How to recognise collateral damage in partial nerve injury models... pain Commentary

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PAIN 153 (2012) 11–12
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Commentary
How to recognise collateral damage in partial nerve injury models of neuropathic
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have identified various neurochemical alterations. For example, increases in levels of messenger RNAs (mRNAs) for several neuropeptides, brain-derived neurotrophic factor (BDNF) and neuronal nitric
oxide synthase have been reported [5,15], as well as changes
involving a subunits of voltage-gated sodium channels, which
might underlie development of spontaneous activity. Electrophysiological studies have directly demonstrated abnormal levels of
spontaneous activity in L4 primary afferents [2,10].
However, the assumption that L4 primary afferents were entirely ‘‘intact’’ following L5 SNL was called into question by studies
that demonstrated expression of activating transcription factor 3
(ATF3) in at least 10% of neurons in the ipsilateral L4 DRG of rats
that had undergone SNL [4,15]. ATF3 is not normally present in primary afferents, but can be detected in the nuclei of virtually all of
those that have undergone axotomy [15]. A specific problem was
whether the changes seen in L4 were really occurring in intact
afferents.
In this issue of Pain, Fukuoka et al. have addressed this problem,
by carrying out a careful anatomical study of neurochemical
changes in the L4 DRG following L5 SNL in the rat, and by relating
these changes to the expression of ATF3 in individual cells [6]. An
important technical point was that they accurately quantified
mRNA levels, in order to look for subtle alterations. Firstly, they
found ATF3 expression in only 5% of L4 DRG neurons, which is
considerably lower than previous estimates of 10–40% [4,15], and
they attributed this to the restricted surgical exposure in their
model, emphasising the need to minimise tissue damage in adjacent segments. Secondly, they found significant differences in the
neurochemical responses of damaged (ATF3+) and undamaged
(ATF3 ) L4 neurons. Specifically, neuropeptide Y was only upregulated in damaged neurons (as reported previously [15]), whereas
BDNF mRNA levels increased in many undamaged neurons.
Because of the potential role of voltage-gated sodium channels
in development of spontaneous activity, and conflicting evidence
from previous studies about changes in expression of their a subunits, Fukuoka et al. also analysed mRNAs for several of these subunits (Nav1.1, Nav1.3, Nav1.6-Nav1.9). They were particularly
interested in Nav1.3, which is dramatically upregulated in axotomised afferents, and has been implicated in the development of
spontaneous firing [2,16]. However, only a barely detectable increase in the mRNA for Nav1.3 was found in the L4 DRG, and this
mainly affected the damaged neurons. The authors concluded that
Nav1.3 expression was unlikely to account for spontaneous discharges in uninjured L4 primary afferents. They were unable to detect significant changes in any of the other Nav mRNAs examined
in the L4 ganglion, apart from a down-regulation of Nav1.8 and
Nav1.9 in damaged C fibre neurons.
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Several animal models of neuropathic pain, involving incomplete lesions of the sciatic nerve or its branches, have been developed since the late 1980s. The first of these, chronic constriction
injury [1], involved loose ligation of the sciatic nerve, resulting in
subsequent strangulation of the nerve. Shortly after this, Seltzer
et al. [12] reported that partial transection of the sciatic nerve could
also be used as a model for neuropathic pain. A limitation with both
of these methods was the inevitable variability in the extent and
severity of the lesion. In order to address this limitation, Kim and
Chung [8] developed the spinal nerve ligation (SNL) model, in which
either L5, or both L5 and L6, spinal nerves on one side were tightly
ligated. These animals rapidly developed signs of mechanical and
cold allodynia, together with heat hyperalgesia, and spontaneous
foot-lifting, which was interpreted as resulting from on-going pain
[8].
Apart from its repeatability, another advantage of SNL over previous models was the anatomical separation of damaged primary
afferent fibres from those that remained intact. It is important to
note that while evoked symptoms of neuropathic pain (allodynia
and hyperalgesia) depend on primary afferents that remain connected to their peripheral receptive fields, ectopic activity in axotomised afferents may give rise to spontaneous pain and, by
inducing central sensitisation in dorsal horn neurons, could also
be involved in initiating the neuropathic pain state.
Several studies exploited the separation of afferents to identify
those responsible for neuropathic pain. There is general agreement
that transection of the ipsilateral L4 dorsal root prevents hyperalgesia and allodynia following SNL, indicating that L4 primary afferents transmit the signals responsible for these phenomena [9,17].
The role of injured primary afferents is controversial. An early
study [13] suggested that surviving central terminals of these
afferents were required for manifestation of the pain. However, it
was subsequently reported that cutting the dorsal root corresponding to the damaged spinal nerve [9], or even removal of the
L5 dorsal root ganglion [14], resulted in mechanical hyperalgesia.
This led to the ‘‘intact nociceptor hypothesis’’, which suggested
that sensitisation of undamaged afferents entering the spinal cord
through nearby dorsal roots was responsible for neuropathic pain
[3]. This sensitisation was thought to result from Wallerian degeneration of the injured afferents, where these lay in close proximity
to intact fibres [3].
These findings have led to considerable interest in the properties of L4 primary afferents in the SNL model. Several studies have
investigated neurons in the L4 dorsal root ganglion (DRG), and
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DOI of original article: 10.1016/j.pain.2011.09.009
0304-3959/$36.00 Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.pain.2011.10.031
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Commentary / PAIN 153 (2012) 11–12
These findings emphasise the need to distinguish between intact and unintentionally damaged primary afferents in neuropathic
models, and the approach used (combining ATF3 detection with
in situ hybridisation histochemistry) can be applied to many other
proteins or peptides whose expression changes after nerve injury.
However, it is important to remember that changes in mRNA levels
may not correlate directly with changes in the level of functional
protein/peptide. For example, although nerve injury causes significant upregulation of the mRNA for preprotachykinin 1 (the precursor for substance P) in large DRG neurons that give rise to
low-threshold mechanoreceptive myelinated afferents [11], it has
not been possible to detect the peptide in central terminals of these
afferents in the dorsal horn in the SNL model, or its release upon
stimulation at Ab fibre strength [7].
[7] Hughes DI, Scott DT, Riddell JS, Todd AJ. Upregulation of substance P in lowthreshold myelinated afferents is not required for tactile allodynia in the
chronic constriction injury and spinal nerve ligation models. J Neurosci
2007;27:2035–44.
[8] Kim SH, Chung JM. An experimental model for peripheral neuropathy
produced by segmental spinal nerve ligation in the rat. Pain 1992;50:355–63.
[9] Li Y, Dorsi MJ, Meyer RA, Belzberg AJ. Mechanical hyperalgesia after an L5
spinal nerve lesion in the rat is not dependent on input from injured nerve
fibers. Pain 2000;85:493–502.
[10] Ma C, Shu Y, Zheng Z, Chen Y, Yao H, Greenquist KW, White FA, LaMotte RH.
Similar electrophysiological changes in axotomized and neighboring intact
dorsal root ganglion neurons. J Neurophysiol 2003;89:1588–602.
[11] Noguchi K, Kawai Y, Fukuoka T, Senba E, Miki K. Substance P induced by
peripheral nerve injury in primary afferent sensory neurons and its effect on
dorsal column nucleus neurons. J Neurosci 1995;15:7633–43.
[12] Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain
disorders produced in rats by partial sciatic nerve injury. Pain
1990;43:205–18.
[13] Sheen K, Chung JM. Signs of neuropathic pain depend on signals from injured
nerve fibers in a rat model. Brain Res 1993;610:62–8.
[14] Sheth RN, Dorsi MJ, Li Y, Murinson BB, Belzberg AJ, Griffin JW, Meyer RA.
Mechanical hyperalgesia after an L5 ventral rhizotomy or an L5
ganglionectomy in the rat. Pain 2002;96:63–72.
[15] Shortland PJ, Baytug B, Krzyzanowska A, McMahon SB, Priestley JV, Averill S.
ATF3 expression in L4 dorsal root ganglion neurons after L5 spinal nerve
transection. Eur J Neurosci 2006;23:365–73.
[16] Waxman SG, Dib-Hajj S, Cummins TR, Black JA. Sodium channels and pain.
Proc Natl Acad Sci USA 1999;96:7635–9.
[17] Yoon YW, Na HS, Chung JM. Contributions of injured and intact afferents to
neuropathic pain in an experimental rat model. Pain 1996;64:27–36.
Conflict of interest statement
None.
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Andrew J. Todd
Institute of Neuroscience and Psychology,
University of Glasgow, Glasgow G12 8QQ, UK
Tel.: +44 141 330 5868.
E-mail address: Andrew.Todd@glasgow.ac.uk
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[6] Fukuoka T, Yamanaka H, Kobayashi K, Okubo M, Miyoshi K, Dai Y, Noguchi K.
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