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High-resolution measurement of electrically-evoked vagus nerve activity in the anesthetized

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IOP PUBLISHING JOURNAL OF NEURAL ENGINEERING
J. Neural Eng. 10 (2013) 026003 (9pp) doi:10.1088/1741-2560/10/2/026003
High-resolution measurement of
electrically-evoked vagus nerve activity in
the anesthetized dog
Paul B Yoo
1,6
, Nathan B Lubock
2
, Juan G Hincapie
3
, Stephen B Ruble
3
,
Jason J Hamann
3
and Warren M Grill
2,4,5
1
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada
2
Department of Biomedical Engineering, Duke University, Durham, NC, USA
3
Cardiac Rhythm Management Division, Boston Scientific Corporation, St. Paul, MN, USA
4
Department of Neurobiology, Duke University, Durham, NC, USA
5
Department of Surgery, Duke University, Durham, NC, USA
E-mail: paul.yoo@utoronto.ca
Received 13 June 2012
Accepted for publication 20 December 2012
Published 31 January 2013
Online at stacks.iop.org/JNE/10/026003
Abstract
Objective. Not fully understanding the type of axons activated during vagus nerve stimulation
(VNS) is one of several factors that limit the clinical efficacy of VNS therapies. The main goal
of this study was to characterize the electrical recruitment of both myelinated and
unmyelinated fibers within the cervical vagus nerve. Approach. In anesthetized dogs,
recording nerve cuff electrodes were implanted on the vagus nerve following surgical excision
of the epineurium. Both the vagal electroneurogram (ENG) and laryngeal muscle activity were
recorded in response to stimulation of the right vagus nerve. Main results. Desheathing the
nerve significantly increased the signal-to-noise ratio of the ENG by 1.2 to 9.9 dB, depending
on the nerve fiber type. Repeated VNS following nerve transection or neuromuscular block (1)
enabled the characterization of A-fibers, two sub-types of B-fibers, and unmyelinated C-fibers,
(2) confirmed the absence of stimulation-evoked reflex compound nerve action potentials in
both the ipsilateral and contralateral vagus nerves, and (3) provided evidence of stimulus
spillover into muscle tissue surrounding the stimulating electrode. Significance. Given the
anatomical similarities between the canine and human vagus nerves, the results of this study
provide a template for better understanding the nerve fiber recruitment patterns associated with
VNS therapies.
(Some figures may appear in colour only in the online journal)
1. Introduction
Vagus nerve stimulation (VNS) is a potential therapy for a
wide range of chronic disorders. The US Food and Drug
Administration approved VNS therapy for intractable epilepsy
and depression, while indications such as chronic heart failure,
Alzheimer’s disease, pain, tinnitus, and anxiety are currently
being investigated (Kirchner et al 2000, Groves and Brown
2005, De Ferrari et al 2011, Engineer et al 2011). Due
in part to the ease of surgical access, the cervical vagus
6
Author to whom any correspondence should be addressed.
nerve is the primary site for implanting nerve stimulating
electrodes (Uthman et al 1993, Ben-Menachem et al 1994).
However, the anatomical and functional complexity of
the vagus nerve presents a significant challenge for both
optimizing the stimulation parameters for clinical therapies
and understanding the neural mechanism(s) of treatment. The
main objective of this study was to investigate the stimulation
thresholds for activating myelinated and unmyelinated fibers
during VNS.
The selection of stimulation amplitude (i.e. electrical
recruitment of different fiber types) for effective or even
optimum VNS therapy has long been a topic of controversy.
1741-2560/13/026003+09$33.00 1 © 2013 IOP Publishing Ltd Printed in the UK & the USA
J. Neural Eng. 10 (2013) 026003 P B Yoo et al
This was considered during the early development of VNS
therapy for epilepsy (Woodbury and Woodbury 1990, Krahl
et al 2001), and has recently been investigated as a potential
key factor for achieving therapeutic outcomes in patients with
chronic heart failure (Schwartz and De Ferrari 2009). While
the notion of setting a pre-determined stimulation intensity
can simplify the clinical implementation of VNS therapy (Li
et al 2004), it is difficult to translate directly the electrical
recruitment of nerve fibers from the rodent model to humans.
The recruitment characteristics of vagus nerve fibers in
rats are well-documented and in fact have been used to define
the unique classification of different fiber types (A-, B-, and C-
fibers) activated during VNS(Woodbury and Woodbury 1990).
In contrast, electrophysiological studies in larger mammalian
species (i.e. size of vagus nerve comparable to humans) have
failed to characterize consistently activation thresholds across
the full range of nerve fiber types, particularly those for very
small myelinated and unmyelinated axons. The thresholds for
activating B-fibers exhibited a wide range of values from0.5 to
2.5 mA (Vuckovic et al 2008, Anholt et al 2011, Castoro et al
2011), while data characterizing the electrical recruitment of
unmyelinated C-fibers has been difficult to interpret (Tosato
et al 2006, Vuckovic et al 2008). In addition, there has been
a recent report of a long-latency reflex (i.e. indirect) response
that is elicited by low intensity stimulation of the vagus nerve
(Ordelman et al 2010).
In this study, we investigated the activation (recruitment)
of all classes of myelinated and unmyelinated fibers
by electrical stimulation of the cervical vagus nerve
in anesthetized dogs. This was achieved by surgically
removing the epineurium from the vagus nerve trunk (i.e.
desheathing), which significantly improved the signal-to-
noise ratio (SNR) of the recorded neural activity. Electrical
nerve stimulation following selective transection of the vagus
nerve and/or neuromuscular block further confirmed the
biological source of all stimulation-evoked responses. Despite
these improvements in signal fidelity, we did not observe
any stimulation-evoked compound nerve action potentials
(CNAPs) that were indicative of a reflex response. This was
consistent in our recordings from either the ipsilateral or
contralateral vagus nerve.
2. Methods
All surgical and experimental protocols were approved by
the Institutional Animal Care and Use Committee of Duke
University. The study included a total of ten mongrel dogs
(female, weight = 18–21 kg).
2.1. Anesthesia
Each animal was sedated (thiopental sodium, 20 mg kg
−1
,
i.v.) and anesthetized with isofluorane (1–5%) during surgical
implantation of nerve and muscle electrodes. Prior to the nerve
stimulation protocol, the anesthesia was switched to propofol
(i.v., 0.2–0.8 mg kg
–1
min
–1
) in five dogs, and to a mixture of
α-chloralose (50 mg kg
−1
, i.v.) +urethrane (500 mg kg
−1
, i.v.)
in the other five dogs. The change of anesthetic agent in the
Figure 1. Experimental setup for recording the neural and muscular
responses evoked by vagus nerve electrical stimulation. Electrical
pulses were delivered to the right vagus nerve trunk using a bipolar
helical nerve cuff electrode implanted at the level of the thyroid
cartilage. The evoked compound nerve action potentials (CNAP)
were recorded from the vagus nerve trunk using tripolar nerve cuff
electrodes. The ipsilateral electroneurogram (ENG) was measured
distal to the stimulating electrode; whereas the contralateral ENG
was measured from the left vagus nerve trunk at the level of the
larynx.
second group of five dogs was due to a secondary experimental
protocol not reported in this manuscript. However, these
changes did not affect the activation characteristics of the vagus
nerve fibers (e.g., stimulation threshold or latencies). Heart
rate, blood pressure, and withdrawal reflexes were monitored
to assess the depth of anesthesia, and additional doses of
anesthesia and analgesia (fentanyl, i.v., 10–15 µg kg
–1
h
–1
)
were provided immediately, as needed. Continuous infusion of
lactated ringer’s solution (i.v., 10 mLkg
–1
h
–1
) was maintained.
At the end of the study, the animals were euthanized with
euthasol (i.v., 0.2 mg kg
−1
).
2.2. Instrumentation
A ventral incision was made to expose the right cervical vagus
nerve, and a bipolar helical nerve stimulating electrode was
implanted at the level of the thyroid cartilage (figure 1). A
tripolar nerve cuff recording electrode was implanted on the
ipsilateral vagus nerve (7.5 to 13.5 cm distal to the stimulating
electrode), and a pair of stainless steel wires to record
electromyogram (EMG) were inserted percutaneously into
the laryngeal musculature. In five of ten dogs, the recording
nerve cuff electrodes were implanted on segments of the
nerve over which the outer connective tissue (epineurium)
of the vagus nerve trunk was surgically removed. In the
remaining five dogs, the recording electrode was placed
around the intact nerve. As confirmed by the measured
neural responses evoked by electrical nerve stimulation, the
2
J. Neural Eng. 10 (2013) 026003 P B Yoo et al
axons remained intact following the desheathing process,
and thresholds for A-fiber activation (0.37 ± 0.18 mA)
were not different than in our prior experiments (0.56 ±
0.16 mA) using a similar preparation without desheathing
(Castoro et al 2011). In four dogs, the contralateral (left)
vagus nerve was dissected at the level of the thyroid
cartilage and instrumented with a tripolar nerve cuff recording
electrode. Constant current pulses (Pulsar-6bp, FHC, Inc.)
were delivered to the right vagus nerve (via a bipolar helical
electrode) and the evoked CNAPs and laryngeal EMG were
recorded. All signals were amplified (CNAP = 100 000,
EMG = 1000) and filtered (CNAP = 100 Hz–30 kHz,
EMG = 30–3 kHz) using a low-noise voltage amplifier
(SR560, Stanford Research Systems). For the purpose of
monitoring depth of anesthesia, the electrocardiogram(surface
electrodes) and arterial blood pressure (femoral artery catheter)
were recorded throughout each experiment. All signals were
sampled (20 kHz) and recorded (DASH 8Xe, Astro-Med or
Powerlab, ADInstruments Inc.).
2.3. Experimental protocol
The stimulus thresholds for activating myelinated (A- and B-
fibers) and non-myelinated C-fibers in the right cervical vagus
nerve were determined in each animal by measuring changes in
both the CNAP and EMG during trains of monophasic current
pulses delivered via the bipolar helical electrode: amplitude =
0.02–50 mA, pulsewidth = 300 µs, frequency = 1 or 2 Hz,
number of pulses = 20. The bipolar stimulating electrode
was connected with the anode proximal and the cathode
distal to the brainstem (Castoro et al 2011). The source(s) of
components of the measured electroneurogram (ENG) were
verified by repeating the stimulation protocol following either
neuromuscular block (succinylcholine, 1 mg kg
−1
i.v., n =
2 experiments) or surgical transection of the vagus nerve
(n = 5 experiments): (i) rostral to stimulating electrodes, (ii)
caudal to the ipsilateral recording electrode, or (iii) between
the stimulating and recording electrodes.
2.4. Tissue processing/image analysis
At the conclusion of the experiment, a 3 cm segment of the
cervical vagus nerve at the level of the thyroid cartilage was
surgically removed in five dogs. The sample was segmented
into 1 cm pieces, immediately placed in 10% formalin, and
stored at 4

C for a minimum of two weeks. Samples were
embedded in paraffin, sectioned at 50 µm thickness, and
stained with Toluidine Blue. Images of the nerve cross section
were digitized using a light microscope (Nikon SMZ1000)
and analyzed (Matlab, Mathworks Inc) to measure the nerve
radius and thickness of epineurium. The algorithm located
and discretized the outer boundaries of both the epi- and
endoneurium. Linear projections from a single point on the
epineuriumwere made to every point on the endoneurium. The
minimum distance of this set of projections was defined as the
epineurium thickness. This procedure was repeated for every
point around the epineurium (>1000 iterations). The radius of
the nerve trunk was approximated by equating the area (i.e.,
total number of pixels) enclosed by the outer boundary of the
epinerium to a circle.
2.5. Data analysis
The evoked ENG and EMG signals were quantified by
calculating the integral of the rectified post-stimulus response
(Castoro et al 2011). The stimulation threshold for activating
each nerve fiber type and laryngeal muscle activity was
defined as the intensity at which a visible increase in evoked
activity was observed. The SNR of each ENG component was
calculated using the peak-to-peak amplitude of the measured
neural signal and compared between experiments with an
intact epineurium and those that were surgically de-sheathed.
All data are expressed as the mean ± standard deviation,
unless otherwise specified. Statistical analysis used a one-way
ANOVA (or student’s t-test), followed by Tukey’s multiple
comparisons test, if necessary.
3. Results
Electrical stimulation of the right vagus nerve trunk in
anesthetized dogs (figure 2) evoked robust responses that
were characteristic of myelinated nerve fiber activity (ENG).
The corresponding laryngeal muscle activity (EMG) exhibited
post-stimulus latencies (9.4 ± 0.72 ms, range =8.6–10.5 ms,
n =10 dogs) that were consistent with previous studies in dogs
(Castoro et al 2011).
3.1. Recording from de-sheathed vagus nerve
In five dogs, the recording nerve cuff electrode was implanted
on the vagus nerve after removal of the epineurium(desheathed
nerve preparation). Electrical stimulation at successively larger
stimulation amplitudes resulted in the initial recruitment
of large myelinated A-fibers, followed by smaller B-fibers
and eventually unmyelinated C-fibers (figures 2 and 3(B)).
The distance between the stimulating and recording nerve
electrodes and the known conduction velocities of different
fiber types (Berthold 1978, Woodbury and Woodbury 1990)
enabled determination of the specific post-stimulus latency
windows for identifying the components of the neural response
(figure 4(B)). The threshold of large-diameter A-fibers (0.37
± 0.18 mA) was similar to that for the laryngeal EMG (0.36
± 0.17 mA) (figure 4(A)). As the intensity was increased,
the recorded ENG displayed two distinct components of B-
fibers—fast (B
f
, 1.6 ± 0.35 mA) and slow (B
s
, 3.8 ±
0.84 mA)—which corresponded to conduction velocities of
15–30 and 3–15 m s
−1
, respectively (figure 2(B)). In four of
five dogs, activation of unmyelinated C-fiber activity occurred
at markedly larger stimulation amplitudes (17 ± 7.6 mA).
In this example (figure 2(B)), the large amplitude response
is indicative of synchronous activity of the large number of
unmyelinated axons within the cervical vagus nerve. As shown
in figure 4(B), the mean conduction velocity of each identified
nerve fiber type was calculated: A-fiber (38.8 ± 4.8 m s
−1
),
fast B-fiber (18.0 ± 4.7 m s
−1
), slow B-fiber (10.5 ±
1.9 m s
−1
), and unmyelinated C-fiber (1.0 ± 0.1 m s
−1
). This
translated to average axon diameters of approximately 7.8, 3.6,
2.1, and 1 µm, respectively (Hursh 1939). It is important to
note that this nerve classification system is based only on fiber
size and does not distinguish, for example, among different
3
J. Neural Eng. 10 (2013) 026003 P B Yoo et al
(A) (B)
Figure 2. High-resolution neural recordings of the canine cervical vagus nerve. In this example, a train of 20 pulses (20 mA and 1 Hz) were
applied following intra-venous injection of succinylcholine (confirmed by loss of laryngeal EMG in A). The overlapped traces of 20 pulses
in (A) show that the neural response of each myelinated fiber type (A, fast B (B
f
), and slow B (B
s
) fibers) occurred within their respective
post-stimulus time widows. Using a longer time scale (B), stimulation-evoked C-fiber activity (averaged ENG trace in gray) was identified
with a post-stimulus latency of 40–50 ms (stimulation-to-recording electrode distance = 7.5 cm). Arrow in (A) indicates stimulation artifact.
(A) (B) (C)
Figure 3. Effects of neuromuscular block and nerve transection. (A) Electrical stimulation of the right vagus nerve (intensity = 6 mA,
overlapped traces of 20 pulses) evoked robust responses in both the measured electroneurogram (ENG) from the desheathed ipsilateral
vagus nerve trunk and the laryngeal electromyogram (EMG). (B) Neuromuscular block resulted in the complete loss of laryngeal EMG
activity and the large-amplitude, short-latency artifact within the vagus ENG (

, 4 ms latency in A). This permitted the identification of A,
fast B (B
f
) and slow B (B
s
) components of the vagus nerve activity. Complete transection of the vagus nerve, both rostral to the stimulating
electrode and caudal to the recording electrode, yielded the same responses. (C) Following transection of the nerve between the stimulating
and recording electrodes, both the ENG and EMG responses were absent during nerve stimulation. In this example, the distance between the
stimulating and recording electrodes was 8 cm.
sensory fibers (e.g., group I afferents or group II afferents
that have conduction velocities similar to A and B efferents,
respectively (Berthold 1978)).
3.2. Increased signal-to-noise ratio (SNR) of recorded neural
activity
The SNR of the neural activity recorded from the desheathed
nerves was significantly higher than the SNRin experiments in
which the epineurium remained intact (figure 5, p <0.002 for
myelinated fibers and p <0.05 for unmyelinated fibers): A(9.9
± 4.3 dB), B (1.2 ± 0.54 dB), and C (1.3 ± 1.8 dB) fibers.
These results show that recording from nerves with surgically
removed epineurium enabled both the differentiation of fast
and slowB-fibers (figure 2) and the recording of unmyelinated
C-fiber activity, the latter of whichwas previouslyunsuccessful
in surgically intact dogs (Vuckovic et al 2008, Castoro et al
2011).
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J. Neural Eng. 10 (2013) 026003 P B Yoo et al
(A) (B)
Figure 4. Recruitment characteristics of both myelinated (n = 5 dogs) and unmyelinated (n = 4 dogs) vagus nerve fibers. (A) Electrical
stimulation of the right vagus nerve exhibited the inverse-physiological recruitment of large-to-smaller diameter axons as the stimulation
intensity was increased. In every experiment, the threshold for activating A-fibers overlapped with that for the laryngeal muscle (EMG). (B)
The mean conduction velocity of each component of the evoked response (distance divided by the time from stimulus pulse to first peak)
exhibited a corresponding decrease with respect to fiber diameter.
Figure 5. The signal-to-noise ratios (SNR) of electrically evoked
CNAPs were calculated in two groups of dogs in which the
epineurium remained intact (n = 5) or was surgically removed (n =
5). Comparison of the SNR among the different fiber types indicate
significant increases in the fidelity of the measured CNAP when the
epineurium was absent (

, p < 0.002;
+
, p < 0.05, paired t-test).
3.3. Absence of reflex vagus nerve activity in anesthetized
dogs
In 2 dogs, a neuromuscular blocking agent was administered
for the purpose of identifying any potential longer-latency
neural (i.e. reflex) responses evoked by vagus nerve
stimulation. Repeated VNS (single pulses at 1 Hz) following
neuromuscular block confirmed that the recorded activity
corresponded to both myelinated and unmyelinated fibers
(figures 2 and 3). After washout of the neuromuscular blocking
agent, electrical stimulation following complete transection of
the nerve either rostral to the stimulating electrode or caudal to
the recording electrode resulted in the same neural responses
(figure 3(B)). Finally, electrical stimulation following nerve
transection between the two electrodes resulted in the complete
loss of all neural activity (figure 3(C)). Taken together, these
results confirmed that the measured neural responses resulted
(A)
(B)
Figure 6. Electrical stimulation of the right vagus nerve (2 mA, 20
pulses, 1 Hz) failed to evoke any reflex neural activity in the
contralateral vagus nerve, which was also desheathed before a
recording nerve cuff electrode was implanted. Instead, there was a
large-amplitude response (A) that temporally coincided with the
laryngeal EMG (B). The absence of both responses during VNS
after IV injection of succinylcholine (i.e. neuromuscular block)
confirmed that the recorded signal (A) was an artifact of laryngeal
muscle activity.
from direct nerve stimulation and not from any brainstem or
end-organ related reflex.
In a total of four dogs (3 intact epineurium + 1 de-
sheathed nerve), the presence of reflex neural activity was
also investigated in the contralateral vagus nerve. In all
experiments, there was no discernible stimulation-evoked
response in the contralateral ENG except for a large amplitude
signal that appeared at a latency of 8–20 ms (figure 6). The
loss of both this signal and the laryngeal EMG following
either neuromuscular block or nerve transection caudal to the
stimulating electrode indicated that this response recorded by
the contralateral nerve cuff electrode was generated by muscles
located within the adjacent larynx (refer to location in figure 1).
The absence of stimulus-evoked reflex responses in both
the ipsilateral and contralateral vagus nerves was consistent
5
J. Neural Eng. 10 (2013) 026003 P B Yoo et al
(A) (B)
Figure 7. Post-mortem analysis of canine cervical vagus nerve. Based on the cross-sectional image of samples obtained from the cervical
vagus nerve (Toluidine blue stain, A), the inner and outer boundaries of the epineurium were used to compute the maximum (solid line) and
minimum (dashed line) thickness of this connective tissue layer. Across five dogs (B), statistical analysis of the minimum, mean, and
maximum values showed significant differences among the three measurements of epineurial tissue thickness (ANOVA with Tukey’s
multi-comparison test,

p < 0.001).
across all anesthetic and analgesic agents used in this study:
isoflurane, propofol, and alpha-chloralose/urethane.
3.4. Stimulation spillover—short latency muscle artifact
In all five experiments in which the nerve was desheathed,
the identification of different fiber types within the CNAP
was validated by repeating stimulation (intensities ranging
from threshold to 50 mA) after i.v. administration of a
neuromuscular blocking agent. This resulted in the same
repeatable electrically-evoked neural responses measured
prior to the neuromuscular block. However, in three of five
dogs (figure 3(A)), notable artifacts within the ENG signal
were observed at stimulation amplitudes above the threshold
for activating fast B-fibers (6.7 ± 3.1 mA, range =4–10 mA,
n =3 dogs). This artifact disappeared following administration
of the neuromuscular blocking agent (figure 2(B)). Given
the short post-stimulus latency (3 to 5 ms), it appeared
that this artifact was generated by a myogenic source—not
related to the recurrent laryngeal nerve that branches off the
vagus nerve—that was activated by current spread from the
stimulating electrode.
3.5. Anatomy of the canine vagus nerve
In five dogs, the cross-sectional image of the cervical vagus
nerve (figure 7) typically exhibited a large single nerve fascicle
surrounded by a layer of connective tissue (i.e. epineurium).
Although this monofascicular configuration indicated the
absence of nerve branches (e.g., recurrent laryngeal nerve)
derived from the right vagus nerve trunk, a small sympathetic
nerve was observed in three of five samples. Electrical
activation of these sympathetic Aδ and C-fibers did not
contribute to our ENG recordings, as the desheathing process
ensured that this nerve was not placed within the recording
nerve cuff electrode.
The mean radius of the vagus nerve (including the
epineurium) in these samples was 1.4 ± 0.1 mm.
Computational analysis of the stained images showed that the
epineuriumthickness varied significantly around the perimeter
of the nerve (figure 7(B), p < 0.001): minimum (0.3 ±
0.1 mm) and maximum (1.3 ± 0.3 mm) thickness. Based
on a minimum of 1000 sample measurements per nerve (i.e.
computed thickness of epineurium around the nerve fascicle),
the average distance between the endoneurium and the outer
surface of the epineurium was 0.7 ± 0.1 mm, range = 0.5 to
0.8 mm (n = 5).
4. Discussion
High resolution recording of stimulation-evoked vagus
nerve activity was achieved by carefully removing the
protective epineural tissue layer from the nerve, but without
compromising the perineurium. This enabled recording of each
individual nerve fiber type recruited during stimulation of the
cervical vagus nerve of anesthetized dogs. Compared to intact
nerves, removal of the epineuriumresulted in an approximately
five-fold increase in the SNR for myelinated fibers, and
over a 12-fold increase in the SNR for unmyelinated fibers.
However, despite these improvements in the recording fidelity,
we were unable to record any reflex neural activity evoked
by vagus nerve stimulation. This was confirmed by repeated
electrical stimulation following either vagus nerve transection
or pharmacological block of neuromuscular transmission.
A comparison of the cervical vagus nerve across different
mammalian species suggests that—in addition to pigs (Tosato
et al 2006, Vuckovic et al 2008)—the canine model represents
a good approximation of the electrical recruitment properties
for human VNS. This is reflected in the notable similarities
in nerve size (nerve diameter = 2.8 mm, figure 7) and
total number of axons. As demonstrated in earlier anatomical
studies, the total number of myelinated axons in the vagus
nerve increases in proportion to body size: 3000 axons in
rabbits (Evans and Murray 1954), 10 000 in cats (Foley and
DuBois 1937, Mei et al 1980), 15 000 in dogs (Satchell et al
1982), and 20 000 in humans (Hoffman and Schnitzlein 1961).
This relationship also applies to unmyelinated C-fibers, the
total number of which is estimated in multiples (scale factor
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J. Neural Eng. 10 (2013) 026003 P B Yoo et al
between 4 and 7) of the number of myelinated axons (Foley
and DuBois 1937, Evans and Murray 1954). Accordingly, the
mammalian cervical vagus nerve consists of a large number of
axons that can range in total from as low as 20 000 axons in
rabbits to over 100 000 in humans (Hoffman and Schnitzlein
1961), with the majority of fibers being very small myelinated
(group III afferents and B fiber efferents) or unmyelinated
C-fibers.
Given the disproportionately greater number of slow
conducting vagal nerve fibers, one would expect electrical
activation of slow B- and C-fibers to yield a robust ENG
signal, particularly in large animals such as the dog. However,
there are few data that characterize the recruitment of these
axons during electrical nerve stimulation (Tosato et al 2006,
Vuckovic et al 2008, Anholt et al 2011, Castoro et al 2011). In
fact, many such studies did not demonstrate the activity of such
small (e.g., unmyelinated) fibers that was repeatedly observed
in rats (Woodbury and Woodbury 1990, Waataja et al 2011).
The results of this study indicate that the electrically-insulating
property of the epineurium—which is mainly comprised of
collagen and elastin fibers, and networks of small blood
vessels (Thomas 1963)—has been the primary limiting factor
for in vivo recording of the vagus nerve in larger animals.
Our histological analysis shows the average thickness of
the epineurium (i.e. nerve-to-electrode distance) can exceed
0.5 mm in adult dogs (figure 7), which is comparable to the
diameter of the intact rat vagus nerve trunk. Given the similar
anatomical properties of the cervical vagus nerve in dogs
and humans, our study further suggests that chronic neural
recording of the human vagus nerve with an intact perineurium
and at physiological SNR levels—as predicted by both in vivo
and computational studies (Sahin et al 2000, Yoo and Durand
2005)—will very likely be limited to measuring only A- or
large B-fiber activity.
The influence of the epineurium may also account for
the marked difference in nerve activation thresholds that are
observed between rats and larger animals. With the stimulus
duration (pulse width = 300 µs) taken into consideration, the
approximate thresholds for activating A-, B- and C-fibers in
the rat are on the order of 10, 30, and 200 µA, respectively
(Woodbury and Woodbury 1990); whereas the present study
demonstrated threshold amplitudes that were up to 100 times
larger (A-fiber = 0.4 mA, B-fiber = 3.8 mA, and C-fiber =
17 mA, refer to figure 4(a)). The range of stimulus intensities
used in this study correspond well to the activation thresholds
of large myelinated fibers in humans (Koo et al 2001), and
also to those reported for activating small diameter fibers
in epileptic canine (Zabara 1992) and non-human primates
(Lockard et al 1990). Interestingly, many clinical applications
of VNS are designed to operate in the range of several mA or
less (Handforth et al 1998, Schwartz and De Ferrari 2009, De
Ferrari and Schwartz 2011). This limitation is primarily due to
side effects including cough or voice hoarseness (Ramsay et al
1994, Cristancho et al 2011) and, in some cases, those related
to significant respiratory-related events (McGregor et al 2005,
Papacostas et al 2007). Further work is needed to clarify
the nerve fiber populations responsible for these undesirable
physiological responses.
Based on the results of this study and the stimulation
parameters typically used in clinical therapies, we can predict
that the reported therapeutic effects of many VNS therapies
are associated with the electrical activation of A- and B-
fibers (Ben-Menachem et al 1994, Schwartz and De Ferrari
2009, Cristancho et al 2011), which correspond to either
motor A-type or sensory group I, II, and III fibers. Although
a distinction between the electrical activation of motor or
sensory nerve was not made, our findings collectively support
the hypothesis that VNS therapies are not dependent on
the activation of unmyelinated C-fibers (Krahl et al 2001).
Given that the vagus nerve consists primarily of sensory fibers
(Foley and DuBois 1937, Evans and Murray 1954), it appears
that the electrical activation of group I, II, and III afferents
could play a critical role in the mechanism of VNS therapy.
Thus, the suppression of epileptic activity (McLachlan 1997),
improvement in heart failure symptoms (De Ferrari et al
2011), or even the hypothesized anti-inflammatory effects of
VNS (Tracey 2002) may be mediated by vagal afferents. It
is noted, however, that efferent vagal nerve stimulation can
also exert physiological effects, such as suppressing systemic
inflammatory responses in rats (Borovikova et al 2000) and
canines (Ruble et al 2010). Whether clinically used VNS
parameters actually recruit these very small diameter fibers
(slow B- and perhaps even C-fibers), which comprise the
abdominal branch of the vagus nerve (Prechtl and Powley
1990), remains to be investigated.
Despite the improved SNR of our nerve recording
method, we did not detect any reflex neural response in
either the ipsilateral or contralateral vagus nerves. This
absence of VNS-evoked reflex activity was consistent both
before and after pharmacologically-induced neuromuscular
block and/or selective nerve transection, and even when
using different anesthetic agents (isoflorane, propofol, and
α-chloralose/urethrane). This absence of stimulation-evoked
reflex activity was unexpected, given that the vagus nerve
is known to mediate various spinal and supraspinal reflexes
(Jansson 1969, McCann and Rogers 1992), and that anesthetic
agents such a alpha-chloralose have been shown to facilitate
(or retain) reflexes in response to peripheral nerve stimulation
(Jonzon et al 1972, Yoo et al 2008). Further work is needed
to determine whether this recording method can detect sub-
populations of asynchronous slow B or C-fiber activity, which
is a likely reflex response elicited by VNS.
Interestingly, electrical stimulation of the vagus nerve
elicited a signal in the contralateral vagus nerve that
consistently exhibited a latency and amplitude (figure 6(a))
that approximated what has been called an ‘indirect’ reflex
response (Ordelman et al 2010). It was present during VNS,
before and after nerve transection rostral to the stimulation
electrode (refer to figure 1), but disappeared following
neuromuscular block and/or nerve transection distal to the
stimulating electrode. Based on these findings, it is clear that
this response was an EMG artifact generated by activation of
the adjacent laryngeal musculature, rather than a stimulation-
evoked reflex neural response.
Another important outcome of this study was the co-
activation of cervical strap muscles (e.g., sternohyoid) not
7
J. Neural Eng. 10 (2013) 026003 P B Yoo et al
directly innervated by the vagus nerve. This stimulation
spillover was observed in over half of the experiments and
occurred at stimulation amplitudes that were above the typical
upper limit current for clinical VNS therapy. Although it is
not clear whether this spread of stimulus current activated
any low-threshold sensory fibers located in close proximity
to the nerve electrode, the emergence of this muscle artifact
suggests a possible source of painful sensations reported
during VNS therapy (Liporace et al 2001). As examined
in several experiments, this spillover was primarily due to
incorrectly fitted stimulating electrodes implanted on the nerve
that, in some cases, was exacerbated by the accumulation of
fluid around the electrode. Completely wrapping the implanted
helical electrode with an insulating material (e.g., thin plastic
sheet) consistently resolved this issue, at least for the duration
of these non-survival studies. In implanted VNS systems,
chronic healing and electrode encapsulation may aide in
preventing spillover of activation to nearby structures.
Acknowledgments
The authors acknowledge Ellen Dixon-Tulloch, Gilda Mills,
Mark Castoro, Haoran Liu and A J Ford for their assistance
during these experiments. JGH, JJH and SBR are full-time
employees of Boston Scientific Corporation which provided
financial support for this work.
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