Surgical Science, 2011, 2, 269-277
doi:10.4236/ss.2011.25059 Published Online July 2011 (http://www.SciRP.org/journal/ss)
Morphological and Electromyogram Analysis for the
Spinal Accessory Nerve Transfer to the Suprascapular
Nerve in Rats
Jun Yan1, Kazuhito Ogino1,2, Jiro Hitomi1
1Department of Anat o my , School of Medicine, Iwate Medical University,
Iwate, Japan
2Department of Plastic Surgery, Dokkyo Medical University,
Tochigi, Japan
Received April 19, 2011; revised June 3, 2011; accepted J une 25, 2011
Abstract
For many years, nerve transfer has been commonly used as a treatment option following peripheral nerve
injury, although the precise mechanism underlying successful nerve transfer is not yet clear. We developed
an animal model to investigate the mechanism underlying nerve transfer between branches of the spinal ac-
cessory nerve (Ac) and suprascapular nerve (Ss) in rats, so that we could observe changes in the number of
motor neurons, investigate the 3-dimensional localization of neurons in the anterior horn of the spinal cord,
and perform an electromyogram (EMG) of the supraspinatus muscle before and after nerve transfer treatment.
The present experiment showed a clear reduction in the number of γ motor neurons. The distributional por-
tion of motor neurons following nerve transfer was mainly within the neuron column innervating the trape-
zius. Some neurons innervating the supraspinatus muscle also survived post-transfer. Compared with the
non-operated group, the EMG restoration rate of the supraspinatus muscle following nerve transfer was 60%
in the experimental group and 80% in a surgical control group. Following nerve transfer, there was a distinct
reduction in the number of γ motor neurons. Therefore, γ motor neurons may have important effects on the
recovery of muscular strength following nerve transfer. Moreover, because the neurons located in regions
innervating either the trapezius or supraspinatus muscle were labeled after Ac transfer to Ss, we also suggest
that indistinct axon regeneration mechanisms exist in the spinal cord following peripheral nerve transfer.
Keywords: Nerve Transfer Treatment, Fluorescent Dye Labeling, Electromyogram, Nerve Axonal
Regeneration, Rat
1. Introduction
Traditionally, direct suture treatment was considered ne-
cessary for the treatment of peripheral nerve injury [1,2].
However, peripheral nerve regeneration was thought to
be impossible because successful cases were very rare
prior to the 18th century. According to Green’s operative
hand surgery (third edition), the earliest successful cases
were demonstrated at the end of the 19th century [3]. The
first case of peripheral nerve transfer was reported by
Balance, and indicated that voluntary movement of mi-
metic muscles could be recovered by suturing the spinal
accessory nerve (Ac) or hypoglossal nerve to the facial
nerve [2]. This cure for peripheral nerve injury was
called nerve transfer, and was commonly used thereafter
for facial nerve paralysis or many other types of periph-
eral nerve injury [4-9].
Nerve transfer as a cure for paralysis of the brachial
plexus was also a classic treatment, and good treatment
results were evident [10-13]. In a commonly used clini-
cal treatment, the branch innervating the trapezius (Ac)
was transferred to the suprascapular nerve (Ss) to recover
paralysis of the supraspinatus muscle and, finally, to re-
cover function of the humeral joint. However, the mor-
phological mechanism underlying this method was not
clarified. A phenomenon was observed under electron
microscopy in which nerve growth cones from the nodes
of Ranvier on damaged nerve fibers could enter into the

J. YAN ET AL. 270
peripheral side of the nerve for re-innervation [14-16].
Following this, many researchers attempted to clarify the
mechanisms underlying nerve fiber regeneration. How-
ever, changes in the motor neurons of the anterior horn,
which are known to play a key role during regeneration
or re-innervation of damaged peripheral nerve fibers, re-
main unaddressed.
In the 1970s, a retrograde method using horseradish
peroxidase (HRP) was the main method used to investi-
gate motor neurons in the anterior horn [17-27]. How-
ever, a major flaw was that HRP leaked, raising concern
over the reliability of such assessments [28].
Fluorescent dye (DiI) proved to be a highly reliable
neuronal marker and for use in pathway tracing [29]. The
fluorescent dye dissolves in the lipids of Schwann cells;
therefore, the reliability of the dye is better than HRP,
which could diffuse into the surrounding tissue and be
absorbed directly into the axon [29,30]. Using the fluo-
rescent dye (DiI), we performed experiments in adult rats
and proved its reliability as a retrograde tracer in the pe-
ripheral nerve system. [31-35].
The objectives of the present experiments were to 1)
use an animal model to confirm the relationship between
nerve transfer between the spinal accessory nerve (Ac)
and suprascapular nerve (Ss) with changes in the number
and distribution of motor neurons; 2) construct a 3-di-
mensional image of motor neurons (including Ac and Ss)
in the anterior horn and compare it to changes in neuron
localization; and 3) to observe changes in the electro-
myogram (EMG) of the supraspinatus muscle before and
after nerve transfer treatment.
2. Materials and Methods
Adult Wistar rats (♂, 9 weeks of age; 105 - 250 g) were
used in all experiments. All animals were obtained from
Japan SLC Inc., and were maintained in the Animal Care
Service Center, School of Medicine, Iwate Medical Uni-
versity. Animals were handled in compliance with ethi-
cal guidelines. Animals were anesthetized with an initial
intraperitoneal dose of sodium pentobarbital (40 mg/kg)
and maintained with additional 2.5 mg doses as needed.
2.1. Nerve Transfer Operations (Ac to Ss, N = 4,
Age 9 Weeks)
As shown in Figures 1 and 2, the muscular branch in-
nervating the branch innervating the trapezius (Ac) and
suprascapular (Ss) nerves were dissected and removed
under surgical microscopy (Olympus Optical Co) in 4
rats. The proximal section of the Ac was then transferred
to the distal section of the Ss using the epineurium sew-
ing method. The animals were maintained for 9 weeks
then killed and their spines removed [36].
2.2. Control Group (Ac to Ac, N = 4)
A comparison of the regeneration results following
transfer of the Ac to the Ss with that of sewing the same
nerve (Ac to Ac) was considered a necessary control for
our study. In order to create appropriate control experi-
ments, the Ac was dissected and amputated in 4 rats then
the proximal and distal sections were sewed together
using the same nerve transfer method.
(a)
(b)
Figure 1. The form of Ac and cervical plexus (sketch) and α,
γ motor neuron in anterior horn (photo). (a): The periph-
eral part of the Ac and cervical nerves of the rat (ventral
view). The Ac has two main branches to innervate the ster-
nocleidomastoideus and trapezius. C2, C3, and C4 send a
branch directly to the Ac mainly. Black arrows: the dyed
regions of the Ac and Ss; (b): The photo showing the α mo-
tor neuron (yellow arrow) and γ motor neuron (green ar-
rowhead) in anterior horn.

J. YAN ET AL.271
(a) (b)
(c) (d)
Figure 2. The photos and sketches showing the operation of
the dye treatment and nerve transfer (Ac to Ss). (a): the Ac
and Ss are in the upper and inferior edge of the omohy-
oideus muscle in rat. (b): the omohyoideus was amputated,
the Ac and Ss has been transferred (black arrowhead). The
right upper is the extended photo of the transferred nerve
(Ac to Ss). (c): the red arrows are showing the dyed regions
of the Ac and Ss, respectively. (d): the red-cross indicates the
transferred point of two nerves, and the red arrow is showing
the dyed point after nerve transfer (after operation 9 weeks).
2.3. Electromyogram (Ss, N = 4; Ac to Ac, N = 4;
Ac to Ss, N = 4; All Animals were 18 Weeks
of Age)
In order to evaluate the regeneration of the nerve fibers
in the transferred nerves, EMGs of the supraspinatus
muscle was examined in the non-operated, nerve transfer,
and control groups. The nerve innervating the muscle
was dissected and the EMG recorded using the electrode
stimulation method (Chart for Windows 5.5.1, ADIn-
struments, Lexington, Australia). In all cases, the stimu-
lation points were changed on the surface of the muscle
and the total number of stimulations was 30. The highest
average value was then calculated.
2.4. Observation of Motor Neurons in the
Anterior Horn of the Ac and Ss (Ac, N = 4;
Ss, N = 4; Ac to Ac, N = 4; Ac to Ss, n = 4)
Under surgical microscopy, the Ac, Ss (non-operated
group, 18 weeks of age), and the nerve transfer group (9
weeks after surgery) were dissected and amputated at a
point near the muscle or on the distal portion of the oper-
ated point, respectively. The fluorescent dye, DiI [1,1’-
dioctadecyl-3,3,3’,3’-tetramethylind carbocyanine per-
chlorate; diI-C18-(3)] was applied to the proximal sec-
tion of the nerves. Proximal sections were wrapped with
the surrounding connective tissue, and the distal sections
were burned to prevent ambiguous labeling. Two weeks
after surgery, animals were anesthetized with 50 mg/kg
intraperitoneal sodium pentobarbital and fixed by intra-
cardiac perfusion with 150 ml physiological salt solution
and 300 ml 4% paraformaldehyde in 0.1 M phosphate
buffer. The spinal cord (C1 to Th1 segments) was surgi-
cally removed along with its dorsal root ganglia. Speci-
mens were post-fixed in the same fixing solution for 24 h
then placed serially into 10%, 20%, and 30% sucrose
solutions in 0.1 M phosphate buffer for 24 h, respectively.
Each spinal cord segment was formed into one block and
cut transversely into 50-μm serial sections using a mi-
crotome. All sections were observed and photographed
using a laser confocal microscope (LSM200GBSU2;
Olympus Optical Co. Ltd., Japan).
We used 4 animals to confirm the restoration of the
peripheral nerves. To do this, peripheral nerve specimens
(distal part of the transferred nerve point) were removed.
Specimens were then embedded in paraffin, cut trans-
versely, and stained using the Masson-Trichrome method
[37].
Morphological assessment of the samples was a valu-
able aspect of our investigation (Figure 1). The diameter
of all stained motor neurons was measured to judge the
proportion of α and γ motor neurons in each segment
(over 30-μm was defined as α and under 25 μm was de-
fined as γ motor neurons [14]).
The number of α and γ motor neurons in each segment
were counted, and the average value of each segment (in
4 spinal cord specimens) was calculated for each. Then
the average number of α and γ motor neurons in the Ac
(non-operated group) and Ac transferred to Ss groups
were treated with the t-test for medical statistics.
In three animals (one from each of the nerve transfer,
control, and un-operated groups), all images were recon-
structed with 3-dimensional reconstruction software
(VoxBlast 3.1, Vaytec, Fairfield, USA) to compare the
localized distribution of the motor neurons in the anterior
horn of the spinal cord.
3. Results
3.1. Electromyogram (EMG)
An EMG (supraspinatus muscle) analysis was conducted

J. YAN ET AL. 272
in the three groups. In the non-operated group (Ss), the
average maximum value of the EMG was 0.045 mv, And
was 0.037 mv in the control group (Ac to Ac), and 0.030
mv in the nerve transfer group (Ac to Ss) (Table 3). The
EMG restoration rate in the control group (Ac to Ac) was
80% of the non-operated group, whilst the restoration
rate in the nerve transfer group (Ac to Ss) was 60% of
the non-operational group.
3.2. Microscopic Observation of Peripheral
Nerve Regeneration Following Nerve
Transfer
As shown in Figure 3, a definite reduction in the number
of nerve fibers on histological sections was not observed
in nerve transfer or non-operational group. Permeation of
inflammatory cells in the nerve transfer group was not
observed. However, the inside diameter of the myeli-
nated nerve fibers was reduced, and atrophy of the mye-
lin sheath was apparent. Moreover, we observed the dis-
appearance of axon(s) in a small number of sections.
3.3. Localization of Neurons in the Anterior
Horn and 3-Dimensional Reconstruction of
the Neurons
The motor neurons of the Ac were distributed from the
C2 to C7 segments, and formed a longitudinal column. In
sections of cranial segments, the neurons were located in
the lateral portion, but in caudal segments the neurons
seemed to be located in the medial portion of the anterior
horn. However, 3-dimensional reconstruction showed that
the motor neurons formed a straight column from the cra-
nial to caudal segments (Figure 4(a)). The neurons of the
Ss were distributed predominantly from the C3 to C7 seg-
ments, and were located in the dorsal-lateral portion of the
anterior horn, forming a longitudinal column (Figure 4(b)).
Results showed that the distribution of motor neurons in
the Ac and Ss slightly overlapped in C3 and C4, but in the
caudal segments the distribution of the two columns was
separate from each other (Figures 4(a), 4(b), and 4(d)).
On the other hand, after nerve transfer the distribution
of motor neurons were broadly consistent with that in the
Ac. We speculate that the motor neurons within the
transferred nerve originated from those belonging to the
Ac. In caudal segments, however, some motor neurons
were located in the dorsal-lateral portion (belonging to
Ss), although with reduced frequency (Figure 4(d)).
3.4. Number and Classification of Labeled
Motor Neurons
In the non-operated rats (including Ac and Ss), the loca-
tion and number of α and γ motor neurons were con-
firmed after retrograde labeling with the fluorescent DiI.
The number of α and γ motor neurons within the Ac, Ss,
control group (Ac to Ac), and the transfer group (Ac to
Ss) are shown in Table 1. The number of γ motor neu-
rons in the nerve transfer group were definitely reduced,
although α neurons were also reduced. The ratio of α : γ
was 4.8 : 1 in Ss, and 7.3 : 1 in the nerve transfer group
(Table 1). In particular, the number of the γ motor neu-
rons in caudal segments were significantly reduced (C5,
C6, and C7) compared with cranial segments.
As shown in Table 2, after nerve transfer the restora-
tion rates of α and γ motor neurons were 79% and 64%,
respectively. In the control group, the restoration rates
of the two neuron groups were 85% and 84%, respec-
tively.
(a) (b)
(c) (d)
(e) (f)
Figure 3. The section photos of peripheral nerves (un-pera-
tional and after nerve transfer). (a) (Ac) and (b) (Ss) are
showing the peripheral nerve sections of un-operational
cases. (d) and (e) are showing the sections which from the
cranial and caudal part of the transferred nerve, respec-
tively. It could be confirmed that the diameter of myeli-
nated fibers was reduced, after compared with (a) and (b).
(c) and (f) are the extended photos of (b) and (e). Note some
of demyelinisation (black arrows) were observed and the
number of fibers were decrease, the inflammatory cells
were observed in (f).

J. YAN ET AL.273
(a) (b)
(c) (d)
Figure. 4. The neurons distribution of Ac, Ss and Ac to Ss.
The neurons of Ac were located in the ventral portion of
anterior horn and that of Ss were in the dorsal-lateral por-
tion (yellow arrow: Ac; green arrow: Ss; in (d). After 3-D
reconstruction, the neuron column of Ac was showing in A,
that of Ss was in B and the column after nerve transfer was
showing in (c). It was clear that the column after nerve
transfer is similar with the Ac, although some dyed neurons
were also observed (d).
Table 1. The average number of the α and γ motor neurons.
The average number of the α and γ motor neurons in Ac
(un-operation), Ss (un-operation), Ac-Ac (control) and Ac-
Ss (nerve transfer) were showing in the table. After nerve
transfer, the numbers of the α and γ motor neurons were
decreased clearly. After analysis by t-test, the signification
difference between the two values were approved (in both α:
Ac-Ac/Ac and γ: Ac-Ss/Ac, p < 0.01).
α γ α/γ
Ac 233.0 39.0 5.9/1
Ss 222.0 45.0 4.8/1
Ac-Ac 199.0 33.0 6.0/1
Ac-Ss 183.0 25.0 7.3/1
4. Discussion
Peripheral nerve transfer has been used for surgical
treatment of brachial plexus injury for many years
[2,11,13,38]. In particular, the “standard” method of
Table 2. The restoration rate of the α and γ motor neurons.
After nerve transfer, the restoration rate of the α and γ
motor neuron were 79% and 64%, respectively. In the con-
trol, the rates of the two neuron group were 85% and 84%,
respectively.
α γ
Ac-Ss/ Ac*100% 79% 64%
Ac-Ac/ Ac*100% 85% 84%
Table 3. The EMG of supraspinatus muscle in nerve trans-
fer cases, control cases and un-operational cases. The graph
is showing the un-operational (a), control (b) and transfer
cases (c) of No.3 animal. The restoration rate of EMG in
control cases (Ac to Ac) is 80% of the un-operational cases,
and the rate of EMG in the nerve transfer (Ac to Ss) is 60%
of the un-operational cases.
Un-operation
(Ss)
control
(Ac-Ac)
transfer
Ac-Ss
1 0.045 0.036 0.031
2 0.044 0.038 0.029
3 0.047 0.035 0.031
4 0.044 0.037 0.029
Aver.0.045 0.037 0.030
(a) (b) (c)
medical treatment for upper root injury of the brachial
plexus employs the transfer of the muscular branch in-
nervating the trapezius to the suprascapular nerve
[5,9,39-41]. In recent years, the quality of surgical mi-
croscopy has improved, not only for nerve transfer but
also in terms of the suturing method used for peripheral
nerve injury [42]. However, there is a clear difference in
the results for individual patients, and it is difficult to
judge treatment results clinically after nerve transfer. On
the other hand, observing the motor neurons in the ante-
rior horn has proven to be effective in animal models for

J. YAN ET AL. 274
estimating peripheral nerve reconstruction [14,21,37].
However, these methods are not sufficient to fully ex-
plain the outcome of the nerve transfer operation. In the
present experiment, we developed an animal model to
evaluate the outcome of peripheral nerve transfer opera-
tions. In the present experiment, we developed an animal
model to order to evaluate the outcome of peripheral
nerve transfer operations. With this animal model, we
investigated the specific number and distribution of mo-
tor neurons in the Ac and Ss groups following nerve
transfer in each cervical segment. We also generated
3-dimensional reconstructions to compare the distribu-
tion of the motor neurons before and after nerve transfer
treatment.
Our results show that the neuron column of Ac begins
from C2 to C7 (mostly in C3 to C6), but that of Ss origi-
nates from C4 to C7 (mostly in C5 and C6). The distri-
bution of motor neurons innervating different muscles
could be used to judge the origin of the muscles [43] and
we believe that the transfer of nerves innervating the
same original muscles could lead to improved treatment
outcomes. In the present experiment, the two columns
were separated and, according to the literature, the two
muscles could originate from different muscle masses
[43]. Consequently, this may explain why the contraction
strength of the supraspinatus muscle was only restored
by 60%.
We investigated the regeneration outcome of nerve
transfer 9 weeks after surgery. This interval (9 weeks)
was considered to represent a suitable term for observing
nerve regeneration because an amputated peripheral nerve
can extend axons by 4.3 mm/day [44]. In this experiment,
regenerated fibers were mostly represented by myeli-
nated fibers. We did not observe the presence of inflam-
matory cells. Consequently, nerve regeneration occurred
in a satisfactory manner, although reportedly 90% of
neurons in humans can degenerate in just six months
following surgery [28,45,46].
In the present experiments, the restoration rate of the α
and γ motor neurons were 85%, 84% (Ac to Ac) and
79%, 64% (Ac to Ss), and in reference to the EMG we
believe our results are exact. We did not find any reports
showing a 90% restoration of contraction of the su-
praspinatus muscle after nerve transfer treatment. There-
fore, we believe that our animal model is an important
and successful model. Misdirection of α motor neurons
in the anterior horn after nerve transfer has been ob-
served with the HRP method [18,37]. However, in the
present experiment it is not yet clear why some motor
neurons innervating the supraspinatus muscle survived
(in this model, the axons of the Ss were amputated, and
the foundation for regeneration was lost). The present
experiment focused on changes in motor neuron numbers
and the distributional proportions of α and γ motor neu-
rons before and after peripheral nerve transfer. Our re-
sults clearly show that the number of γ motor neurons
was lower following nerve transfer than before nerve
transfer. These observations agree with previous studies
of α motor neurons; the γ motor neurons have not previ-
ously been included in analyses [18,37]. The number of γ
motor neurons was significantly reduced following nerve
transfer. Therefore, the γ motor neurons could be pre-
sumed to have a very important effect on the functional
restoration of damaged muscles.
We believe the reduction in γ motor neurons is the
main factor underlying restoration of muscle contraction.
If the number of γ motor neurons is reduced, then the
contraction conditions of the intra-fusal fibers in the
spindles could not be transmitted to α motor neurons in a
smooth manner. More specifically, decreased efficiency
of the γ loop would result in reduced contractile strength.
In considering why the number of γ motor neurons was
reduced after nerve transfer, we speculate that several
factors are involved. Firstly, the axons of the γ motor
neurons are thin and non-myelinated. Secondly, the spe-
cific effects of neutrophic factors are not yet proven on
the neurons [47]. Supplementary observation of associ-
ated changes of sensory neurons in spinal ganglia is nec-
essary.
The motor neurons innervating the trapezius and su-
praspinatus muscle were observed together in C3-C6,
and the two groups of neurons were identified in a
somewhat ventral-dorsal arrangement. Following nerve
transfer, the surviving motor neurons were observed
predominantly in the same distributional field of Ac, and
this phenomenon could not be explained by the theory of
“motor neuron misdirection” [37]. On the other hand, the
effects of homeobox genes are very important when ax-
ons extend out of the spinal cord in early embryos
[48-51]. However, these early molecular studies did not
consider whether the neurons could survive (or not) fol-
lowing nerve transfer. Therefore, it is necessary to clarify
why the motor neurons innervating the supraspinatus
muscle survived after nerve transfer treatment; i.e., by
adopting a genetic or molecular approach.
5. Conclusions
The distributional portion of motor neurons following
nerve transfer was mainly within the neuron column in-
nervating the trapezius. Some neurons innervating the
supraspinatus muscle also survived post-transfer. The
EMG restoration rate of the supraspinatus muscle fol-
lowing nerve transfer was 60%, and the rate of the con-
trol group was 80% of that in the non-operated group.
Following nerve transfer, there was a distinct reduction

J. YAN ET AL.275
in the number of γ motor neurons. Therefore, γ motor
neurons may have important effects on the recovery of
muscular strength following nerve transfer treatment.
After nerve transfer treatment, the functional restora-
tion of damaged muscle may be related to the develop-
mental origin of the donor nerve.
6. Acknowledgments
We thank Dr. S. Kobayashi, and Dr. S. Kimura (Iwate
Medical University) for their technical advice.
This work was supported by a research grant from the
Ministry of Education, Culture, and Science of Japan
(No. 22590178), and it was also supported financially by
the Advanced Medical Science Center of Iwate Medical
University.
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List of Abbreviations
Ac: the branch innervating trapezius;
Am: auricularis magnus;
C: cranial;
Cb: communicating branch to C7;
C1: the first cervical segment.
Cs: cleidomastoideus (in rat);
D: dorsal branch;
Oh: omohyoideus;
Sb: the branch innervating sternocleidomastoideus;
Sm: scalenus medius;
Ss: suprascapular nerve;
Tc: transverses colli;
Tr: trapezius;
V: ventral.