J Neurosurg 120:502–508, 2014
©AANS, 2014
Assessment of the rate of spinal motor axon regeneration by
choline acetyltransferase immunohistochemistry following
sciatic nerve crush injury in mice
Laboratory investigation
Qiuju Yuan, Ph.D.,1 Huanxing Su, Ph.D., 5 Kin Chiu, Ph.D., 2 Zhi-Xiu Lin, Ph.D.,1
and Wutian Wu, M.D., Ph.D. 2–4,6
School of Chinese Medicine, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T.,
Hong Kong SAR, China; 2Department of Anatomy, 3State Key Laboratory of Brain and Cognitive Sciences,
4
Research Center of Reproduction, Development and Growth, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Pokfulam, Hong Kong SAR, China; 5State Key Laboratory of Quality Research in
Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China; and
6
GHM Institute of CNS regeneration, Jinan University, Guangzhou, China
1
Object. The purpose of this study was to examine whether choline acetyltransferase (ChAT) staining can be used
for assessing the rate of motor neuron regeneration at an early phase of axon outgrowth.
Methods. The authors developed a new sciatic nerve crush model in adult mice. In this model, in addition to
performing a sciatic nerve crush injury, the authors excised the ipsilateral lumbar L3–6 dorsal root ganglion (DRG),
which resulted in degeneration of the sensory fibers entering into the sciatic nerve. Crushed nerve sections obtained
at Day 3 or Day 7 postinjury were analyzed by means of immunostaining.
Results. The immunostaining showed that ChAT, a motor axon–specific antigen, was totally co-localized with
growth-associated protein 43 (GAP-43), which is expressed in regenerating nerves and transported into growth cones.
Conclusions. Our results suggest that measuring the length of motor axon outgrowth by ChAT immunostaining
is reliable. ChAT staining provides a more convenient method for evaluating the rate of motor axon outgrowth in a
mixed nerve.
(http://thejns.org/doi/abs/10.3171/2013.8.JNS121648)
Key Words • nerve injury • axon regeneration • motor neuron • ChAT staining • peripheral nerve
I
njury to the peripheral nervous system is often followed by poor functional recovery, especially when
the injury is inflicted on large nerve trunks like the
brachial or lumbar plexus,1,5 although peripheral axons
have the capacity to regenerate through the injury site toward distal territories. This phenomenon may be due to
compromise of motor axon regeneration by chronic distal
nerve stump denervation, delayed repair, or prolonged regeneration distance, with the ability for regeneration being progressively impaired with time and/or distance.5,8,26
Discovering new therapies for accelerating the rate of
motor axon growth after peripheral nerve injury is of
clinical significance. Accordingly, finding convenient
Abbreviations used in this paper: CGRP = calcitonin gene–
related peptide; ChAT = choline acetyltransferase; GAP-43 =
growth-associated protein 43; NF200 = neurofilament-200; PBS =
phosphate-buffered saline.
502
and reliable methods for measuring the rate of motor regeneration is also important.
Immunostaining nerve sections against axon-specific
antigens is one of the quantitative methods used to measure axon regeneration.2,13,23,27,28,30 Assessing the rate of
motor axon regeneration is usually accomplished through
immunostaining facial nerve sections against antigens of
calcitonin gene–related peptide (CGRP) and galanin,28 because these peptides are synthesized in 2 apparently nonoverlapping motoneuron populations16 and undergo anterograde transport into the axon growth cones. However, this
method is not suitable for assessing motor axon regeneration in large nerve trunks such as the brachial or lumbar
plexus, although their motor axons also contain galanin
or CGRP.3,22 Unlike the facial nerve, which is mostly moThis article contains some figures that are displayed in color
on­line but in black-and-white in the print edition.
J Neurosurg / Volume 120 / February 2014
Assessment of motor axon outgrowth
tor, the sciatic nerve is a mixed nerve, composed of both
sensory and motor axons. Galanin and CGRP are widely
expressed in the sensory nervous system.21,29 Thus, when
a sciatic nerve is injured, both sensory and motor neurons
will regenerate their axons. Because CGRP, galanin, and
GAP-43 are expressed by both sensory and motor neurons, immunostaining for these antigens is not suitable for
specifically measuring motor axon regeneration in mixed
nerves.
Spinal motor neurons use acetylcholine as their neurotransmitter. Choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, is now generally
accepted as a definitive marker for normal spinal motor
neurons and their axons.24 However, immunostaining
nerves against ChAT is usually used for assessing motor
axon regeneration after onset of nerve reinnervation following nerve injury.4,12 Assessing the rate of motor axon
regeneration at an early stage of axon growth through immunostaining of peripheral nerve sections against ChAT
has so far not been established. In this mouse study, we
used a sciatic nerve crush model, in which the dorsal root
ganglion was also removed, to demonstrate whether ChAT
is suitable for assessing short-term motor axon regeneration. We compared immunostaining for ChAT with immunostaining for GAP-43, which can be transported in an
anterograde manner into the axon growth cones.
Animals
Methods
Young adult male C57/BL6 mice (2–3 months old)
were used in this study. All surgical interventions and
subsequent care and treatment procedures were approved by the Committee on the Use of Live Animals for
Teaching and Research of the University of Hong Kong.
Lesion Model and Surgical Procedures
The mice were anesthetized with ketamine (80 mg/
kg) and xylazine (8 mg/kg) and placed on the surgical
table. The sciatic nerve of the right hind leg was subjected
to a procedure to cause a crush lesion following a previously described experimental protocol.10,33,34 Briefly, following incision of the skin between the knee and thigh,
the sciatic nerve was carefully exposed and then crushed
for 10 seconds with a forceps with a fine tip. The nerve
was crushed at the sciatic notch point immediately distal
from where it emerges from beneath the gluteus maximus
muscle. The wound was closed by suturing the muscles
and skin. Following the crush injury, we further excluded
sensory nerve sprouts in the sciatic nerve using a previously described procedure.31 Briefly, in addition to the
sciatic nerve crush injury, a dorsal laminectomy was carried out. The L3–6 spinal roots were exposed under a
surgical microscope, and the dorsal root ganglia of L3–6
were excised (Fig. 1). For a negative control experiment
for GAP-43 staining, the L3–6 dorsal root ganglia were
excised from 5 mice without sciatic nerve crush injury.
After surgery, the mice were kept on a heating pad for an
hour to maintain their body temperature before being returned to their cage. The animals were allowed to survive
for 3 or 7 days (n = 8 for each time point).
J Neurosurg / Volume 120 / February 2014
Fig. 1. Schematic drawing depicting the sciatic nerve crush model
and the removal of a dorsal root ganglion.
Perfusion and Tissue Processing
At the end of the postoperative survival period, the
mice were deeply anesthetized with ketamine and xylazine
and perfused intracardially with normal saline, followed by
4% paraformaldehyde in 0.1 M phosphate-buffered saline
(PBS) (pH 7.4). A 1-mm–long sciatic nerve segment was
dissected from a point 2 or 8 mm distal to the crush site at
3 or 7 days postinjury, respectively; it was immersion-fixed
in the same fixative for 6 hours and then placed into 30%
sucrose in 0.1-M PBS overnight. Transverse or longitudinal serial sections (10 μm thick) of the sciatic nerves were
obtained using a Leica cryotome. The sections were then
mounted on gelatin-coated microscope slides.
Immunocytochemistry
For single immunostaining of ChAT (1:800, Chemi­
con), standard immunofluorescence was performed on
the sciatic nerve sections according to a procedure described previously.32 Briefly, the sections were incubated
with the primary antibody against ChAT in 0.1-M PBS
(pH 7.4) containing 10% normal goat serum and 0.2%
Triton X-100 overnight at room temperature. Following
incubation in the primary antibody, antigens were visualized using Alexa 568-conjugated secondary antibody
(1:800, Molecular Probes). Double immunofluorescence
analysis was performed by sequential incubation in 2 primary antibodies. Briefly, the sections were incubated with
antibody to ChAT and then the Alexa 568-conjugated
secondary antibody. After being washed thoroughly, the
sections were incubated with antibody to either neurofilament 200 (NF200) (1:3000, Chemicon International) or
GAP-43 (1:500, Chemicon International) and then Alexa488-conjugated secondary antibody (1:800, Molecular
Probes). Finally, the sections on gelatin-coated glass
slides were coverslipped in mounting medium (Dako)
and fluorescent images were captured with a Zeiss microscope equipped with a SPOT digital camera (Diagnostic
Instruments).
Results
Presence of ChAT- and non–ChAT-Immunostained Axons in
Normal Sciatic Nerves
Double immunostaining for ChAT/NF200 (Fig. 2A–
C) showed mixed response in sections of normal sciatic
nerve; some areas stained positive for ChAT (arrows in
503
Q. Yuan et al.
Fig. 2C) and some did not (short arrows in Fig. 2C), indicating that the sciatic nerve contains both motor and
nonmotor (sensory) axons.
Detection of ChAT Immunostaining in Motor Axon Sprouts
ChAT immunostaining was detected in nonlesional sciatic nerve (Fig. 3A). At Day 3 following the crush
injury and 2 mm distal to the crush site, ChAT-positive
Fig. 2. Photomicrographs of transverse sections of sciatic nerve
from the nonlesional side stained for ChAT (A, red) and NF200 (B,
green) and after double-staining for ChAT and NF200 (C). The sciatic
nerve contains ChAT-positive (slender arrows in upper part of C) and
ChAT-negative (thick, short arrows in lower part of C) axons. Yellow
indicates neurons that are positive for both ChAT and NF200. Scale
bar = 30 µm.
504
staining of the newly formed axon sprouts was seen (Fig.
3B). Similarly, at Day 7 following the crush injury and 8
mm distal to the crush site, ChAT-positive staining of the
newly formed axon sprouts was observed (Fig. 3C).
Co-Localization of ChAT and GAP-43 Expression in Motor
Axon Sprouts
To evaluate whether the method is reliable for assessing the regeneration rate of motor fibers in the sciatic
nerve, we compared the expression of ChAT and GAP-
Fig. 3. Photomicrographs of ChAT-immunostained transverse sections of sciatic nerve from the nonlesional side (A) and the lesional
side obtained on Day 3 (B) or Day 7 (C) postinjury. Newly formed axons could be stained by ChAT immunostaining at the 2 examined time
points. Scale bar = 30 µm.
J Neurosurg / Volume 120 / February 2014
Assessment of motor axon outgrowth
Fig. 4. Longitudinal sections of lesional sciatic nerve obtained on Day 3 (A-C) or Day 7 (D-F) postinjury immunostained for
ChAT (red) and GAP-43 (green). All ChAT-labeled newly formed axons expressed GAP-43 (yellow in C and F) in sciatic nerves.
Scale bar = 40 µm.
43 by double immunostaining. ChAT was completely
co-localized with GAP-43 in the crushed sciatic nerve in
motor axon sprouts at the 2 examined time points of Day
3 and Day 7 after crush injury (Fig. 4A–C, D–F, Fig. 5A–
C, D–F), indicating that ChAT can be transported into
growth cones of motor sprouts as can GAP-43 even at the
early stages of axon regeneration after nerve crush. The
results of the negative control experiments for GAP-43
staining in the absence of the crush injury but in the presence of dorsal root ganglionectomy are shown in Fig. 6.
Discussion
The most widely used model of peripheral nerve injury is sciatic nerve injury.1,11,16–20 This model is useful
for testing the efficacy of new therapies for accelerating
and enhancing axon growth and regeneration after injury.
When compared with other models of peripheral nerve
injury, sciatic nerve crush has several advantages, including easy access to the sciatic nerve. However, the sciatic
J Neurosurg / Volume 120 / February 2014
nerve is a mixed nerve, including both motor and sensory
fibers. If adult sciatic nerve is injured, both motor and
sensory injured neurons will regenerate their axons, and
both will express GAP-43.10,14,31 Thus, the use of GAP43 immunostaining, which is usually used for evaluating
axon outgrowth of optic nerve,6,9,15,25 is not suitable for
specifically evaluating motor or sensory axon outgrowth
in sciatic nerve. Spinal motor neurons use acetylcholine
as their neurotransmitter, and ChAT, the enzyme that
synthesizes acetylcholine, is now generally accepted as
a definitive marker for normal spinal motor neurons and
their axons.4,5,24 However, previous studies have shown
that ChAT protein expression is transiently reduced after
axon injury.7 So far, ChAT immunostaining has only been
used for long-term assessment of motor axon regeneration following peripheral nerve injury, sometimes 60 days
after such injury, when ChAT protein expression is fully
recovered.12 To evaluate whether ChAT immunostaining
can be used to label motor axons for assessing the rate
of motor axon outgrowth at the early stage of axon re505
Q. Yuan et al.
Fig. 5. Transverse sections of lesional sciatic nerve obtained on Day 3 (A–C) or Day 7 (D–F) postinjury immunostained for
ChAT (red) and GAP-43 (green). All ChAT-labeled newly formed axons expressed GAP-43 (yellow in C and F) in sciatic nerves.
Scale bar = 100 µm.
generation after peripheral nerve injury, we compared the
expression of ChAT and GAP-43 staining in motor axon
sprouts in sciatic nerve. We purified sciatic nerve by removing the dorsal root ganglia so as to exclude the input
of GAP-43 from dorsal root ganglia into sensory nerve.
Thus, all GAP-43–positive axon sprouts are motor neurons. We found that ChAT was totally colocalized with
GAP-43 in motor axon sprouts, indicating that ChAT
can be transported into nerve terminals, as can GAP-43.
Thus, measuring the length of motor axon outgrowth by
ChAT immunostaining is reliable.
Conclusions
This newly developed experimental method in our
present study, which used ChAT staining to detect motor
506
axon sprouts, provides a more convenient and reliable approach for evaluating motor axon outgrowth in a mixed
nerve. It is anticipated that this protocol would be useful
for evaluating the efficacy of a therapy for accelerating the
rate of motor axon growth after peripheral nerve injury.
Disclosure
This study was supported by a Direct Grant of The Chinese
University of Hong Kong and the University of Hong Kong.
Author contributions to the study and manuscript preparation
include the following. Conception and design: Wu, Yuan, Su, Lin.
Acquisition of data: Yuan, Su, Lin. Analysis and interpretation of
data: Su, Lin. Drafting the article: Yuan, Lin. Critically revising
the article: Wu, Yuan, Su, Lin. Reviewed submitted version of
manuscript: Wu, Yuan, Su, Lin. Statistical analysis: Yuan, Su, Lin.
Administrative/technical/material support: Wu, Chiu, Lin. Study
supervision: Wu, Lin.
J Neurosurg / Volume 120 / February 2014
Assessment of motor axon outgrowth
Fig. 6. Negative control experiments for ChAT and GAP-43 staining
in the absence of crush injury but in the presence of dorsal root ganglionectomy. Only ChAT, but not GAP-43 (arrow in C), was observed.
Scale bar = 100 µm.
References
1. Bain JR, Mackinnon SE, Hunter DA: Functional evaluation of
complete sciatic, peroneal, and posterior tibial nerve lesions in
the rat. Plast Reconstr Surg 83:129–138, 1989
2. Boyd JG, Gordon T: A dose-dependent facilitation and inhibition of peripheral nerve regeneration by brain-derived neurotrophic factor. Eur J Neurosci 15:613–626, 2002
3. Brumovsky P, Mennicken F, O’Donnell D, Hökfelt T:
Differential distribution and regulation of galanin receptors- 1
J Neurosurg / Volume 120 / February 2014
and -2 in the rat lumbar spinal cord. Brain Res 1085:111–120,
2006
4. Castro J, Negredo P, Avendaño C: Fiber composition of the rat
sciatic nerve and its modification during regeneration through
a sieve electrode. Brain Res 1190:65–77, 2008
5. Eggers R, Tannemaat MR, Ehlert EM, Verhaagen J: A spatiotemporal analysis of motoneuron survival, axonal regeneration
and neurotrophic factor expression after lumbar ventral root
avulsion and implantation. Exp Neurol 223:207–220, 2010
6. Feigenspan A, Dedek K, Schlich K, Weiler R, Thanos S:
Expression and biophysical characterization of voltage-gated
sodium channels in axons and growth cones of the regenerating
optic nerve. Invest Ophthalmol Vis Sci 51:1789–1799, 2010
7. Hoang TX, Nieto JH, Tillakaratne NJ, Havton LA: Autonomic
and motor neuron death is progressive and parallel in a lumbosacral ventral root avulsion model of cauda equina injury. J
Comp Neurol 467:477–486, 2003
8. Höke A, Gordon T, Zochodne DW, Sulaiman OA: A decline
in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann
cell denervation. Exp Neurol 173:77–85, 2002
9. Kaneda M, Nagashima M, Nunome T, Muramatsu T, Yamada
Y, Kubo M, et al: Changes of phospho-growth-associated protein 43 (phospho-GAP43) in the zebrafish retina after optic
nerve injury: a long-term observation. Neurosci Res 61:281–
288, 2008
10. Kato K, Liu H, Kikuchi S, Myers RR, Shubayev VI: Immediate
anti-tumor necrosis factor-alpha (etanercept) therapy enhances axonal regeneration after sciatic nerve crush. J Neurosci
Res 88:360–368, 2010
11. Kirsch M, Campos Friz M, Vougioukas VI, Hofmann HD:
Wallerian degeneration and axonal regeneration after sciatic
nerve crush are altered in ICAM-1-deficient mice. Cell Tissue
Res 338:19–28, 2009
12. Lago N, Navarro X: Correlation between target reinnervation
and distribution of motor axons in the injured rat sciatic nerve.
J Neurotrauma 23:227–240, 2006
13. Leung JY, Bennett WR, Herbert RP, West AK, Lee PR, Wake
H, et al: Metallothionein promotes regenerative axonal sprouting of dorsal root ganglion neurons after physical axotomy.
Cell Mol Life Sci 69:809–817, 2012
14. Liu Z, Cai H, Zhang P, Li H, Liu H, Li Z: Activation of ERK1/2
and PI3K/Akt by IGF-1 on GAP-43 expression in DRG neurons with excitotoxicity induced by glutamate in vitro. Cell
Mol Neurobiol 32:191–200, 2012
15. Ma TC, Campana A, Lange PS, Lee HH, Banerjee K, Bryson
JB, et al: A large-scale chemical screen for regulators of the
arginase 1 promoter identifies the soy isoflavone daidzeinas a
clinically approved small molecule that can promote neuronal
protection or regeneration via a cAMP-independent pathway.
J Neurosci 30:739–748, 2010
16. Moore RY: Cranial motor neurons contain either galaninor calcitonin gene-related peptidelike immunoreactivity. J
Comp Neurol 282:512–522, 1989
17. Ngeow WC, Atkins S, Morgan CR, Metcalfe AD, Boissonade
FM, Loescher AR, et al: A comparison between the effects of
three potential scar-reducing agents applied at a site of sciatic
nerve repair. Neuroscience 181:271–277, 2011
18. Ngeow WC, Atkins S, Morgan CR, Metcalfe AD, Boissonade
FM, Loescher AR, et al: The effect of Mannose-6-Phosphate
on recovery after sciatic nerve repair. Brain Res 1394:40–48,
2011
19. Ngeow WC, Atkins S, Morgan CR, Metcalfe AD, Boissonade
FM, Loescher AR, et al: Histomorphometric changes in repaired mouse sciatic nerves are unaffected by the application
of a scar-reducing agent. J Anat 219:638–645, 2011
20. Noorafshan A, Omidi A, Karbalay-Doust S, Aliabadi E, Deh­
ghani F: Effects of curcumin on the dorsal root ganglion
structure and functional recovery after sciatic nerve crush in
rat. Micron 42:449–455, 2011
507
Q. Yuan et al.
21. Pope RJ, Holmes FE, Kerr NC, Wynick D: Characterisation
of the nociceptive phenotype of suppressible galanin overexpressing transgenic mice. Mol Pain 6:67, 2010
22. Ringer C, Weihe E, Schütz B: Calcitonin gene-related peptide
expression levels predict motor neuron vulnerability in the
superoxide dismutase 1-G93A mouse model of amyotrophic
lateral sclerosis. Neurobiol Dis 45:547–554, 2012
23. Saito H, Dahlin LB: Expression of ATF3 and axonal outgrowth
are impaired after delayed nerve repair. BMC Neurosci 9:88,
2008
24. Su H, Wu Y, Yuan Q, Guo J, Zhang W, Wu W: Optimal time
point for neuronal generation of transplanted neural progenitor cells in injured spinal cord following root avulsion. Cell
Transplant 20:167–176, 2011
25. Su Y, Wang F, Teng Y, Zhao SG, Cui H, Pan SH: Axonal
regeneration of optic nerve after crush in Nogo66 receptor
knockout mice. Neurosci Lett 460:223–226, 2009
26. Sulaiman OA, Midha R, Munro CA, Matsuyama T, Al-Majed
A, Gordon T: Chronic Schwann cell denervation and the presence of a sensory nerve reduce motor axonal regeneration.
Exp Neurol 176:342–354, 2002
27. Van der Zee CE, Man TY, Van Lieshout EM, Van der Heijden
I, Van Bree M, Hendriks WJ: Delayed peripheral nerve regeneration and central nervous system collateral sprouting in
leucocyte common antigen-related protein tyrosine phosphatase-deficient mice. Eur J Neurosci 17:991–1005, 2003
28. Werner A, Willem M, Jones LL, Kreutzberg GW, Mayer U,
Raivich G: Impaired axonal regeneration in alpha7 integrindeficient mice. J Neurosci 20:1822–1830, 2000
29. Wu ZM, Chen YF, Qiu PN, Ling SC: Correlation between the
distribution of SP and CGRP immunopositive neurons in dor-
508
sal root ganglia and the afferent sensation of preputial frenulum. Anat Rec (Hoboken) 294:479–486, 2011
30. Yi C, Dahlin LB: Impaired nerve regeneration and Schwann
cell activation after repair with tension. Neuroreport 21:958–
962, 2010
31. Yuan Q, Hu B, Wu Y, Chu TH, Su H, Zhang W, et al: Induction
of c-Jun phosphorylation in spinal motoneurons in neonatal
and adult rats following axonal injury. Brain Res 1320:7–15,
2010
32. Yuan Q, Scott DE, So KF, Wu W: A subpopulation of reactive
astrocytes at affected neuronal perikarya after hypophysectomy in adult rats. Brain Res 1159:18–27, 2007
33. Yuan Q, Su H, Guo J, Tsang KY, Cheah KS, Chiu K, et al:
Decreased c-Jun expression correlates with impaired spinal
motoneuron regeneration in aged mice following sciatic nerve
crush. Exp Gerontol 47:329–336, 2012
34. Zickler P, Küry P, Gliem M, Hartung HP, Jander S: Differential
patterns of local gene regulation in crush lesions of the rat optic and sciatic nerve: relevance to posttraumatic regeneration.
Cell Physiol Biochem 26:483–494, 2010
Manuscript submitted November 12, 2012.
Accepted August 8, 2013.
Please include this information when citing this paper: published
online September 13, 2013; DOI: 10.3171/2013.8.JNS121648.
Address correspondence to: Wutian Wu, M.D., Ph.D., Department
of Anatomy, Li Ka Shing Faculty of Medicine, The University
of Hong Kong, 21 Sassoon Rd., Hong Kong SAR, China. email:
[email protected].
J Neurosurg / Volume 120 / February 2014