Background
The current treatment for brachial plexus root avulsion is mainly based on nerve transfers
and nerve grafts directly implanted into the spinal cord. The results of brachial
plexus reconstruction are poor, despite the sophistication of the various methods
used [[1 ]]. In animals, nerve regeneration into a peripheral nerve (PN) graft after root avulsion
was demonstrated in a series of experiments in rats, cats and primates [[2 ],[3 ],[4 ],[5 ],[6 ],[7 ]]. We have previously shown that spinal motoneurons in adult rats can regenerate
and reinnervate muscles to recover partial function [[8 ],[9 ],[10 ],[11 ]]. However, avulsed motoneurons in neonatal rats are unable to regenerate into a
PN graft [[12 ]], which indicates that intrinsic neuronal factors also determine the regenerative
capabilities.
Successfully regenerating neurons in mammalian peripheral nervous system (PNS) undergo
a variety of changes in gene expression, for example, the prominent upregulation of
growth-associated proteins [[13 ],[14 ]]. This regeneration-associated gene (RAG) expression is believed to enhance the
growth potential of injured neurons. Sensory neurons exhibit little regeneration of
their central axon into a peripheral nerve transplant unless their peripheral axon
is also axotomized [[15 ]], correlating with the stimulation of RAG expression, such as GAP-43 after axotomy
of the peripheral but not of the central axon [[16 ]]. In central nervous system, brain-derived neurotrophic factor (BDNF) but not neurotrophin-3
(NT-3) was found to increase the number of axotomized rubrospinal tract neurons that
regenerated into grafts of sciatic nerve implanted into the spinal cord at the level
of spinal transaction, also correlating with the stimulation of GAP-43 expression
after application of BDNF but not of NT-3. Expression of GAP-43 has also been investigated
in spinal motoneurons following axonal injury [[17 ]]. However, the correlation between GAP-43 expression and regenerative capacity of
injured motoneurons has not been well established. The present experiment was designed
to study the expression of GAP-43 following unilateral avulsion and implantation of
cervical 7 (C7) of brachial plexus in neonatal and adult rats. The potential role
of such expression for axonal regeneration of avulsed motoneurons after root avulsion
was discussed.
Materials and methods
Female Sprague-Dawley postnatal day 1 (PN1), and adult rats (220-250 g) were used.
Animals were anesthetized under deep hypothermia (for PN1) or with ketamine (80 mg/kg)
and xylazine (8 mg/kg) (for adult rats). All surgical interventions and subsequent
care and treatment were approved by the Committee on the Use of Live Animals for Teaching
and Research of the University of Hong Kong.
Anesthetized animals were placed on the surgical table and a dorsal laminectomy was
carried out. The dura was opened and the ventral root and dorsal root with the ganglion
of C7 were selectively avulsed from the spinal cord by traction under a surgical microscope
following procedures described previously [[18 ]]. The site was checked visually to confirm complete avulsion.
For animals received PN reimplantation, the avulsed ventral root was reimplanted following
the procedure described in a previous study [[10 ]]. Briefly, after avulsion and dorsal root ganglion removing, the ventral root was
carefully reimplanted into the ventrolateral aspect of spinal segment C7 with a fine
glass probe. Care was taken not to injure the spinal white matter. The dura was closed.
The muscles, subcutaneous tissues and skin were closed in separate layers. Following
the operation, the animals were allowed to survive for 1, 3, 7, 14 and 28 days, with
five rats in each postoperative time period.
At the end of the postoperative survival period, the rats were deeply anesthetized
with a lethal dose of ketamine (160 mg/kg) and xylazine (16 mg/kg) and were perfused
intracardially with normal saline, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered
(PB) (pH 7.4). A 5 mm segment of C7 spinal nerve was dissected before its first branch.
The C7 spinal segments were carefully dissected under a dissection microscope in order
to avoid damage the implantation area. Tissues were immersion-fixed in the same fixative
for 6 h. They were then placed into 30% sucrose in 0.1 M PB overnight. Transverse
serial sections of spinal cord at 40 μm were cut and collected in wells containing
0.1 M PB.
The sections were incubated overnight at room temperature with a rabbit polyclonal
antibody against GAP-43 (1:500, Chemicon International, Temecula, Calif). After rinsing
with PB, they were incubated for 2 hours at room temperature with a goat-anti-rabbit
secondary antibody conjugated with Alexa-488 (1:400, Molecular Probes, Eugene, USA).
The primary and secondary antibodies were diluted in PBS containing 1% normal goat
serum and 0.2% Triton X-100.
After reaction, the sections were mounted on gelatin-coated glass slides and coverslipped
in mounting medium (Dako, Denmark). Fluorescent images were captured with Zeiss microscope
(Zeiss, Gottingen, Germany) equipped with Spot digital camera (Diagnostic Instruments,
Sterling Heights, MI, USA). Numbers of GAP-43-IR motoneurons in every alternate section
were counted. All results are expressed as mean ± SD.
Sections immunostained with antibody against GAP-43 were counterstained with neutral
red. The number of surviving motoneurons was counted on both the intact and the lesioned
sides as described previously [[19 ]]. The total number of surviving motoneurons on the lesioned side was expressed as
a percentage of the number of motoneurons on the contralateral side.
Results
Age-dependent GAP-43-IR expression in avulsed motoneurons
No GAP-43-IR motoneurons could be found in normal neonatal or adult rats ([Fig 1A, C ] respectively). Following root avulsion in neonatal animals, GAP-43-IR motoneurons
could not be seen in lesion side of ventral horn at all examined post-injury time
points following avulsion ([Table 1 ], [Fig 1B ]). In this age of animals, avulsion induced marked motoneuron death within 1 week
post-injury ([Table 2 ]).
Figure 1 . No GAP-43-IR was detected in ventral horn of the lesion side in neonatal at 3 days
post-injury (B), which was comparable to the age-matched normal control (A). B1 is
the enlargement of the square area in the ventral horn of image B showing negative
GAP-43-IR of motoneurons (arrows). In contrast, GAP-43-IR was induced in many avulsed
motoneurons at 14 days post-injury in the adult animals (D) compared with the adult
normal control (C). D1 is the enlargement of the square area in the ventral horn of
image D showing positive GAP-43-IR of motoneurons (arrows). Scale bar = 400 μm in
A-D, 100 μm in B1 and D1.
Table 1
GAP-43-IR motoneurons in neonatal and adult rats after avulsion.
Survival day(s)
neonatal
adult
1
–
+/–
3
–
+
7
–
+
14
–
++
28
–
–
The staining induction (–, absent; +, moderate; ++, intense) was assessed as compared
to the non-operated side using criteria as (–) no GAP-43-IR motoneuron, (+/–) 1-30
GAP-43-IR motoneurons, (+) 31-150 GAP-43-IR motoneurons and (++) > 150 GAP-43-IR motoneurons.
Table 2
Survival of motoneurons after root avulsion in neonatal and adult rats.
Day(s) after avulsion
neonatal
adult
1
99.3 ± 5.9
98.1 ± 7.5
3
63.4 ± 4.7
99 ± 4.9
7
5.2 ± 0.9
102.2 ± 6.1
14
89.2 ± 5.7
28
46.4 ± 4.1
Data are expressed as a percentage (mean ± SEM) of the number of motoneurons on the
contralateral side, which represent 100%.
In contrast, following spinal root avulsion in adult animals, GAP-43-IR motoneurons
in the avulsed ventral horn were present at 1 day post-injury, subsequently increased
from 3 to 7 days and peaked at 14 days post-injury ([Table 1 ], [Fig 1D ]). Expression of GAP-43 decreased at 4 week post-avulsion ([Table 1 ]). In adult rats, avulsion did not lead to significant motoneuron death until 2 weeks
post-injury ([Table 2 ]).
Age-dependent motor axon regeneration following reimplantation of avulsed roots
To assess whether there is also an age-dependent motor axon regeneration, fiber growth
into the implanted ventral roots was investigated. As shown in [Fig 2A ] and [2B ] (arrow), reimplanted ventral roots contact well with the ventral root exit zone
3 days following reimplantation in the neonatal and adult. No GAP-43-IR fibers were
seen in ventral root exit zone and implanted ventral roots in the neonatal rats ([Fig 2A ]). In contrast, numerous GAP-43-IR fibers were found towards and into the reimplanted
ventral root from the ventral root exit zone in the adult animals ([Fig 2B ]). At 2 weeks post-implantation, no regenerating axons revealed by GAP-43 immunostaining
were observed in reimplanted C7 spinal nerve in the neonatal ([Fig 2C ]). In contrast, many GAP-43-IR axons were found in the adult ([Fig 2D ]).
Figure 2 . No GAP-43-IR regenerative axons were found in the reimplanted area (A) and the C7
spinal nerve (C) following root avulsion and reimplantation in the neonatal. Numerous
GAP-43-IR regenerative motor axons were found in the reimplanted area (B) and the
C7 spinal nerve (D) following root avulsion and reimplantation in the adult. Insertion
in B is the enlargement of the rectangle area in B showing GAP-43 positive fibers
grow into the re-implanted root (small arrows). Scale bar = 100 μm.
Discussion
This study showed that 1) adult but not neonatal motoneurons expressed GAP-43 following
root avulsion, 2) GAP-43 was transiently expressed in adult avulsed motoneurons, 3)
adult but not neonatal motoneurons could regenerate their avulsed axons into the reimplanted
peripheral nerve.
Age-dependent upregulation of GAP-43 in avulsed motoneurons
It has previously been reported that regenerative capacity for avulsed motoneurons
is age-dependent [[12 ]]. For example, neonatal motoneurons are unable to regenerate their axons into the
transplanted PN graft following root avulsion [[12 ]] whereas in adult animals motoneurons are able to regenerate axons into the PN graft
[[8 ],[10 ]]. In this study, we used root avulsion and reimplantation model and found that adult
but not neonatal motoneurons could regenerate their axons into the reimplanted ventral
root and spinal nerve. This result further confirms that regenerative capacity for
avulsed motoneurons is age-dependent. The poor regeneration in the neonatal rats following
root avulsion is in contrast with the situation observed in human. Previous clinical
observations have showed that a better functional recovery from the brachial plexus
injury at birth compared with that in the adult [[20 ]]. However, the extrapolation of experimental data to human situation will have to
confront the issue of age comparison between humans and the animals. Although there
is no simple answer to making age comparisons between humans and the animals used
in animal models [[21 ]], Romijn et al [[22 ]] uses a variety of measurements and determines that the nervous system of a newborn
human is developmentally most comparable to that of a PN13 rat pup. If so, the result
observed in a newborn human would be consistent with that in PN13 rat pup. In fact,
previous studies have shown that avulsed motoneurons in around PN13 rats can regrow
their axons into PN graft [[12 ]]. Whether the difference in age-dependent motoneuron regenerative capacity between
rats and human is due to different mature stages of rats and human beings needs further
investigation.
Successful regeneration depends on upregulation of some molecules [[23 ],[24 ]]. Identification of molecules involved in regenerative processes is a key step toward
development of therapeutic tools in order to promote functional recovery.
Although many molecules appear to correlate with the neuron’s regenerative competence,
the most prominent molecular involved in regeneration is GAP-43 [[14 ]]. GAP-43 is extensively investigated in CNS and PNS following axonal injury, however,
GAP-43 expression in avulsed spinal motoneurons, which are destined to die ultimately,
is not investigated.
In this study, we have found that expression of GAP-43 was upregulated in spinal motoneurons
and such expression is age-dependent. No GAP-43 expression could be found in neonatal
motoneurons following root avulsion. The coincident expression of GAP-43 with robust
axonal regeneration in adult and the absence of GAP-43 expression and axonal regeneration
in neonatal suggest that GAP-43 plays an important role in regeneration of avulsed
spinal motoneurons. The failure of GAP-43 expression in neonatal avulsed motoneurons
may be due to the fact that a more rapid motoneuron loss occurs in neonatal rats compared
with that in adult rat following root avulsion. However, the fact that GAP-43 was
induced in the avulsed spinal motoneurons in adult rats 1 day onward after avulsion
implies that 1 day may be a sufficient time interval for a GAP-43 induction. After
avulsion at neonatal, although most motoneurons still survived for 1 day after injury,
no GAP-43-positive motoneuron was observed. This may exclude the possibility that
there was not sufficient time to allow GAP-43 to become manifest in avulsed motoneurons
in neonatal rats.
Age-dependent GAP-43 expression in avulsed motoneurons may result from age-dependent
expression of calcitonin gene related peptide, which is responsible for encoding growth-associated
protein following nerve injury [[17 ]]. Calcitonin gene related peptide is upregulated in adult motoneurons after injury,
whereas it is downregulated following the same injury in developing animals [[17 ]].
Transient expression of GAP-43 in adult animals
Unlike nerve crush, which preserves the endoneural tube and the continuity of basal
lamina, providing neurotrophic support and a physical guide for the proximal axonal
ends [[25 ],[26 ]], avulsion injury separates motoneurons from all peripheral axons and associated
glia. Clinically, it was noted that patients with PN graft transplantation early after
the injury had a better outcome than later [[7 ]]. Thus, an optimal timing for surgery is an important factor for optimal functional
recovery after root avulsion injury. Based on the role of GAP-43 in axonal regeneration,
a better understanding of time course of GAP-43 expression in avulsed motoneurons
may be essential to develop an optimal time window for surgery repair in order to
accelerate the re-connection of the axons with their targets. In the present study,
we found that GAP-43 was transiently expressed in adult rats following root avulsion
within two weeks and returned to minimal level four weeks post-injury. Therefore,
we suggest that optimal timing for surgery repair is around 2 weeks post-injury. Delayed
implantation of a PN graft up to 3 weeks post-injury does not significantly affect
regeneration even if motoneuron survival is reduced at those surgery time points following
spinal root avulsion in adult rats [[11 ],[27 ]]. Delayed implantation of a PN graft at 4 weeks post-injury results in a poor regeneration
of avulsed motoneurons (data not shown). The fact that avulsed spinal motoneurons
have duration for retaining the ability to regenerate may be due to transient expression
of GAP-43 of avulsed motoneurons.
Conclusion
Close association of GAP-43 expression and capacity of regeneration in reimplanted
spinal nerve of avulsed motoneurons suggests that GAP-43 is a potential therapeutic
target for treatment of root avulsion of brachial plexus.
Abbreviations
PNS:
peripheral nervous system
RAG:
regeneration-associated gene
BDNF:
brain-derived neurotrophic factor
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QY performed experiments, collected and analyzed data, was involved in study design
and wrote the manuscript; BH. collected and analyzed data; HS. collected and analyzed
data; KFS analyzed data; ZL analyzed data; WW designed the study, collected and analyzed
data, wrote the manuscript. All authors read and approved the final manuscript.
