multiple
comparisons between groups at each time point. All statistical
analyses were performed with use of JMP software (version 13; SAS
Institute). The level of significance was set at p < 0.05. The data in
the figures are expressed as the mean and the standard deviation
(SD), whereas the data in the Results section are expressed as the
mean difference and the 95% confidence interval (CI).

Muscle Wet Weight Source of Funding

The tibialis anterior and gastrocnemius muscles were dissected This work was funded by an industry-sponsored research
and weighed 56 days postoperatively. The ipsilateral:contra- agreement from Checkpoint Surgical to Washington Uni-
lateral ratio was calculated. versity through one of the authors (M.D.W.).

Statistical Analysis Results

The primary outcome during experimental planning was mye-
linated axon counts26. A power analysis (alpha = 0.05, power =
0.8, effect size = 0.86) was performed considering the effect of ES
on regenerated myelinated axon counts27 with use of G*power
3.1.9.7 (Heinrich Heine University). Five mice per group were
Gene Analysis of Dorsal Root Ganglia
D orsal root ganglia harvested at 24 hours from all 3 groups
revealed that nerve injury elevated the expression of
regeneration-associated genes, Bdnf and Atf3, as well as inflam-
matory genes, Il6, Csf1, and Ccl2 (Fig. 3). However, both the

TABLE I Histomorphometric Data of Distal Repaired Sciatic Nerves at 14 Days Postoperatively*

Outcome Metric Uninjured No ES 10-Minute ES 60-Minute ES

Myelinated axon count (no.) 4,521 ± 468 496 ± 218 1,055 ± 378§ 835 ± 239‡
Myelinated axon density (axons/mm2) 29,822 ± 2,685 3,133 ± 1,377 6,344 ± 3,024† 5,531 ± 2,029
Percent nerve area (%) 72.14 ± 2.04 3.22 ± 1.62 7.13 ± 3.65† 5.15 ± 1.89
Fiber width (mm) 4.78 ± 0.24 3.21 ± 0.41 3.32 ± 0.14 3.02 ± 0.22
G-ratio 0.60 ± 0.02 0.51 ± 0.06 0.49 ± 0.03 0.54 ± 0.03

*All data are expressed as the mean and the standard deviation (n = 10 for each group). ‡p < 0.05, †p < 0.01, §p < 0.001 compared with the no-ES
group (1-way ANOVA followed by a post hoc Tukey-Kramer test).
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Fig. 6
Figs. 6-A through 6-D Illustrations showing that ES improved motor
recovery. Foot faults represent steps that miss the grid rungs and enter the
grid pattern. Preoperative values are represented at Day 0. Fig. 6-A
Schematic of the grid-walking test, which involves motor coordination as
well as proprioceptive and other sensory feedback. Figs. 6-B, 6-C, and 6-D
Quantification of foot faults (proportion of foot fault steps to total steps for
the affected limb). All data are expressed as the mean and the standard
deviation (n = 10 per group). Fig. 6-B †P < 0.05 between Day 7 and all time
points from Day 28 onward in the no-ES group.^P < 0.05 between Day 7 and
all time points from Day 17 onward in the 10-minute-ES group. ‡P < 0.05
between Day 7 and all time points from Day 17 onward in the 60-minute-ES
group. #P < 0.05 between the no-ES group and both ES groups from Day 17
onward. The arrows indicate that significant differences continued until
56 days postoperatively. Figs. 6-C and 6-D Bar graphs showing the
quantification of foot faults in the no-ES and ES groups at Days 17 and 56,
respectively. *P < 0.05. **P < 0.01.
10-minute-ES and 60-minute-ES groups revealed significantly
elevated expression of Bdnf (mean difference, 1.04 [95% CI, 0.44
to 1.64] and 1.54 [95% CI, 0.94 to 2.13], respectively), Atf3 (mean
difference, 22.9 [95% CI, 3.6 to 42.1] and 32.3 [95% CI, 13.0 to
51.6], respectively), and Il6 (mean difference, 13.4 [95% CI, 4.5 to
22.2] and 11.1 [95% CI, 2.3 to 19.9], respectively), in comparison
with the no-ES group. Expression levels of Csf1 in the 60-minute-
ES group (mean difference, 1.78 [95% CI, 0.83 to 2.72]) and Ccl2
in the 10-minute-ES group (mean difference, 2.38 [95% CI, 0.47
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to 4.28]) were significantly higher than that in the no-ES group.
Gene expression levels were not significantly different between
the 10-minute-ES and 60-minute-ES groups.
Motor Nerve Regeneration
Retrograde labeling of motoneurons regenerating their axons
from the repair site without ES revealed staggered axon regen-
eration. There were significantly greater numbers of labeled
motoneurons 14 days compared with 7 days postoperatively
(mean difference, 355.4 [95% CI, 217.7 to 493.1]) (Figs. 4-A, 4-B,
and 4-C), but there were no differences after 14 days. Based
on these results, we assessed the number of motoneurons re-
generating axons in the ES groups at 7 days postoperatively. The
numbers of labeled motoneurons in the 10-minute-ES and 60-
minute ES groups were significantly greater than those in the no-
ES group (mean difference, 202.0 [95% CI, 17.5 to 386.5] and
219.4 [95% CI, 34.9 to 403.9], respectively). No significant dif-
ference was observed between the 10-minute-ES and 60-minute-
ES groups (Figs. 4-D and 4-E).
Histomorphological Analysis
Histomorphological analyses of the sciatic nerve 3 mm distal to
the repair site at 14 days revealed ongoing axon regeneration, as
normal sciatic nerve contains ;4,500 myelinated axons. Mye-
linated axon regeneration in the ES groups was increased com-
pared with the no-ES group (Fig. 5, Table I). The 10-minute-ES
and 60-minute-ES groups had significantly greater myelinated
axon counts than the no-ES group (mean difference, 559.3 [95%
CI, 241.1 to 877.5] and 339.4 [95% CI, 21.2 to 657.6], respec-
tively). Myelinated axon density and percent nerve area in the
10-minute-ES group were significantly higher than those in the
no-ES group (mean difference, 3,211.3 axons/mm2 [95% CI,
718.7 to 5,703.9 axons/mm2] and 3.91% [95% CI, 1.08% to
6.74%], respectively). No significant differences were observed
among the 3 groups in terms of fiber width and g-ratio.
Fig. 7
Figs. 7-A and 7-B Bar graphs illustrating that ES improved mechanical
sensitivity but not cold sensitivity. All data are expressed as the mean and the
standard deviation (n = 5 per group). **P < 0.01. Fig. 7-A Mechanical
sensitivity of the affected foot based on von Frey monofilament tests at
56 days postoperatively. In uninjured nerve (dashed line), there is no ipsi-
lateral/contralateral difference in the paw-withdrawal threshold ratio (ratio =
;1). Fig. 7-B Cold allodynia responses to the application of cold acetone to
the affected foot, measured at 56 days postoperatively. Mean data for the
uninjured (contralateral) nerve (n = 15) are represented by the dashed line.
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Fig. 8
Figs. 8-A and 8-B Bar graphs illustrating that ES increased muscle recovery as
expressed as relative muscle weight (i.e., the ratio of affected muscle weight
to the contralateral muscle weight). All data are expressed as the mean and
the standard deviation (n = 10 per group). *P < 0.05. **P < 0.01. ***P <
0.001. Fig. 8-A Gastrocnemius muscle. Fig. 8-B Tibialis anterior muscle.
Grid-Walking Test
All animals demonstrated few foot faults prior to injury as
measured on Day 0 but then showed impaired function based
on increased foot faults after the nerve injury (Figs. 6-A and 6-
B). Recovery, while incomplete, was appreciated in all animals
during the observation period following surgery. Compared
with Day 7, the 10-minute-ES and 60-minute-ES groups
demonstrated significantly fewer foot faults from Day 17
onward (mean difference, 14.8% [95% CI, 5.6% to 24.0%] and
13.3% [95% CI, 4.3% to 22.3%], respectively). In contrast, the
no-ES group demonstrated significantly fewer foot faults from
Day 28 onward (mean difference, 13.1% [95% CI, 2.5% to
23.7%]) (Fig. 6-B). By Day 17, the 10-minute-ES and 60-
minute-ES groups demonstrated significantly fewer foot faults
(mean difference, 10.5% [95% CI, 1.1% to 19.9%] and 10.9%
[95% CI, 1.5% to 20.3%]) compared with the no-ES group
(Fig. 6-C). At Day 56, this improved functional recovery based
on foot faults was maintained, with the 10-minute-ES and 60-
minute-ES groups being significantly different (mean differ-
ence, 11.9% [95% CI, 3.8% to 20.0%] and 10.9% [95% CI,
2.9% to 19.0%], respectively) as compared with the no-ES
group (Fig. 6-D). There was no significant difference between
the 2 ES groups on the grid-walking test.
Mechanical and Cold Sensitivity
Prior to nerve injury, the groups demonstrated no ipsilateral/
contralateral difference in terms of the paw-withdrawal thresh-
old on mechanical sensitivity analysis (ratio, ;1). After nerve
injury, the groups demonstrated decreased mechanical sensi-
tivity (ratio, >1). Fifty-six days postoperatively, the 10-minute-
ES and 60-minute-ES groups demonstrated significant differ-
ences in the ipsilateral/contralateral ratio of the paw-withdrawal
threshold as compared with the no-ES group (mean difference,
2.21 [95% CI, 0.90 to 3.52] and 2.16 [95% CI, 0.85 to 3.48],
respectively) (Fig. 7-A).
Prior to nerve injury, all groups had some sensitivity to
cold acetone (;3.2 seconds of response). After nerve injury,
the groups demonstrated increased cold sensitivity. No signif-
icant differences among the groups in terms of cold sensitivity
were found by Day 56 (Fig. 7-B).
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Muscle Wet Weight
The relative gastrocnemius muscle weight was significantly
greater for the 10-minute-ES and 60-minute-ES groups as
compared with the no-ES group (mean difference, 0.08 [95%
CI, 0.01 to 0.16] and 0.10, [95% CI, 0.03 to 0.18]). Similarly,
the relative tibialis anterior muscle weight was significantly
greater for the 10-minute-ES and 60-minute-ES groups as
compared with the no-ES group (mean difference, 0.16 [95%
CI, 0.07 to 0.24] and 0.14 [95% CI, 0.05 to 0.22] respectively)
(Fig. 8).
Discussion
T he present study demonstrated that both the 10-minute and
60-minute ES protocols accelerated axon regeneration,
thereby promoting improved functional recovery, compared
with that in the no-ES group. Furthermore, there were no
obvious differences between the ES groups in any measured
outcome.
To our knowledge, only 4 clinical trials have investigated
the use of ES as an intervention in nerve surgery since 2010, all
of which utilized a 60-minute ES protocol14-17, which was re-
viewed in detail by Ransom et al33. Those results suggested a
slow adoption of ES in the clinic, which could be improved by
simplifying the application of ES to the treatment of nerve
injuries. The present study is the first to demonstrate that ES
protocols of <60 minutes can be effective for promoting
regeneration and recovery22-24,34,35. A 10-minute ES protocol
involving the use of direct current has been previously shown
to promote improved axon regeneration in vitro and in vivo22-24.
The present study was different from those studies in that we
considered a 10-minute pulsed-current ES protocol using a
clinically available nerve stimulator and compared the results
with the more common 60-minute ES protocol.
The findings observed in association with the 10-
minute ES protocol are supported by previous literature6-10.
Pulsed-current ES protocols applied for 60 minutes increase
regeneration-associated gene expression earlier than that after
injury alone11 and accelerate the rate at which axons cross a
nerve repair site6,7, similar to our observations. These out-
comes have been associated with improved functional
recovery6,7. Therefore, our findings suggest that ES facilitating
earlier axon regeneration results in axons reaching end-organ
targets earlier and thereby improves functional recovery.
The mechanism promoting the regenerative effects for
the 10-minute ES protocol is unclear. The present study sug-
gests that regeneration was improved via a similar mechanism
as that associated with 60-minute ES, with ES-generated action
potentials leading to increased expression of growth-associated
genes and, in turn, accelerated axon regeneration11,13,36. While
this direct connection cannot yet be proven, studies applying
optogenetics have offered insights. In optogenetic studies, the
number of pulses generating action potentials, rather than
duration, was the predominant cue for promoting axon growth
from neurons37. Optical stimulation of neurons generating far
fewer pulses than 1 hour at 20 Hz still resulted in increased
axon growth in vitro compared with no pulses37. Therefore, a
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10-minute ES protocol may stimulate sufficient evoked-action
potentials to elicit neurons to enhance axon growth.
Our results also suggest that ES could involve non-
neuronal cells that may play a role in nerve regeneration, as
inflammatory genes became elevated relative to the no-ES
group. ES therapies are associated with an increase in macro-
phage accumulation38,39, and increased macrophage accumu-
lation and the responses of the macrophages can promote
nerve regeneration40,41. However, the current study is limited
in terms of our ability to draw conclusions as the histomor-
phometry techniques that were utilized to describe nerve
regeneration could not quantify inflammatory cells. In addi-
tion, the assessors of the histomorphometry were not blinded
to treatment group, representing a potential source of
detection bias.
The present study had several additional limitations. Only
male sex was considered, and future studies will need to consider
any sex effects due to ES. Additionally, the animal injury and
repair model represents a “best-case scenario” in terms of re-
generation as nerve transection followed by immediate repair
and then application of ES is ideal. Future studies would benefit
from considering clinical scenarios, such as blunt trauma, which
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Junichi Sayanagi, MD1
Jesús A. Acevedo-Cintrón, BS1
Deng Pan, BS1
Lauren Schellhardt, BA1
Daniel A. Hunter1
Alison K. Snyder-Warwick, MD1
Susan E. Mackinnon, MD1
Matthew D. Wood, PhD1
1Divisionof Plastic and Reconstructive Surgery, Department of Surgery,
Washington University School of Medicine, St. Louis, Missouri
Email for corresponding author: woodmd@wustl.edu
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