Our overarching hypothesis is that the signals resulting in nociceptor sensitization are not separated, since the PKA, PKC, and MAPK cascades, once activated, partially or completely converge to induce hyperalgesia
. The JNK pathway is believed to be a critical signaling pathway in the processing of neuropathic pain via distinct mechanisms in the DRGs and spinal cord
[9, 10, 24]. In this study, we used single JNK knockout mice to analyze the contribution of individual JNK isoforms to the development of neuropathic pain after unilateral SNT. We also studied the relationship between JNK activity and the expression of GAP-43 and CGRP, in order to relate changes at the molecular and cellular levels to the development of sensory alterations.
Our findings document, for the first time, that: (1) JNK is involved in pain–related behavior and in the modulation of GAP43 and CGRP expression in lumbar DRG neurons following SNT; (2) administration of a JNK inhibitor partially prevents these effects; (3) the deletion of a single specific JNK isoform partially prevents the maintenance of the sensory alteration, but not the onset of neuropathic pain symptoms such as mechanical hyperalgesia; (4) JNK inhibition in wt mice, as well as deletion of JNK1 or JNK3, abolishes SNT-induced autotomy behavior.
The experimental model
Animal models of neuropathic pain are mostly based on peripheral nerve injury for reproducibility and simplicity
. Axotomy was the first widely used model, since it simulated the clinical condition of amputation
. Complete nerve transection is associated with spontaneous pain-related behaviors, allodynia and hyperalgesia, and with autotomy, or the self-mutilation of the injured foot. These three behavioral signs are used as indices to measure the degree of neuropathic pain, and to evaluate the effects of therapy
. In 2008, the International Association for the Study of Pain (IASP) modified many of the definitions in the pain field including that: i) the term allodynia should be used only when the test stimulus, e.g. thermal or mechanical, does not normally activate nociceptors; ii) when this is not clearly the case, “hyperalgesia” is the preferred term
. For this reason, in this study we analyzed the onset and maintenance of neuropathic pain in terms of mechanical hyperalgesia, since we tested the animal’s behavior by means of anautomated Von Frey test, which uses mechanical stimulation.
Modulation by JNK of the molecular mechanisms involved in neuropathic pain
In this study we have focused our attention on the primary sensory neurons of the lumbar tract, which are the first neurons involved in pain transmission from the hind limb periphery. Following peripheral axotomy, reorganization of central DRG projections to the spinal cord are thought to mediate neuropathic pain
. Earlier studies proposed different explanations of how neuronal perikarya can receive information about a distant axon injury, including via retrograde transport of injury-induced proteins and/or via decreased target-derived trophic support
It has been shown, both in vitro and in vivo, that axotomy of sensory neurons causes a rapid, massive and transient increase in JNK expression; this is followed by the activation of c-Jun, which supports axonal outgrowth and neuron survival
[34, 35]. In fact, JNK blockade by D-JNKI-1 and SP600125 reduces c-Jun phosphorylation in DRGs, and hampers axonal outgrowth, both in terms of the length and number of regenerating axons
Many JNK scaffold proteins interact with kynesin 1, which is involved in vesicular trafficking within axons
. JNK is activated locally in the axon as part of a damage signal, which is retrogradely transported in a molecular assembly called a signalosome
. In cultured cortical neurons, JNK-interacting protein (JIP)1 has been shown to be involved in axonal elongation, since it is localized at the tip of the growing axon, and co-labels with the axonal marker Tau-1
. Chang et al.
 demonstrated that JNK1 KO mice exhibit progressive degeneration of neurites, associated with shorter microtubules,reduced Microtubules Associated Protein (MAP)1B, and MAP2 phosphorylation. MAP1-B is implicated in axonal regeneration, as seen in DRG cultures
 and its phosphorylation is increased in the rearrangement of axonal circuitry following spinal cord injury, together with JNK activation
. All three JNK isoforms seem to contribute to neurite re-growth in PC12 cell culture
. The absence of the JNK target c-Jun impairs the expression of cluster of differentiation (CD)44, galanin or alpha7beta1 integrins, molecules known to be involved in regeneration
To assess the correlation between JNK activity and axonal regeneration, we analysed GAP43-IR in DRGs after SNT. JNK expression and its activation persist for at least 30 days after axotomy if regeneration is blocked
; JNK activation in DRG neurons would thus initially promote survival after injury, and later would operate as part of a growth program
. In agreement with this, we document here that GAP43 immunoreactivity increases in axotomized primary sensory DRG neurons, and that this increase is partially prevented by D-JNKI-1 administration, or by deleting the genes for JNK1 and JNK3.
In primary somato-sensory neurons, CGRP is localized in peripheral axons, in their somata in the DRG, and in unmyelinated and thin myelinated central afferent fibers, where it has been implicated in the transmission/modulation of pain
. The role of GCRP, as well as that of substance P in neuropathic pain has been poorly investigated, partly because they are depleted after peripheral nerve lesion
. Impaired information-processing in the spinal cord may play an important role in the development of sensory abnormalities that result from peripheral nerve injury. It is likely that the lesion-induced downregulation of substance P and CGRP attenuates the transmission of peptide-related information at the first synapse in the dorsal horn; such changes may represent adaptive responses that limit the consequences of peripheral nerve damage to the organism as a whole, and that promote survival and recovery of individual neurons
. In animal models that investigate pathways that regulate inflammatory responses, a relationship between the JNK pathway and CGRP has been documented
[22, 23]. In agreement with previous studies
, we report here that in axotomized primary sensory DRG neurons, CGRP activity is down-regulated. Moreover, our results following D-JNKI-1 treatment further underscore a relationship between JNK pathway activation and CGRP expression. We propose that, following nerve injury, activation of JNK pathway may influence CGRP regulation, mediated via JNK1 and JNK2 isoforms.
Studies on pain have primarily focused on neurons; however Zhuang et al.
 demonstrated that spinal nerve ligation (SNL) also activates JNK1 in spinal astrocytes, and that such activation is essential for the maintenance of neuropathic pain. Glial changes, associated with an increased sprouting of primary nociceptive afferents (C and A-δ fibers) that enter the spinal cord, suggest a strict correlation between neuro-glial plasticity changes and peripheral sensitization
[45, 46]; these findings highlight the role of glia in pain transmission and suggests that JNK could also participate in this mechanism. In support, intratechal administration of TNF-α- activated astrocytes induces mechanical allodynia, which is preserved by D-JNKI-1 treatment
. Related to this, the relationship between NGF expression and JNK activation should be further investigated: on one hand, several reports claim that NGF, whose levels are increased in inflamed tissues, induces the activation of JNK in DRGs
. On the other hand, NGF administration prevents the facilitation of pain transmission at the level of the superficial laminae of the spinal cord
Role of JNK in pain behavior
Even though studies of knockout mice have some limitations, such as the occurrence of compensatory changes for the knocked out gene or the dependence of the phenotype on the background of mice strain
, our findings exclude the possibility that the deletion of a specific JNK isoform results in abnormalities of locomotion or motor coordination, or in the alteration of the mechanical pain threshold. On the contrary, following SNT, KO mice displayed a decreased mechanical pain threshold compared to wild type animals: inhibition of JNK by D-JNKI-1 also had similar effects. However, JNK inhibition by D-JNKI-1 does not necessarily act as analgesic: since a JNK inhibitor can dramatically affect the production of cytokines
, we suppose that it can function as an anti-hyperalgesic and/or as a neuromodulator, by restoring the baseline or normal pain threshold of the injured mice to which it is administered. Thus D-JNKI-1, instead of diminishing the stimulus-dependent response, would prevent that response, either by suppressing the pain-associated suffering or by directly reducing the responsiveness of nociceptors. D-JNKI-1 could thus be used to prevent and treat the onset of SNT-induced neuropathic pain.
D-JNKI-1 treated mice exhibit hypoalgesic responses to neuropathic pain within 30 days post- surgery, suggesting that JNK isoforms play a critical role in pain perception. These findings are consistent with recent results obtained using intrathecal or systemic delivery of D-JNKI-1 in another animal model for the study of neuropathic pain
[9, 27], in which the authors showed that JNK blockade prevents the development of neuropathic pain. Even though our work was aimed at investigating the role of JNK in modulating injury-induced cellular changes of DRGs in relation to concomitant pain behavior, we are aware of the involvement of supraspinal centers in the control and in the genesis of neuropathic pain. In fact, several reports refer changes in the expression of MAPKs, and namely of JNK, in somatosensory
 and cingulate
 cortex and in the hippocampus
[53, 54]. On the contrary, no changes were reported in the thalamus in a model of chronic pain in rats
. Although the parallel changes of DRG markers and pain behavior that we observed suggest that these phenomena may be causally related, in our experimental design, both in transgenic and in D-JNKI-1-treated mice single or all JNK isoforms were blocked in the entire brain. Therefore, further studies will be necessary to dissect the specific contribution of supraspinal centers in neuropathic pain and the relevant role played by JNK. Similarly, specific analysis and targeted manipulaitions of JNK expression or activity would be required to elucidate functional changes of intra- and supra spinal circuits involved in the pathogenesis of neuropathic pain.
Autotomy, or self mutilation can be interpreted either as a response to a persistent irritative stimulus, the failure to recognize the limb as self, or both. Our strain of mice, C57BL/6, displays a high autotomy score following sciatic nerve tight ligature
. Whereas JNK2 KO and wt mice displayed this behavior following SNT, mice with the JNK1 or JNK3 genes deleted, also mice treated with D-JNKI-1, displayed no autotomy lesions. This finding further supports a role for JNK in neuropathic pain. However, the increase in autotomy lesions which we observed in operated JNK2 KO mice apparently contradicts this view. Indeed, JNK2 KO mice show both GAP43 upregulation and the autotomy phenotype, whereas the other two strains failed to activate GAP43 and did not develop autotomy. Thus, GAP43 upregulation correlates with autotomy, which has been related to spontaneous pain
. This observation was somewhat surprising, since all JNK isoforms appear to be involved in the maintenance of neuropathic pain, as assessed by plantar aesthesiometer stimulation. However, it is likely that the appearance of autotomy and mechanical hyperalgesia following noxious plantar stimulation are elicited by different mechanisms that involve distinct JNK isoforms. In fact, autotomy score and Von Frey filament test (which is comparable to the automated Von Frey test we used in our study) reflect two different aspects of nociception: the first one measures a spontaneous behavior, whereas the second measures a cutaneous hypersensitivity to applied stimuli
This is also consistent with our data on the prolonged effect of multiple D-JNKI-1 treatment on autotomy behavior, which does not occur with single injection, whereas both treatment protocols have the same results on mechanical hyperalgesia. Autotomy behavior might be elicited at higher levels as well: for example, it has been shown that autotomy behavior correlates with the expression of different dopamine receptors in the cingulate cortex