JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE
RESEARCH ARTICLE
J Tissue Eng Regen Med 2015; 9: 415–423.
Published online 10 December 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1833
Motor function recovery during peripheral nerve
multiple regeneration
Shuai An, Peixun Zhang*, Jianping Peng, Lei Deng, Zhenwei Wang, Zhiyong Wang, Yanhua Wang,
Xiaofeng Yin, Yuhui Kou, Na Ha and Baoguo Jiang*
Peking University People’s Hospital, Beijing, China
Abstract
Neuronal functional compensation and multiple regenerating axon sprouting occur during peripheral nerve
regeneration. Sprouting nerve buds were quantitatively maintained and had matured when multiple injured
distal nerves were anastomosed to smaller number of proximal nerve stumps; this has positive clinical
significance for proximal stump damage. This study investigated whether sprouting axon buds would
reinnervate the distal neuromuscular junction and maintain the function of the target organ under compensation conditions. The results showed that the sprouting axon buds maintained the numbers and morphology of motor end plates repaired by a smaller number of proximal nerve stumps, and recovered 80.0%
tetanic muscle force compared with the normal side. Meanwhile, nerve conduction velocity, compound
muscle action potential and diameter of muscular fibres declined 72.7%, 73.2% and 61.8%, respectively, compared with normal. This observation indicates the potential functional reserve of neurons and that it is feasible to repair nerve fibre injury through anastomosis of multiple distal nerve stumps with a smaller number
of proximal nerve stumps, within the limits of compensation. Copyright © 2013 John Wiley & Sons, Ltd.
Received 8 January 2013; Revised 19 April 2013; Accepted 2 September 2013
Keywords
functional recovery; motor end plate; multiple regeneration
1. Introduction
Peripheral nerve injury is common in clinical practice.
Mangled nerve injury and avulsion often result in significant
damage to proximal nerves, which render the nerve repair
difficult (Lundborg et al., 1994; Allodi et al., 2012). Current
repair methods, such as nerve transfer or nerve implantation, usually need sacrifice of another normal nerve as the
donor nerve. Previous studies have revealed that regeneration and compensation occurs in the regeneration of peripheral nerves (Redett et al., 2005; Jiang et al., 2007; Wang
et al., 2009). Furthermore, these studies have provided
preliminary evidence for the maturation of sprouting nerve
fibres (Redett et al., 2005; Yin et al., 2011). These findings
provide a possible novel strategy for repairing serious damage to proximal nerve stumps through direct anastomosis
to smaller number of proximal nerve stumps. This would
avoid injury to donor nerve and not have the disadvantages
of allograft transplant reactions. However, the recovery
outcome has many contributing factors. Correlations have
been observed between the functionality of repaired nerves
and the maturity of nerve fibres, the quantity of motor end
plates and the thickness of muscle fibres (Wood et al., 2011).
In order to explore the possibilities of this repair method
as a strategy, it should be considered from several aspects,
such as nerve fibres, neuromuscular junctions and muscle fibres. A rat tibial nerve model was adopted in this study to further examine whether collateral axon buds could completely
re-establish their control of motor end-plates and maintain
the diameter and function of the original muscle fibres when
multiple injured distal nerves were repaired through anastomosis to a smaller number of proximal nerve stumps.
2. Materials and methods
2.1. Animals
* Correspondence to: B. Jiang and P. Zhang, Department of
Orthopedics and Trauma, Peking University People’s Hospital,
No. 11, South Xi-Zhi-Men Street, Beijing, China, 100044. E-mail:
[email protected]; E-mail: [email protected]
Copyright © 2013 John Wiley & Sons, Ltd.
Experiments were performed using 36 specific pathogenfree (SPF) female Sprague–Dawley rats with a body weight
of 200 g. The animals were randomly divided into three
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S. An et al
experimental groups, groups A, B and C, for the subsequent surgical treatments. Experimental procedures
were reviewed and approved by the Ethics Committee
of the People’s Hospital, Peking University.
2.2. Materials
Biodegradable chitin conduits (People’s Hospital of Peking
University and Chinese Textile Academy, Patent Number
01136314.2) with a length of 8 mm, an inner diameter of
1.5 mm and a wall thickness of 0.1 mm were used.
2.3. Animal model preparation
The surgery was performed under a microscope. The rats
were anaesthetized through intraperitoneal (i.p.) injection of sodium pentobarbital (30 mg/kg). Following complete anaesthesia, skin preparation and disinfection were
carried out in the right hind limb. The right sciatic nerve
and its two main branches (common peroneal nerve and
tibial nerve) were isolated until fully exposed. The tibia
nerve was severed at 5 mm below the bifurcation site of
the sciatic nerve. The animals in each group (n = 12)
were treated with the following procedures (Figure 1):
Group A: The severed tibial nerve was repaired through
anastomosis with the small-gap conduits. The distance
between stumps was 2 mm.
Group B: The common peroneal nerve was severed at the
same level, and the proximal stump was anastomosed to the
distal stump of the severed tibial nerve through a conduit
(with a gap of 2 mm). The other two stumps were sutured
to the muscle in opposite directions to avoid self-repair.
Group C: The tibial nerve was severed, and the resulting
two stumps were sutured to the muscle in opposite directions to avoid self-repair.
Nerve anastomosis through conduits was carried out
with 10–0 nylon suture, and the incision was subsequently
closed with 4–0 suture.
2.4. Observed parameters
2.4.1. Gross morphology and behavioural
observation
The animals (n = 36) were observed to evaluate wound
healing, muscle morphology of the hind limb, and
behavioural changes at different time-points (weeks 4,
6, 8 and 12).
2.4.2. Neuroelectrophysiological examination
A Medlec Synergy electrophysiological system (Oxford
Instrument Inc., Oxford, UK) was used for the examination. The repaired sciatic nerve was exposed at week 12
after the surgery. The stimulating electrodes were placed
on the distal and proximal nerve trunks, on the anastomotic
Copyright © 2013 John Wiley & Sons, Ltd.
Figure 1. (a) Proximal tibial nerve and distal tibial nerve at a
nerve fibre ratio of 1:1; repairing the nerve through anastomosis
with small-gap conduits. (b) Proximal common peroneal nerve
and distal tibial nerve at a nerve fibre ratio of about 1:3 to 1:2;
repairing the nerve through anastomosis with small-gap
conduits. (c) The two stumps of the severed tibial nerve were
sutured to the muscle in opposite directions to avoid self-repair,
which led to denervation of the gastrocnemius. Tp, proximal
tibial nerve stump; Td, distal tibial nerve stump; CPp, proximal
common peroneal nerve stump
plane in the sciatic nerve, while the recording electrode was
inserted into the middle of gastrocnemius; the reference
electrode was placed in the thigh muscle on the same side.
Paraffin oil was applied around the nerve trunk to reduce
bypass conduction through the liquid. The stimulation
signal was a square wave, with an intensity of 0.9 mA, a
wave width of 0.1 ms and a frequency of 1 Hz. The conduction velocity of the regenerated nerve fibres was recorded
by measuring the latent period. The stimulation intensity
was gradually strengthened until the amplitude of the
compound muscle action potential (CMAP) wave ceased
to progressively increase and a generally identical shape
for the CMAP wave was formed from the stimulation at
both the distal and proximal stumps. The amplitude of the
distal CMAP was recorded, which was the distance from
the initiation point to the negative peak of the wave.
2.4.3. Tetanic muscle contraction strength
The rats were fully anaesthetized before sample collection
at week 12 after surgery; this involved dissection and
J Tissue Eng Regen Med 2015; 9: 415–423.
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Changes in peripheral nerve multiple regeneration
isolation of the gastrocnemius. The hind limb was fixed on
a specially made holding frame, with the distal end of the
gastrocnemius connected to a tension sensor and then using
the holding frame kept the gastrocnemius and the tension
sensor aligned. The initial tension was maintained at a
chosen level (0 < F < 0.1 N). An electrophysiological system
was used to generate an initial electric stimulation with an
intensity of 0.9 mA, a wavelength of 0.1 ms and a frequency
of 1 Hz. The stimulation electric current was subsequently
strengthened until the waveform of the tetanic contraction
induced stopped increasing. A PCLAB-UE biomedical signal
acquisition and processing system (Beijing Microsignal star
Inc., Beijing, China) was used to record the waveform of the
tetanic contraction of the gastrocnemius on both sides. The
amplitudes of the waves were measured and the ratio of the
wave amplitudes of experimental side to the untreated
normal control side was used as the overall recovery rate
of muscle strength (Shin et al., 2008).
2.4.4. Wet muscle weight measurement and
diameter of muscle fibres by haematoxylin and
eosin (H&E) staining
The gastrocnemius was isolated by severing it at its
starting and ending point immediately after the aforementioned parameters were measured, and the weight
of the muscle was measured. Transverse sectioning of
the muscle samples was performed for H&E staining after
fixing with paraformaldehyde, dehydrating with graded
ethanol and embedding in paraffin wax. The crosssections of the muscle fibres were photographed under a
magnification of 10 × 20 and five fields were selected in
the upper left, lower left, upper right, lower right and
centre of the cross-section of the muscle fibres for quantification of the diameter of muscular fibres in each field
and for measurements using IMAGE PRO PLUS 6.0 software
(Media Cybernetics Inc., Rockville, MD, USA).
2.4.5. Osmium tetroxide staining of the tibial
nerve and quantification of nerve fibres
Twelve samples of each group were post-fixed in 1%
osmium tetroxide for 1 day, after which the specimen
was sliced into 2 μm cross-sections. The cross-section of
the nerve was photographed under a magnification of
10 × 20 and five fields were selected in the upper left,
lower left, upper right, lower right and centre of the nerve
for quantification of the myelinated nerve fibres in each
field and the measurement of the area of each field using
a combination of manual measurements and measurements
using IMAGE PRO PLUS 6.0 software. The numbers of myelinated nerve fibres in each field were calculated manually and
the area of the field and total area were measured using
IMAGE PRO PLUS. The average number of myelinated nerve
fibres per unit area were then calculated. The total number
of myelinated nerve fibres (N) = the number of myelinated
nerve fibres per unit area (n/ds) × area of the cross-section
(s). The total number of myelinated nerve fibres was calculated using this above equation.
Copyright © 2013 John Wiley & Sons, Ltd.
2.4.6. Immunohistochemical staining of
motor end plates
Using the cupric-ferricyanide staining method developed
by Kamovsky and Roots, acetylthiocholine iodide was
added to freshly prepared incubation buffer as the
enzyme’s substrate, which was hydrolysed into thiocholine
by acetylcholinesterase (AChE) in tissue (Karnovsky and
Roots 1964). Thiocholine reduced the ferricyanide in the incubation buffer into ferrocyanide and this reacted with copper ions to form cupric ferrocyanide, which was deposited
as a brown precipitate at sites with AChE activity. The entire
muscle was divided into three equal parts along its longitudinal axis, and consecutive sagittal cryosectioning (10 μm)
was performed. One section was collected every 100 sections for staining. The selected sections were washed with
phosphate-buffered saline (PBS) three times for 20 min
each time, followed by incubation in Kamovsky–Roots
(KR) solution for 6 h. The sections were then washed with
PBS three more times for 20 min each (Ma et al., 2002).
The sections were finally mounted and observed for quantification of the motor end plates under a 10 × 20 light microscope. The total number of motor end-plates (N) = the sum
of motor end-plates per section (n1 + n2 + …… + n) × 100.
2.4.7. Immunofluorescence staining of
acetylcholine receptors
Sagittal cryosectioning was performed with the muscle
specimen, and one section was collected every 100 sections.
The sections were 20 μm thick. After being air dried, the
sections were washed with PBS three times for 20 min each
time and blocked with serum. The sections then underwent
specific staining of the acetylcholine receptor using
tetramethylrhodamine-labelled α-bungarotoxin (T-BTX).
The T-BTX stock solution (1 mg/ml in PBS) was diluted
1:400. The sections were incubated with diluted T-BTX at
4°C overnight (12 h) and then were washed with PBS three
times for 20 min each time. The sections were mounted
with anti-quenching mounting media. The morphology of
the motor end plates was observed and recorded under a
magnification of 10 × 40 with fluorescence excitation at
620 nm (Ma et al., 2002; Magill et al., 2007). Five fields were
observed in the upper left, lower left, upper right, lower right
and centre areas for each section, and the perimeter and area
of a motor end plate were measured by drawing the maximal
smooth perimeter around the image of each endplate
according to their grey level and then calculated under image
scale using IMAGE-PRO PLUS automatically.
2.5. Statistical analysis
Statistical analyses was performed using SPSS 11.0 (SPSS
Inc., Chicago, IL, USA). The measurement data are expressed
as mean ± SD. An independent t-test was adopted for
two-group comparison, and analysis of variance (ANOVA)
was used for multi-group comparison. Comparison between
the groups was made by analysing data with a post-hoc
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418
S. An et al
method Student-Newman-Keuls (S-N-K). The S-N-K test
is a pairwise comparison of every combination of group
pairs. This test calculates a q test statistic for each pair,
and displays the P value for that comparison. Enumeration
data were analysed using a chi-square test. Statistical
significance was established as p < 0.05.
3. Results
3.1. General observation of the animals
Claudication of the right hind limb, and gradual muscular
atrophy in the lower leg were observed in the right hind
limb in each group after the surgery. At 10 days after
repair, the rats in all groups exhibited discoloration of the
toenails, sparse and dull hair coat on the lower leg and foot
and an insensitivity to pain stimuli on the experimental
side. Foot ulcers were observed at 6 weeks postoperatively
in the untreated group (n = 2), without significant improvement of gait, while the other two groups displayed
nearly normal gait and gradual improvement coordination.
3.2. Quantification of nerve fibres and
electrophysiological examination
The number of normal tibial nerve fibres was 3313 ± 204
at 12 weeks after the surgery. The numbers of proximal
and distal nerve fibres were 3384 ± 273 and 3197 ± 242,
respectively, in group A and 1159 ± 151 and 2909 ± 189,
respectively, in group B. The distal nerve fibres in group C
showed evident degeneration, and some even disappeared
(Table 1). No statistically significant difference was
observed in the number of distal nerve fibres between
group A and group B.
Electrophysiological examination was performed with
fully anaesthetized rats. The nerve conduction velocity
of a normal tibial nerve was 49.9 ± 7.1 m/s. The nerve
conduction velocity was 43.4 ± 11.7 m/s and 36.3 ± 8.1
m/s in group A and group B, respectively. The nerve
conduction velocity in group B was lower than that of
the intact nerve; the difference was statistically significant. The compound muscle action potential indicated
that the wave amplitude of the experimental side was
88.4 ± 5.6% and 73.2 ± 18.9% of the contralateral side
in group A and group B, respectively, but this was not
statistically significant.
3.3. Muscle morphology and strength
Haematoxylin and eosin staining showed that the crosssectional diameter of a normal muscle fibre was approximately 31.9 ± 5.6 μm, with distinct borders and uniform
staining (Figure 2). The muscle fibres of group C displayed
marked atrophy caused by prolonged denervation, with
indistinct borders and uneven staining. The muscle fibre
diameters of the three groups were 21.2 ± 6.9 μm,
19.7 ± 6.4 μm and 13.6 ± 4.8 μm for groups A, B and C,
respectively. The muscle fibre diameter of the untreated
normal control group was greate than those of the
experimental three groups; that of group C was significantly lower than those of the other two groups.
There were no significant differences among the body
weights of rats of all groups before the surgery and there
were also no significant differences in the body weights of
the rats among all groups 4 weeks after the surgery. At 12
weeks after the surgery, the body weights of the rats in
group C were slightly greater than those of the other
two groups, but this was not significantly different. The
gastrocnemius was collected for wet weight measurement
after the rats were euthanized. The wet weight ratios of
the experimental side to the normal side were
64.75 ± 13.5%, 54.66 ± 12.5% and 28.27 ± 10.9% in
groups A, B, and C, respectively; the ratio of group C was
significantly lower than those of the other two groups.
Under anaesthesia, the ratio of the tetanic contractility
of the experimental side to the contralateral side was
93.7 ± 21.4% and 80.0 ± 10.1% for groups A and B,
respectively. The muscles from group C did not show
significant tetanic contraction when the reverse sutured
distal nerve was stimulated.
3.4. Morphology and quantity of neuromuscular
junctions
After the staining of AChE, the normal neuromuscular junction appeared as spherical or clostridial form with a brown
colour and the centre was shallow while the peripheral
region was saturated (Figure 3). The numbers of motor
end-plates in the gastrocnemius nerve fibres of the normal
control and the three experimental groups (A, B and C)
were 27400 ± 6698, 22950 ± 8817, 20433 ± 7187 and
9283 ± 1653, respectively; the numbers in group C were
significantly lower than those of the other groups.
Table 1. Comparison of Myelinated axon numbers, MCV, Peak of CAMP, muscle force and diameter of muscular fibers for all group
Group
Myelinated axon numbers
Proximal
Normal
Group A
Group B
Group C
MCV (m/s)
Peak of CAMP (%)
Muscle force (%)
Diameter of muscular
fibers (μm)
49.2±7.1
43.4±11.7
36.3±8.1*
NA
NA
88.4±5.6
73.2±18.9
NA
NA
93.7±21.4
80.0±10.1
NA
31.9±5.6
21.2±6.9*
19.7±6.4*
13.6±4.8*
Distal
3313±204
3384±273
3197±242
1159±151*
2909±189
NA
NA
*p<0.05, with significant difference.
Copyright © 2013 John Wiley & Sons, Ltd.
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DOI: 10.1002/term
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Changes in peripheral nerve multiple regeneration
Figure 2. (a) Cross-section of normal muscle, showing distinct borders, uniform staining and large diameter. (b,c) Distinct borders
and uniform staining, but with shortened diameter, after anastomosis with small-gap conduits and nerve repair in group B. (d)
Indistinct borders, uneven staining and reduced diameter in group C
Figure 3. The staining of acetylcholinesterase (AChE) 12 weeks postoperatively (×20 objective lens): (a) normal group, (b) group A,
(c) group B, (d) group C. The normal end plate appeared as spherical or clostridial form with a brown colour and the centre of the end
plate was shallow while the peripheral region was saturated. End plates in group B were observed with nearly normal shape and
numbers, which is better than the condition of end plates in group C (denervated group) ( p < 0.001) and similar to the end plates
in group A (p = 0.600)
To further evaluate morphometry of the motor end
plates in the postsynaptic membrane, specific staining of
the acetylcholine receptor was performed, and the perimeter and area of the staining parts were measured
(Figure 4). No significant differences were observed in
the area and perimeter of the stained motor end plates
between the normal control and group A with group B.
In contrast, group C showed a significantly reduced
perimeter compared with the other groups (Figure 5).
Copyright © 2013 John Wiley & Sons, Ltd.
4. Discussion
Injuries to the peripheral nerves result in partial or total
loss of motor, sensory functions in the denervated
segments of the body because of the interruption of
axons, degeneration of distal nerve fibres, and eventual
death of axotomized neurons. Functional deficits caused
by nerve injuries can be compensated by reinnervation
of denervated targets by regenerating injured axons buds.
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S. An et al
Figure 4. Immunofluorescence staining of motor end plates: (a) normal group, (b) group A, (c) group B, (d) group C, (e) single end
plate, (f) the outline of end plate according to grey level. The perimeter and area of a motor end plate were measured by drawing the
maximal smooth perimeter around the image of each endplate according to their grey level and the area was calculated from the
image scale automatically using IMAGE-PRO PLUS software
This phenomenon utilizes the functional reserve of
peripheral nerve, the essence of which is the result of
extensive reinnervation by functional nerve fibres after
the cross-mixing of polyneurons in the proximal sites of
peripheral nerve. Thus, exploring the potential of axon
buds will be one of the essential points in the process of
peripheral regeneration.
The multiple regenerating axons sprouting exist in the
process of peripheral nerve regeneration. Previous studies
indicated that the amount of regenerating axon buds will
obviously be greater than the amount of proximal nerve
fibres after transected peripheral nerve injuries. It is
broadly accepted that most of these regenerating axon
buds could not extend into distal tubules of the basement
membrane and instead turn into neuromas or ultimately
degenerate. The possible reasons for the failed extension
include the mechanical blockade of scar tissue, interference from soft tissues and other factors, and incorrect
growth direction of the axon buds (Morris et al., 1972;
Ito and Kudo, 1994; Skouras et al., 2011). Recently, some
studies have shown that the number of regenerating axon
buds grown into distal stumps could maintain about two to
three times the number of the proximal fibre. Furthermore,
these axon buds could become mature sprouting nerve
fibres, which are considered as multiple regenerations of
Copyright © 2013 John Wiley & Sons, Ltd.
axon sprouting (Redett et al., 2005; Jiang et al., 2007).
Therefore, for the proximal damaged nerve, it is possible
to repair multiple injured distal nerves through anastomosis
to smaller number proximal nerve stumps. However, under
these conditions, whether collateral axon buds could utilize
their potential functional reserve and compensate for the
original function of muscle fibres is unclear.
This study investigated the changes in motor end plates
and muscle function after the repair of multi-nerve
injuries through anastomosis with a smaller number of
nerve stumps. According to the experimental results, at
a certain ratio (35.0%) of distal and proximal nerve
stumps in anastomosis repair, the number of distal axon
buds could reach 87.8% of that of normal tibial fibres,
the amount and morphology of motor end plates could
be basically maintained and the tetanic force of muscle
fibres could be 80.0% of the force of the normal untreated
side. It is concluded that the sprouting axon buds of
peripheral nerves can extend into distal stumps, grow to
the neuromuscular junction and control the connected
muscles to achieve the recovery of motor function.
In the current study, more than one lateral bud sprouted
from a single neuron and was retained to reach the neuromuscular junction and play the role of compensation under
the condition of less proximal fibre amount. From the
J Tissue Eng Regen Med 2015; 9: 415–423.
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Changes in peripheral nerve multiple regeneration
Figure 5. (a) Comparison of the number of motor end plates. (b)
Comparison of the area of motor end plates. (c) Comparison of
the perimeter of motor end plates. The denervated group exhibited
a significantly reduced number and perimeter of motor end plates
than the other two experimental groups and the normal control.
Asterisk indicates a significant difference at p < 0.05
results, the amount of proximal nerve fibres in group B is
obviously less than that of group A and the untreated normal control group, but the amount of distal stumps in group
B is close to the latter. This phenomenon is likely related to
the number of the tubules of the basement membrane at
the distal nerve stump (Jiang et al., 2007; Moradzadeh
et al., 2008; Nichols et al., 2004). As the tubules of the basement membrane are more than merely proximal nerve
fibres, the multiple regenerating axon buds could grow into
distal tubules and be surrounded by proliferative Schwann
cells in the process of bidirectional myelinisation. In addition, nerve regeneration chambers formed in the biological
Copyright © 2013 John Wiley & Sons, Ltd.
421
conduits provide spaces for selective growth. In contrast,
traditional epineurium anastomosis lacks space for selective
growth and, considering directional changes of nerve stumps
during surgery, regenerated axons often grow into soft tissue
between nerve bundles instead of the end organ, which
might be partly responsible for poor functional recovery.
In the present study, the motor end plates were counted
using AChE staining. A neuromuscular junction is composed of three components: nerve endings, muscular fibres
and Schwann cells. A motor end plate is actually referred to
as the postsynaptic membrane of a neuromuscular junction,
which is located on a muscular fibre and faces the nerve
ending (Ma et al., 2002). Acetylcholinesterase staining is
an effective method for locating motor end plates. The
numbers of motor end plates in group B and group A were
compared with that of normal rats, and the differences
were not statistically significant. However, the number
of motor end plates in group C was significantly reduced
12 weeks after surgery. These results indicate that the two
treatment methods maintained the number of motor end
plates. In previous studies, reduced activity of motor end
plates was determined based on lighter AChE staining, the
absence of staining at the centre of the motor end plates
and vacuolar changes. However, both the morphology and
the intensity of AChE staining are affected by a variety of
factors such as staining time and temperature and the
storage time of the staining solution. Thus, AChE staining
can only be used for qualitative analysis. Accordingly, this
study adopted specific fluorescence staining of the
acetylcholine receptor for the semi-quantification of
morphological changes in motor end plates (Deschenes
et al., 2003, 2006, 2010). After measuring the perimeter
and area of motor end plates, no significant changes were
found in the motor end plate areas of the three groups
compared with motor end plates in normal muscles,
suggesting that the activity of the motor end plates was
maintained at a normal level. However, with prolonged
denervation, the perimeter of the motor end plates in group
C showed significant reduction, indicating a trend towards
gradual atrophy.
In addition, considering the slight reduction in the
numbers of nerve fibres and motor end plates, as well as
in muscle size and strength, the electrophysiological
parameters and muscle cross-sectional diameter were
significantly lower in group B than in those of normal
muscle; this may be related to the decreased activity of
the axon buds. Previous studies have shown that during
the process of motor end plate reinnervation, neurons
could give rise to collaterals, resulting in a single motor
end plate being controlled by multiple nerve endings
(Keller-Peck et al., 2001; Buffelli et al., 2003; Livet et al.,
2007; Magill et al., 2007). In the process of multiple
collaterals competing for a motor end plate, the collaterals
exhibited varied activity and competitiveness. In general,
with fewer collateral branches from same neuron and a
larger diameter, the collateral will show higher activity
for material transport and neurotransmitter release
(Kasthuri and Lichtman, 2003). In this study, the use of
a smaller number of nerve stumps to repair a larger
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S. An et al
number of injured nerve fibres was evaluated. The potential
functional reserve of neurons was explored and the activity
of each axon bud was found to significantly reduce.
Therefore, although the number and morphology of motor
end plates were maintained, the electrophysiological
parameters and muscle fibre diameter were decreased.
This phenomenon of compensation and multiple
regeneration was also reported in previous studies of endto-side neurorrhaphy and nerve transposition. Studies have
proven that donor nerves could grow into the recipient
nerve through end-to-side neurorrhaphy, suggesting that
the nerve axon buds have the capability of growth and maturation (Kostakoglu, 1999; Okajima and Terzis, 2000; AlQattan, 2001; Lowe et al., 2002; Geuna et al., 2006). Similar reports of using thin nerves to repair some essential
thick nerves were also found in studies of nerve transposition in which the function of the nerves for muscle control
was greatly recovered (Rohde and Wolfe, 2007, Schalow
et al., 1992). Although these evaluations use clinical functional scores, their results are fundamentally consistent
with our findings. The results of this study demonstrate
that when the proportion of anastomosis is in the range
one-half to one-third of the nerve fibres at the proximal
stumps and at the distal stumps in nerve anastomosis, the
repair might achieve a satisfactory functional recovery.
Therefore, it is suggested that a small number of nerve
fibres can be separated from the adjacent stump of nerve
trunk and used as donor nerve fibres. This will not only
maintain the original function of the adjacent nerve to a certain degree, but it will also restore the function of the region
controlled by the injured nerve. This method may become a
new strategy for the repair of high-level nerve injuries.
Some limitations of this study should be noted. In this
study, the observations are in specific special animal
models, and therefore considering topographic specificity
and species differences during peripheral nerve regeneration, the results should be further confirmed in other
models. Further, owing to the differences and variations
in nerve position and distribution of regeneration,
although the operation and observation was completed
according to unified standard methods, the result was
inevitably influenced by subjective cognizance and system
bias. Whether muscle function can be further restored
through neuronal compensation, or whether muscle
function will decline with long-term compensation, requires
further study.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors thank Zhang Hong-Bo, Li Qing-Tian, Xu Chun-Gui
and Chen Xu-Hong for their kind help and Dong Jian-Qiang
for guidance. This research project was funded by the
Chinese National Natural Science Fund for Outstanding
Youth (30625036), the Chinese 973 Project Planning
(2005CB522604), the Chinese National Natural Science
Youth Fund (30801169), the Beijing City Science & Technology New Star Classification A-2008-10, the Chinese National
Natural Science Fund (31171150, 81171146, 30971526,
31040043) and the Chinese Educational Ministry New Century
Excellent Talent Support Project (2011).
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