Ò PAIN 153 (2012) 11–12 www.elsevier.com/locate/pain Commentary How to recognise collateral damage in partial nerve injury models of neuropathic pain po r CD R 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. Co pi aa ut or iza da 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 q 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 04/04/2014 Ò 12 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. CD po r da Andrew J. Todd Institute of Neuroscience and Psychology, University of Glasgow, Glasgow G12 8QQ, UK Tel.: +44 141 330 5868. E-mail address: [email protected] Co pi aa ut or iza [1] Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87–107. [2] Boucher TJ, Okuse K, Bennett DL, Munson JB, Wood JN, McMahon SB. Potent analgesic effects of GDNF in neuropathic pain states. Science 2000;290:124–7. [3] Campbell JN, Meyer RA. Mechanisms of neuropathic pain. Neuron 2006;52:77–92. [4] Djouhri L, Koutsikou S, Fang X, McMullan S, Lawson SN. Spontaneous pain, both neuropathic and inﬂammatory, is related to frequency of spontaneous ﬁring in intact C-ﬁber nociceptors. J Neurosci 2006;26:1281–92. [5] Fukuoka T, Noguchi K. Contribution of the spared primary afferent neurons to the pathomechanisms of neuropathic pain. Mol Neurobiol 2002;26:57–67. [6] Fukuoka T, Yamanaka H, Kobayashi K, Okubo M, Miyoshi K, Dai Y, Noguchi K. Re-evaluation of the phenotypic changes in L4 DRG neurons following L5 spinal nerve ligation. Pain 2011;153:68–79. R References 04/04/2014