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Some individuals recover from the pain of nerve trauma within 12 months or less whereas others experience life-long intractable pain. This transition between reversible pain and the establishment of chronic neuropathic pain is poorly understood. We examined the role of persistent inflammation in the dorsal root ganglia (DRG) in the long-term maintenance of mechanical allodynia; an index of neuropathic pain. Male Sprague-Dawley rats underwent chronic constriction injury (CCI), spared nerve injury (SNI) or sham surgery. Both CCI and SNI animals displayed robust mechanical allodynia in the ipsilateral paw at 7 d post-surgery; however, only SNI animals maintained mechanical allodynia at 42 d post-surgery. DRGs were extracted at 7 d or 42 d post-surgery to assess inflammation via rt-qPCR or immunohistochemistry to measure colony stimulating factor 1 (CSF1) expression, satellite glial cell (SGC) activation, presence of Iba1 positive macrophages and interleukin1 β (IL-1β) mRNA levels. Whereas DRGs from SNI animals continued to display inflammatory markers at 42 d, those from CCI animals did not. Moreover, the level of allodynia displayed by each individual animal correlated with the extent of DRG inflammation. These data support the hypothesis that the amount of CSF1 immunoreactivity and the persistence of inflammation in ipsilateral DRGs contribute to the difference between transient and persistent mechanical allodynia observed in the CCI and SNI models. We also suggest that feedback loops involving cytokines and neurotransmitters may contribute to increased DRG activity in chronic neuropathic pain. Consequently, targeting persistent CSF1 production and peripheral neuroinflammation may be an effective approach to the management of chronic neuropathic pain., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Brachial plexus avulsion (BPA) represents the most devastating nerve injury in the upper extremity and is always considered as a sophisticated problem due to its resistance to most standard pain relief medications or neurosurgical interventions. There is also a lack of understanding on the underlying mechanisms. Our study aimed to investigate whether spinal CCL2-CCR2 signaling contributed to the development of neuropathic pain following BPA via modulating glutamate N-methyl-d-aspartate receptor (NMDAR). A rat model of BPA on lower trunk (C8-T1) was established, and the sham- and BPA-operated animals were intrathecally injected with saline, C-C chemokine receptor type 2 (CCR2) inhibitor INCB3344 and NMDAR antagonist DL-AP5 one week postoperatively, the behavioral performance of the treated animals and expressions of C-C motif ligand 2 (CCL2), CCR2, and N-methyl-D-aspartic acid receptor 2B (NR2B) in spinal cord sections of each group were examined. It was shown that BPA injury significantly reduced mechanic withdrawal thresholds the next day after surgery until the end of the observation. Both CCL2 and CCR2 expressions increased in BPA rats compared to those in sham rats. CCL2 was mainly localized in astrocytes, and CCR2 was preferably expressed on astrocytes and neurons. Besides, NMDAR subunit NR2B increased in BPA-operated rats, which was reversed in response to CCR2 and NR2B inhibition. However, these inhibitors didn't change the spinal NMDAR level in sham rats. CCR2 and NMDAR inhibition efficiently alleviated mechanical allodynia caused by BPA either at early or late phase of neuropathic pain. Collectively, CCL2-CCR2 axis is associated with mechanical pain after BPA by elevating NMDAR signaling., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Synaptosomal-associated protein 25 (SNAP-25) plays an important role in neuropathic pain. However, the underlying mechanism is largely unknown. Vesicular glutamate transporter 2 (VGluT2) is an isoform of vesicular glutamate transporters that controls the storage and release of glutamate. In the present study, we found the expression levels of VGluT2 correlated with the upregulation of SNAP-25 in the spinal cord of rats following chronic constriction injury (CCI)-induced neuropathic pain. Cleavage of SNAP-25 by Botulinum toxin A (BoNT/A) attenuated mechanical allodynia, downregulated the expression of VGluT2 and reduced glutamate release. Overexpression of VGluT2 abolished the antinociceptive effect of BoNT/A. Upregulation of SNAP-25 in naive rats increased VGluT2 expression and induced pain-responsive behaviors. In pheochromocytoma (PC12) cells, the expression of VGluT2 was also depended on SNAP-25 dysregulation. Moreover, we found VGluT2 was involved in SNAP-25-mediated regulation of astrocyte expression and activation of the PKA/p-CREB pathway mediated the upregulation of SNAP-25 in neuropathic pain. The findings of our study indicate that VGluT2 contributes to the effect of SNAP-25 in maintaining the development of neuropathic pain and suggests a novel mechanism underlying SNAP-25 regulation of neuropathic pain., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuropathic pain is a complication after a spinal nerve injury. The inflammasomes are now identified to be responsible for triggering inflammation in neuropathic pain. Autophagy participates in the process of neuropathic pain and can regulate the inflammasome activation in different diseases. Our previous research reported that hydrogen exerted a protective effect against neuropathic pain. Therefore, we focused on the mechanism and role of autophagy and inflammasome, by which hydrogen alleviated the hyperpathia induced by neuropathic pain. The results showed that neuropathic pain stimulated activation of inflammasome NLRP3 and autophagy pathway in the microglial cells of the spinal cord. The inhibition of NLRP3 inhibited the hyperpathia induced by spinal nerve litigation surgery. The absence of autophagy aggravated the inflammasome activity and hyperpathia. Hydrogen promoted autophagy related protein expression, inhibited the inflammasome NLRP3 pathway activation, and relieved the hyperpathia induced by neuropathic pain. Hydrogen treatment could alleviate hyperpathia by autophagy-mediated NLRP3 inactivation., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Nerve damage leads to the development of disabling neuropathic pain in susceptible individuals, where patients present with pain as well as co-morbid affective behavioural disturbances, such as anhedonia, decreased motivation and depression. In this study we aimed to characterise changes in neuroinflammation in the medial prefrontal cortex (mPFC) and hippocampus (HP) in a rat model of neuropathic pain (NP) and behavioural changes. 53 rats underwent sciatic nerve chronic constriction injury (CCI) and were characterised as either, No effect, Acute effect or Lasting effect on the basis of changes in exploration behaviour in a radial-arm maze. Microglial and astrocyte morphology, as well as IL-1β, IL-6, IL-10, MCP-1, p38 MAPK and BDNF expression was quantified throughout the mPFC and HP using protein multiplex assays and immunofluorescence. All behavioural groups of CCI rats displayed equal levels of mechanical allodynia; however, the characteristic withdrawal from pellet-seeking observed in Lasting effect rats was accompanied by neuroimmune activation within the contralateral ventral HP and mPFC. This includes increased expression of IL-1β, IL-6 and MCP-1, increased phospho-p38 MAPK expression in neurons and microglia, and a shift to a reactive microglial morphology in the caudal PL and IL, ventral CA1 and DG. Therefore, neuroinflammation in the mPFC and ventral HP may influence individual differences in radial-arm maze behaviour following CCI. Our data provide further evidence that individual differences in neuroimmune activation in the interconnected ventral HP-mPFC circuitry may play a role in the divergent behavioural trajectories following nerve injury, with neuroinflammation being coincident with affective behavioural changes in susceptible individuals., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

p53 and parkin are involved in mitochondrial quality control. The present study aimed to characterize the functional significance of parkin/p53 in the development of mitochondrial dysfunction and the pathophysiology of neuropathic pain in type I diabetes. Type I diabetes was induced in mice (N = 170) using streptozotocin (STZ). Pifithrin-α, a selective p53 inhibitor, was administered to assess its effects on diabetic pain hypersensitivity, parkin expression and mitochondrial function. Expressions of parkin and p53, mitochondrial number and level of reactive oxygen species (ROS) in the dorsal root ganglion (DRG) were analyzed by immunohistochemistry, western blotting and real time PCR. Separately, mice were treated using intravenous methylglyoxal, then pain hypersensitivity and p53/parkin expression in the DRG were assessed. Mitochondrial membrane potential was also analyzed in cultured DRG neurons treated with methylglyoxal. Mice developed pain hypersensitivity for 3 weeks after STZ treatment. p53 expression was significantly increased (control, 0.68 ± 0.122; STZ, 1.88 ± 0.21) whereas parkin expression was significantly reduced (control, 1.02 ± 0.17; STZ, 0.59 ± 0.14), in the DRG after STZ treatment. Inhibition of p53 by pifithrin-α prevented STZ-induced pain hypersensitivity and parkin downregulation. Pifithrin-α also inhibited STZ-induced reductions in mitochondrial number and accumulation of mitochondrial ROS. Methylglyoxal elicited pain hypersensitivity and alteration of p53/parkin expression, similar to STZ. Methylglyoxal also decreased mitochondrial membrane potential in cultured DRG neurons. Alteration of p53/parkin expression produces mitochondrial dysfunction and ROS accumulation, leading to pain hypersensitivity in diabetic or methylglyoxal treated mice. Methylglyoxal produces neurological derangements similar to diabetes, via direct mechanisms on DRG neurons., (Copyright © 2019. Published by Elsevier Ltd.)

Trigeminal neuropathic pain (TGN) is an attacking, abrupt, electric-shock headache involving abnormal cortical activity. The neural mechanism underlying TGN remains elusive. In this study, we explored the role of microglia in the primary somatosensory barrel cortex (S1BF), which is a critical region for TGN, of a mouse model of TGN that displayed significant pain-related behaviors. Using electrophysiological recordings, we found robust neuronal hyperactivity in glutamatergic neurons of S1BF (Glu S1BF ). Chemogenetic inhibition of Glu S1BF neurons significantly relieved mechanical allodynia in TGN mice. In naïve mice, chemogenetic activation of Glu S1BF neurons induced pain sensitization. In addition, we found that microglia in the S1BF (microglia S1BF ) were significantly activated, with density and morphology changes. Intraperitoneal administration of minocycline, a microglia inhibitor, attenuated pain sensitization, and decreased Glu S1BF neuronal activity. Together, these findings demonstrate the putative importance of microglia as a key regulator in TGN through actions on Glu S1BF neuronal adaptation., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cortical reorganization has been proposed as a major factor involved in phantom pain with prior nociceptive input to the deafferented region and input from the non-deafferented cortex creating neuronal activity that is perceived as phantom pain. There is substantial evidence that these processes play a role in neuropathic pain, although causal evidence is lacking. Recently it has been suggested that a maintenance of the cortical representation of the former hand area is related to phantom pain. Although interesting, evidence for this process is so far scarce. In addition, peripheral factors have been proposed as important for phantom limb pain. Although often introduced as contradictory, we suggest that cortical reorganization, preserved limb function and peripheral factors interact to create the various painful and nonpainful aspects of the phantom limb experience. In addition, the type of task (sensory versus motor), the interaction of injury- and use-dependent plasticity, the type of data analysis, contextual factors such as the body representation and psychological variables determine the outcome and need to be considered in models of phantom limb pain. Longitudinal studies are needed to determine the formation of the phantom pain experience., (Copyright © 2017 IBRO. All rights reserved.)

Neuropathic pain affects multiple brain functions, including motivational processing. However, little is known about the structural and functional brain changes involved in the transition from an acute to a chronic pain state. Here we combined behavioral phenotyping of pain thresholds with multimodal neuroimaging to longitudinally monitor changes in brain metabolism, structure and connectivity using the spared nerve injury (SNI) mouse model of chronic neuropathic pain. We investigated stimulus-evoked pain responses prior to SNI surgery, and one and twelve weeks following surgery. A progressive development and potentiation of stimulus-evoked pain responses (cold and mechanical allodynia) were detected during the course of pain chronification. Voxel-based morphometry demonstrated striking decreases in volume following pain induction in all brain sites assessed - an effect that reversed over time. Similarly, all global and local network changes that occurred following pain induction disappeared over time, with two notable exceptions: the nucleus accumbens, which played a more dominant role in the global network in a chronic pain state and the prefrontal cortex and hippocampus, which showed lower connectivity. These changes in connectivity were accompanied by enhanced glutamate levels in the hippocampus, but not in the prefrontal cortex. We suggest that hippocampal hyperexcitability may contribute to alterations in synaptic plasticity within the nucleus accumbens, and to pain chronification., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Accumulated evidences suggest important roles of glial GAP-junctions in pain. However, only a few studies have explored the role of neuronal GAP-junctions or electrical synapses in neuropathic pain (NP). Therefore, the present study explores the role of connexin 36 (Cx36) in NP using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model in rat. A significant increase in Cx36 labeling was observed in the medullary dorsal horn (MDH) of CCI-IoN-lesioned compared to sham rats. The expression of Cx36 in CCI-IoN-lesioned rats revealed a rostroventral gradient of punctuate labeling within lamina IIo of the MDH. Cx36-positive somata and processes were also observed in MDH laminae IIi and III-V. These somata were mostly of the Gamma aminobutyric acid (GABA) and occasionally Glycine transporter 2 (GlyT2) cell subtypes. Moreover the GABA cell subtypes are highly coupled in lamina IIo as revealed by the intense Cx36 staining in this lamina. Pharmacological Cx36 blockade by intracisternal administration of mefloquine decreased significantly the mechanical allodynia observed in CCI-IoN-lesioned rats. Altogether, our findings demonstrated that Cx36 play an important role in mechanical allodynia by coupling GABA cells. Increasing cell coupling by enhancing Cx36 expression favors neuropathic pain while disrupting this coupling alleviates it. This mechanism may constitute a novel target for the treatment of orofacial mechanical allodynia., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Alteration in gene expression along the pain signaling pathway is a key mechanism contributing to the genesis of neuropathic pain. Accumulating studies have shown that epigenetic regulation plays a crucial role in nociceptive process in the spinal dorsal horn. In this present study, we investigated the role of enhancer of zeste homolog-2 (EZH2), a subunit of the polycomb repressive complex 2, in the spinal dorsal horn in the genesis of neuropathic pain in rats induced by partial sciatic nerve ligation. EZH2 is a histone methyltransferase, which catalyzes the methylation of histone H3 on K27 (H3K27), resulting in gene silencing. We found that levels of EZH2 and tri-methylated H3K27 (H3K27TM) in the spinal dorsal horn were increased in rats with neuropathic pain on day 3 and day 10 post nerve injuries. EZH2 was predominantly expressed in neurons in the spinal dorsal horn under normal conditions. The number of neurons with EZH2 expression was increased after nerve injury. More strikingly, nerve injury drastically increased the number of microglia with EZH2 expression by more than sevenfold. Intrathecal injection of the EZH2 inhibitor attenuated the development and maintenance of mechanical and thermal hyperalgesia in rats with nerve injury. Such analgesic effects were concurrently associated with the reduced levels of EZH2, H3K27TM, Iba1, GFAP, TNF-α, IL-1β, and MCP-1 in the spinal dorsal horn in rats with nerve injury. Our results highly suggest that targeting the EZH2 signaling pathway could be an effective approach for the management of neuropathic pain., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuropathic pain is absent in infants and emergent years after injury. Adult spinal cord microglia play a key role in initiating neuropathic pain, and modulation of microglia is a potential target for treating neuropathic pain. In this study, we evaluated the role of microglia after infant peripheral nerve injury and the effect of exercise on the delayed-onset neuropathic pain. Rat pups received spared nerve injury, and behavior tests were performed to evaluate their pain threshold. qPCR, immunohistochemistry, and Western blot were used for M1 and M2 marker expression analysis. In contrast to the microglial polarization to the M1 phenotype observed in the adult spinal cord, in infant nerve injury, microglial polarization immediately shifted to the M2 phenotype. In adolescence, microglia polarized to the M1 phenotype, which was concomitant with the emergence of neuropathic pain. Exercise shifted spinal cord microglia polarization to the M2 phenotype and reduced neuropathic pain. In addition, IL-10 increased and TNF-α decreased after exercise, and intrathecal injection of the IL-10 antibody reduced the exercise-induced analgesia. Our study found that infant nerve injury induced delayed spinal cord microglia polarization to the M1 phenotype and that exercise was effective in the treatment of delayed adolescent neuropathic pain via the modulation of microglial polarization., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuropathic pain encompasses a broad range of conditions associated with a lesion or disease of the peripheral or central somatosensory system and its prevalence in the general population may be as high as 7-8%. The interest in the pathophysiology of neuropathic pain has increased over the last two decades with an exponential increase in the number of experimental studies. However, despite the hopes raised by scientific discoveries, there has been no rational development of a truly new class of drugs. This situation revealing the limitations of certain experimental models, also results of limitations in clinical research. One of the reasons for the therapeutic difficulties in these patients is probably due to the fact that treatments are used in a uniform fashion whatever the clinical picture, while these syndromes are in fact highly heterogeneous. Clinical advances have recently been made in this field, following the validation of new specific clinical tools and the standardization of quantitative sensory testing paradigms facilitating improvements in the clinical characterization of these syndromes. It has been clearly demonstrated that neuropathic pain is a consistent clinical entity, but it is multidimensional in terms of its clinical expression, with different sensory profiles, potentially reflecting specific pathophysiological mechanisms. This new conceptualization of neuropathic pain should improve the characterization of the responder profiles in clinical trials and provide valuable information for the development of new and more clinically sound translational approaches in experimental models in animals., (Copyright © 2016. Published by Elsevier Ltd.)

Neuropathic pain is treated using serotonin norepinephrine reuptake inhibitors with mixed results. Pain facilitation mediated by α1-adrenoceptors may be involved, but whether norepinephrine (NE) is tonically released is unclear. The aim of this study was to determine whether NE is tonically released from A7 cells following chronic constriction injury (CCI), and if the lateral hypothalamus (LH) plays a role in this release in male and female rats with nociceptive and neuropathic pain types. Neuropathic groups received left CCI while nociceptive groups remained naïve to injury. Fourteen days later, rats were given intrathecal infusion of either the α1-adrenoceptor antagonist WB4101, the α2-adrenoceptor antagonist yohimbine (74 μg), or normal saline for control. Paw withdrawal latency (PWL) from a thermal stimulus was measured. The generalized estimated equation method was used for statistical analysis. Nociceptive rats given WB4101 had a PWL significantly longer than saline control (7.89 ± 0.63 vs. 5.87 ± 0.52 s), while the PWL of neuropathic rats given WB4101 was 13.20 ± 0.52 s compared to 6.78 ± 0.52 s for the saline control rats. Yohimbine had no significant effect. Microinjection of cobalt chloride (CoCl) in the A7 catecholamine cell group to prevent synaptic transmission blocked the effect of WB4101 in all groups, supporting the notion that spinally descending A7 cells tonically release NE that contributes to α1-mediated nociceptive facilitation. Microinjection of CoCl into the left LH blocked the effect of WB4101 in nociceptive and neuropathic male rats, but had no effect in female rats of either pain type, suggesting differential innervation. These findings indicate that tonic release of NE acts at pronociceptive α1-adrenoceptors, that this effect is greater in rats with nerve damage, and that, while NE comes primarily from the A7 cell group, LH innervation of the A7 cell group is different between the sexes., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Maladaptive plasticity within the dorsal horn (DH) of the spinal cord is a key substrate for development of neuropathic pain following peripheral nerve injury. Advances in genetic engineering, tracing techniques and opto-genetics are leading to a much better understanding of the complex circuitry of the spinal DH and the radical changes evoked in such circuitry by nerve injury. These changes can be viewed at multiple levels including: synaptic remodeling including enhanced excitatory and reduced inhibitory drive, morphological and electrophysiological changes which are observed both to primary afferent inputs as well as DH neurons, and ultimately circuit-level rewiring which leads to altered connectivity and aberrant processing of sensory inputs in the DH. The DH should not be seen in isolation but is subject to important descending modulation from the brainstem, which is further dysregulated by nerve injury. Understanding which changes relate to specific disease-states is essential, and recent work has aimed to stratify patient populations in a mechanistic fashion. In this review we will discuss how such pathophysiological mechanisms may lead to the distressing sensory phenomena experienced by patients suffering neuropathic pain, and the relationship of such mechanisms to current and potential future treatment modalities., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Inflammatory response induced by protrused nucleus pulposus (NP) has been shown to play a crucial role in the process of radicular pain. Lipoxins represent a unique class of lipid mediators that have anti-inflammatory and pro-resolving action. The present study was undertaken to investigate if intrathecal lipoxin A4 (LXA4) could alleviate mechanical allodynia in the rat models of application of NP to the L5 dorsal root ganglion (DRG). Non-compressive models of application of NP to L5 DRG were established and intrathecal catheterization for drug administration was performed in rats. Daily intrathecal injection of vehicle or LXA4 (10ng or 100ng) was performed for three successive days post-operation. Mechanical thresholds were tested and the ipsilateral lumbar (L4-L6) segment of spinal dorsal horns were removed for the determination of tumor necrosis factor-α (TNF-α), IL-1β, transforming growth factor-β1 (TGF-β1) and IL-10 expression and NF-κB/p65, extracellular signal-regulated kinase (ERK), C-Jun N-terminal kinase (JNK) and P38 expression. Application of NP to DRG in rats induced mechanical allodynia, increased the expression of pro-inflammatory factors (TNF-α and IL-1β), NF-κB/p65, the phosphorylated-ERK (p-ERK), -JNK (p-JNK) and -P38 (p-p38) and decreased the expression of anti-inflammatory cytokines (TGF-β1 and IL-10) in the ipsilateral lumbar (L4-L6) segment of spinal dorsal horns. Intrathecal injection of LXA4 alleviated the development of neuropathic pain, inhibited the upregulation of pro-inflammatory cytokines (TNF-α and IL-1β), upregulated the expression of anti-inflammatory cytokines (TGF-β1 and IL-10) and attenuated the activation of NF-κB/p65, p-ERK, p-JNK, but not p-p38, in a dose-dependent manner. In this study, we have demonstrated that LXA4 potently alleviate radicular pain in a rat model of non-compressive lumbar disc herniation. The anti-inflammatory and pro-resolution properties of LXA4 have shown a great promise for the management of radicular pain caused by intervertebral disc herniation., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

In the spinal nerve ligation (SNL) model of neuropathic pain, as in other pain models, abnormal spontaneous activity of myelinated sensory neurons occurs early and is essential for establishing pain behaviors and other pathologies. Sympathetic sprouting into the dorsal root ganglion (DRG) is observed after SNL, and sympathectomy reduces pain behavior. Sprouting and spontaneous activity may be mutually reinforcing: blocking neuronal activity reduces sympathetic sprouting, and sympathetic spouts functionally increase spontaneous activity in vitro. However, most studies in this field have used nonspecific methods to block spontaneous activity, methods that also block evoked and normal activity. In this study, we injected small inhibitory (si) RNA directed against the NaV1.6 sodium channel isoform into the DRG before SNL. This isoform can mediate high-frequency repetitive firing, like that seen in spontaneously active neurons. Local knockdown of NaV1.6 markedly reduced mechanical pain behaviors induced by SNL, reduced sympathetic sprouting into the ligated sensory ganglion, and blocked abnormal spontaneous activity and other measures of hyperexcitability in myelinated neurons in the ligated sensory ganglion. Immunohistochemical experiments showed that sympathetic sprouting preferentially targeted NaV1.6-positive neurons. Under these experimental conditions, NaV1.6 knockdown did not prevent or strongly alter single evoked action potentials, unlike previous less specific methods used to block spontaneous activity. NaV1.6 knockdown also reduced pain behaviors in another pain model, chronic constriction of the sciatic nerve, provided the model was modified so that the lesion site was relatively close to the siRNA-injected lumbar DRGs. The results highlight the relative importance of abnormal spontaneous activity in establishing both pain behaviors and sympathetic sprouting, and suggest that the NaV1.6 isoform may have value as a therapeutic target., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

EphB receptors and their ephrinB ligands are implicated in modulating of spinal nociceptive information processing. Here, we investigated whether protein kinase A (PKA), acts as a downstream effector, participates in the modulation spinal nociceptive information related to ephrinB-EphB signaling. Intrathecal injection of ephrinB2-Fc caused thermal hyperalgesia and mechanical allodynia, which were accompanied by increased expression of spinal PKA catalytic subunit (PKAca) and phosphorylated cAMP-response element-binding protein (p-CREB). Pre-treatment with H89, a PKA inhibitor, prevented the activation of CREB by ephrinB2-Fc. Inhibition of spinal PKA signaling prevented and reversed pain behaviors induced by the intrathecal injection of ephrinB2-Fc. Furthermore, blockade of the EphB receptors by intrathecal injection of EphB2-Fc reduced formalin-induced inflammatory, chronic constrictive injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, which were accompanied by decreased expression of spinal PKAca and p-CREB. Overall, these results confirmed the important involvement of PKA in the modulation of spinal nociceptive information related to ephrinBs-EphBs signaling. This finding may have important implications for exploring the roles and mechanisms of ephrinB-EphB signaling in physiologic and pathologic pain., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Aspirin-triggered Lipoxin A4 (ATL), as a Lipoxin A4 (LXA4) epimer, is endogenously produced by aspirin-acetylated cycloxygenase-2 (COX-2) and plays a vital role in endogenous anti-inflammation via the LXA4 receptor (ALX). Recent investigations have indicated that spinal neuroinflammation and the activation of the Janus Kinase 2 (JAK2)/Signal Transducers and Transcription Activators 3 (STAT3) signaling pathway are involved in neuropathic pain states. However, the effect of ATL on neuroinflammation and JAK2/STAT3 signaling in chronic constriction injury (CCI)-induced neuropathic pain in rats has not been well-studied. The present study demonstrated the anti-inflammatory and analgesic effect of ATL on neuropathic pain and assessed the role of spinal JAK2/STAT3 signaling on the effect of ATL. Intrathecal administration of ATL significantly attenuated mechanical allodynia via spinal ALX and inhibited the upregulation of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) on day 7 of CCI surgery. In addition, ATL markedly suppressed the upregulation of p-STAT3 induced by the neuropathic pain. Blockade of JAK2-STAT3 signaling with intrathecal administration of the JAK2 inhibitor AG490 or the STAT3 inhibitor S3I-201 clearly reduced mechanical allodynia and the upregulation of pro-inflammatory cytokines in CCI rats. Interestingly, inhibition of JAK2/STAT3 signaling via ATL or the specific signaling inhibitor (AG49, S3I-201) further promoted the increased expression of suppressor of cytokine signaling 3 (SOCS3) mRNA in the spinal cord induced by CCI surgery. Taken together, our results suggested that the analgesic effect of ATL was mediated by inhibiting spinal JAK2/STAT3 signaling and hence the spinal neuroinflammation in CCI rats., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Lateral hypothalamic (LH) stimulation produces antinociception in female rats in acute, nociceptive pain. Whether this effect occurs in neuropathic pain or whether male-female sex differences exist is unknown. We examined the effect of LH stimulation in male and female rats using conditions of nociceptive and neuropathic pain. Neuropathic groups received chronic constriction injury (CCI) to induce thermal hyperalgesia, a sign of neuropathic pain. Nociceptive rats were naive for CCI, but received the same thermal stimulus following LH stimulation. To demonstrate that CCI ligation produced thermal hyperalgesia, males and females received either ligation or sham surgery for control. Both males and females demonstrated significant thermal hyperalgesia following CCI ligation (p<0.05), but male sham surgery rats also showed a significant left-right difference not present in female sham rats. In the second experiment, rats randomly assigned to CCI or nociceptive groups were given one of three doses of the cholinergic agonist carbachol (125, 250, or 500 nmol) or normal saline for control, microinjected into the left LH. Paw withdrawal from a thermal stimulus (paw withdrawal latency; PWL) was measured every 5 min for 45 min. Linear mixed models analysis showed that males and females in both pain conditions demonstrated significant antinociception, with the 500-nmol dose producing the greatest effect across groups compared with controls for the left paw (p<0.05). Female CCI rats showed equivalent responses to the three doses, while male CCI rats showed more variability for dose. However, nociceptive females responded only to the 500-nmol dose, while nociceptive males responded to all doses (p<0.05). For right PWL, only nociceptive males showed a significant carbachol dose response. These findings are suggestive that LH stimulation produces antinociception in male and female rats in both nociceptive and neuropathic pain, but dose response differences exist based on sex and pain condition., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Acute and chronic pain resulting from injury, surgery, or disease afflicts >100 million Americans each year, having a severe impact on mood, mental health, and quality of life. The lack of structural and functional information for most ion channels, many of which play key roles in the detection and transmission of noxious stimuli, means that there remain unidentified therapeutic targets for pain management. This study focuses on the transient receptor potential canonical subfamily 4 (TRPC4) ion channel, which is involved in the tissue-specific and stimulus-dependent regulation of intracellular Ca²⁺ signaling. Rats with a transposon-mediated TRPC4-knockout mutation displayed tolerance to visceral pain induced by colonic mustard oil (MO) exposure, but not somatic or neuropathic pain stimuli. Moreover, wild-type rats treated with a selective TRPC4 antagonist (ML-204) prior to MO exposure mimicked the behavioral responses observed in TRPC4-knockout rats. Significantly, ML-204 inhibited visceral pain-related behavior in a dose-dependent manner without noticeable adverse effects. These data provide evidence that TRPC4 is required for detection and/or transmission of colonic MO visceral pain sensation. In the future, inhibitors of TRPC4 signaling may provide a highly promising path for the development of first-in-class therapeutics for this visceral pain, which may have fewer side effects and less addictive potential than opioid derivatives., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The neurobiological mechanisms underlying the suppression of neuropathic pain by spinal cord stimulation (SCS) are still incompletely known. The present study aims at exploring whether the descending pain control system in the rostroventromedial medulla (RVM) exerts a role in the attenuation of neuropathic pain by SCS. Experiments were performed in the rat spared nerve injury (SNI) pain model. The effects of SCS on neuronal activity of pronociceptive ON-like, antinociceptive OFF-like, and neutral cells, including 5-HT-like cells, in the RVM were analyzed in SCS responding and SCS non-responding SNI animals as well as in naïve controls. Decreased spontaneous activities in OFF-like cells and increased spontaneous activities in ON-like cells were observed in SNI animals, whereas the spontaneous activities of 5-HT-like and neutral cells were unchanged. SCS produced a prominent increase in the discharge of OFF- and 5-HT-like cells in SCS responding, but not in non-responding SNI animals or controls. Discharge rates of ON-like and neutral cell were not affected by SCS. In awake SNI animals, microinjection of a GABAA receptor agonist, muscimol, into the RVM significantly attenuated the antihypersensitivity effect induced by SCS while a non-selective opioid receptor antagonist, naltrexone, was ineffective. It is concluded that SCS may shift the reciprocal inhibitory and facilitatory pain modulation balance controlled by the RVM in favor of inhibition. This increase in the descending antinociceptive effect operates in concert with segmental spinal mechanisms in producing pain relief., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

We demonstrated previously that the lysophosphatidic acid-1 (LPA1) receptor plays a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. The present study revealed that mild cerebral ischemia by left transient middle cerebral artery occlusion (tMCAO) for 15min causes the hypersensitive responses (paw withdrawal) to the nociception by electrical stimuli to the paw by the use of Neurometer Current Perception Threshold/C (CPT/C). The hypersensitivity or neuropathic pain was only observed by the stimulation with 250 and 2000, but not 5Hz, which are the characterized sine-wave frequencies of Aδ-, Aβ- or C-fibers, respectively. The significant neuropathic pain was observed from day 2 through week 2 on the right paw after tMCAO, while there was slight but significant pain sensitivity on the left paw at day 7. The neuropathic pain on the contralateral side at week 2 after tMCAO was completely abolished in LPA1(-/-) mice. These results suggest that LPA1 receptor signaling plays key roles in the development of central neuropathic pain following cerebral ischemia as well as the peripheral neuropathic pain following partial sciatic nerve injury., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Hyperexcitability of dorsal horn neurons has been shown to play a key role in neuropathic pain following chronic experimental spinal cord injury. With a neonatal in vitro spinal cord injury model, we show that a chemically-induced lesion leads to rapid gain-of-function of sublesional dorsal horn networks biased to hyperexcitation. The expression of the GABA synthetic enzyme GAD65 was significantly reduced at the same level of the spinal cord, suggesting a compromised inhibitory system. We propose that our model could be useful to test early approaches to contrast spinal cord injury-induced central sensitization of dorsal horn circuits., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Spinal cord stimulation (SCS) may alleviate certain forms of neuropathic pain; its mechanisms of action are, however, not fully understood. Previous studies have mainly been focused onto segmental spinal mechanisms, though there is evidence indicating a supraspinal involvement. This study aims to evaluate the relative importance of segmental and supraspinal mechanisms related to the activation of the dorsal columns (DCs). Rats were used to induce the spared nerve injury neuropathy and simultaneously subjected to chronic bilateral DC lesions at the C6-C8 level. Two pairs of miniature electrodes were implanted in each animal, with a monopolar system placed in the dorsal epidural space at a low thoracic level (below lesion) and a bipolar system placed onto the dorsal column nuclei (above lesion). Stimulation (50 Hz, 0.2 ms, 2-4V, 5 min) was applied via either type of electrodes, and tests for sensitivity to tactile and thermal stimuli were used to assess its inhibitory effects. Various receptor antagonists {bicuculline (GABA(A)), saclofen (GABA(B)), ketanserine (5HT(2)), methysergide (5HT(1-2)), phentolamine (α-adrenergic), propranolol (β-adrenergic), sulpiride (D(2)/D(3) dopamine) or saline were injected prior to the SCS. Rostral and caudal stimulations produced a comparable inhibition of neuropathic manifestations, and these effects were attenuated by about 50% after DC lesions. Pretreatment with the various receptor antagonists differentially influenced the effects of rostral and caudal stimulation. Our findings suggest that both supraspinal and segmental mechanisms are activated by SCS, and that in this model with DC lesions, rostral and caudal stimulations may activate different synaptic circuitries and transmitter systems., (Copyright © 2012 IBRO. All rights reserved.)

Anti-neoplastic agents in the platinum-complex, taxane, vinca alkaloid, and proteasome-inhibitor classes induce a dose-limiting, chronic, distal, symmetrical, sensory peripheral neuropathy that is often accompanied by neuropathic pain. Clinical descriptions suggest that these conditions are very similar, but clinical data are insufficient to determine the degree of similarity and to determine if they share common pathophysiological mechanisms. Animal models do not have the limitations of clinical studies and so we have characterized a rat model of chronic painful peripheral neuropathy induced by a platinum-complex agent, oxaliplatin, in order to compare it with a previously characterized model of chronic painful peripheral neuropathy induced by a taxane agent, paclitaxel. The oxaliplatin model evokes mechano-allodynia, mechano-hyperalgesia, and cold-allodynia that have a delayed onset, gradually increasing severity, a distinct delay to peak severity, and duration of about 2.5 months. There is no effect on heat sensitivity. Electron microscopy (EM) analyses found no evidence for axonal degeneration in peripheral nerve, and there is no upregulation of activating transcription factor-3 in the lumbar dorsal root ganglia. There is a statistically significant loss of intraepidermal nerve fibers in the plantar hind paw skin. Oxaliplatin treatment causes a significant increase in the incidence of swollen and vacuolated mitochondria in peripheral nerve axons, but not in their Schwann cells. Nerve conduction studies found significant slowing of sensory axons, but no change in motor axons. Single fiber recordings found an abnormal incidence of A- and C-fibers with irregular, low-frequency spontaneous discharge. Prophylactic dosing with two drugs that are known to protect mitochondria, acetyl-l-carnitine and olesoxime, significantly reduced the development of pain hypersensitivity. Our results are very similar to those obtained previously with paclitaxel, and support the hypothesis that these two agents, and perhaps other chemotherapeutics, produce very similar conditions because they have a mitotoxic effect on primary afferent neurons., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

There is increasing evidence that pain transmission on one side of the body is influenced by a painful state on the other side. We have investigated this phenomenon by studying the activation pattern (using C-fos labeling) of spinal glycinergic and GABAergic (Gly/GABA) neurons after capsaicin injection in the ipsilateral hind paw of rats that were preconditioned with an acute or chronic pain stimulus in the contralateral hind paw or rats that were not preconditioned (control). For this purpose, fluorescent in situ hybridization with GlyT2 and GAD67 mRNA probes was combined with fluorescent C-fos immunohistochemistry. Rats were preconditioned with acute (capsaicin, Complete Freund's Adjuvant (CFA) 1.5 h), chronic inflammatory (CFA 20 h and 4 days), neuropathic (spared nerve injury (SNI) 2 weeks), or control pain stimuli (saline 20 h and 4 days; sham-SNI 2 weeks). We found that after capsaicin injection in rats preconditioned with CFA inflammation (4 days), sham-SNI or with SNI neuropathic pain, the numbers (27 ± 3, 21 ± 2, and 21 ± 2, respectively) and percentages (55% ± 4, 43% ± 2, and 42% ± 2, respectively) of C-fos activated neurons that were Gly/GABA increased significantly as compared with control (10 ± 1 and 25% ± 2). The increase in the total number of C-fos activated Gly/GABA neurons was present primarily in the superficial dorsal horn (laminae I and II; control: 9%; CFA 4 days: 56%; SNI 2 weeks: 42%). This increase in C-fos activation of Gly/GABA neurons occurred without significant changes in the total number of C-fos activated neurons, and without any significant changes in the mechanical thresholds in the hind paws after capsaicin injection. The results showed that one-sided chronic pain, especially inflammation, significantly increases the C-fos activation pattern of spinal Gly/GABA neurons on the other side of the spinal cord. This further underlines the existence of a dynamic interaction between ipsi- and contralateral spinal neurons in the processing of nociceptive information., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Paclitaxel chemotherapy is limited by a long-lasting painful neuropathy that lacks an effective therapy. In this study, we tested the hypothesis that paclitaxel may release mast cell tryptase, which activates protease-activated receptor 2 (PAR2) and, subsequently, protein kinases A and C, resulting in mechanical and thermal (both heat and cold) hypersensitivity. Correlating with the development of neuropathy after repeated administration of paclitaxel, mast cell tryptase activity was found to be increased in the spinal cord, dorsal root ganglia, and peripheral tissues in mice. FSLLRY-amide, a selective PAR2 antagonist, blocked paclitaxel-induced neuropathic pain behaviors in a dose- and time-dependent manner. In addition, blocking downstream signaling pathways of PAR2, including phospholipase C (PLC), protein kinase A (PKA), and protein kinase Cε (PKC), effectively attenuated paclitaxel-induced mechanical, heat, or cold hypersensitivity. Furthermore, sensitized pain response was selectively inhibited by antagonists of transient receptor potential (TRP) V1, TRPV4, or TRPA1. These results revealed specific cellular signaling pathways leading to paclitaxel-induced neuropathy, including the activation of PAR2 and downstream enzymes PLC, PKCε, and PKA and resultant sensitization of TRPV1, TRPV4, and TRPA1. Targeting one or more of these signaling molecules may present new opportunities for the treatment of paclitaxel-induced neuropathy., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Peripheral nerve injury often results in neuropathic pain that is manifested as hyperalgesia, and allodynia. Several studies suggest a functional role for neuronal nitric oxide synthase (nNOS) in the development or maintenance of neuropathic pain, but such a contribution remains unclear. In our current study, we found that intraplantar injection of the NOS substrate L-arginine or NO donor 3-morpholino-synonimine (SIN-1) produced mechanical hypersensitivity that lasted more than 24 h. Following L5 spinal nerve ligation (L5 SNL), immunoreactivity for nNOS in the ipsilateral L5 but not L4 dorsal root ganglion (DRG) was dramatically increased in mainly small- and medium-sized neurons and non-neuronal cells. L5 SNL caused increased nNOS immunoreactivity in the ipsilateral sciatic nerve, mainly in Schwann cells and the ipsilateral glabrous hind paw skin, mainly on the basement membrane. Furthermore, total nNOS protein and mRNA in the ipsilateral sciatic nerve and hind paw skin were markedly upregulated following nerve injury. Intraplantar injection of the NOS inhibitor 7-nitroindazole (7-NI) or the non-specific NOS inhibitor L-N(G)-nitro-arginine methyl ester (L-NAME) effectively suppressed SNL-induced mechanical allodynia. Collectively, these data suggest that in the periphery nNOS upregulation induced by peripheral nerve injury contributes to mechanical hypersensitivity during the maintenance phase of neuropathic pain. Blocking nNOS signaling in the periphery may thus be a novel therapeutic strategy for the treatment of neuropathic pain., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The expression and role of monocyte chemoattractant protein-1 (MCP-1) in the rat dorsal root ganglion (DRG) and spinal cord was evaluated in the lumbar 5 ventral rhizotomy (L5 VR) model of neuropathic pain. MCP-1 protein expression in the L4/L5 DRG neurons following L5 VR peaked after 3 days, and then declined. Immunohistochemistry showed that no MCP-1 immunoreactivity was observed in the spinal cord after L5 VR, while enzyme-linked immunosorbent assay (ELISA) revealed a small but significant increase in MCP-1 protein content. L5 VR resulted in robust and prolonged mechanical allodynia and thermal hyperalgesia. Administration of anti-MCP-1 neutralizing antibody before and at early time points after L5 VR resulted in a significant attenuation of mechanical allodynia and thermal hyperalgesia, while post-treatment had a weaker effect on established neuropathic pain. Extensive colocalization of tumor necrosis factor receptor 1 (TNFR1) and MCP-1 was observed in the L5 DRG following L5 VR, and treatment with TNFR1 antisense oligonucleotide reduced L5 VR-induced MCP-1 expression in L5 DRG neurons and neuropathic pain behaviors. MCP-1/chemokine (C-C motif) receptor 2 signaling has been proposed as a major regulator of macrophage trafficking. In contrast to the effect on pain behaviors, however, intrathecal administration of anti-MCP-1 neutralizing antibody had no effect on the L5 VR-induced increase in ED-1-immunoreactive macrophages in the L5 DRG and the distal stump of the transected L5 ventral root. These data indicate that increased MCP-1 in DRG neurons might participate in the initiation, rather than the maintenance, of neuropathic pain induced by L5 VR. Furthermore, increased MCP-1 in the DRG is induced by TNF-α/TNFR1 and has no effect on the infiltration of macrophages into the DRG following L5 VR., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

An impaired spinal GABAergic inhibitory function is known to be pivotal in neuropathic pain (NPP). At present, data concerning time-dependent alterations within the GABAergic system itself and post-synaptic GABA(A) receptor-mediated inhibitory transmission are highly controversial, likely related to the experimental NPP model used. Furthermore, it is unknown whether the severity of NPP is determined by the degree of these GABAergic disturbances. In the present study we therefore examined in one experimental animal model whether anatomical changes within the spinal GABAergic system and its GABA(A) receptor-mediated inhibitory function are gradually aggravated during the development of partial sciatic nerve injury (PSNL)-induced NPP and are related to the severity of PSNL-induced hypersensitivity. Three and 16 days after a unilateral PSNL (early and late NPP, respectively), GABA-immunoreactivity (GABA-IR) and the number of GABA-IR neuronal profiles were determined in Rexed laminae 1-3 of lumbar spinal cord cryosections. Additionally, the efficiency of dorsal horn GABA(A) receptor-induced inhibition was examined by cation chloride cotransporter 2 (KCC2) immunoblotting. NPP-induced hypersensitivity was only observed at the ipsilateral side, both at early and late time points. During early NPP, a decrease in ipsilateral dorsal horn GABA-IR was observed without alterations in the number of GABA-IR neuronal profiles or KCC2 protein levels. In contrast, bilateral increases in spinal GABA-IR accompanied by an unchanged number of GABA-IR interneurons were observed during late NPP. This was furthermore attended with decreased ipsilateral KCC2 levels. Moreover, the degree of hypersensitivity was not related to disturbances within the spinal GABAergic system at all time points examined. In conclusion, our anatomical data suggest that a dysfunctional GABA production is likely to be involved in early NPP whereas late NPP is characterized by a combined dysfunctional GABA release and decreased KCC2 levels, the latter suggesting an impaired GABA(A) receptor-mediated inhibition., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Sensory neurons express a variety of voltage-gated Ca2+ channel subtypes, but reports differ on their proportionate representation, and the effects of painful nerve injury on each subtype are not established. We compared levels of high-voltage activated currents in medium-sized (30-40 μm) dorsal root ganglion neurons dissociated from control animals and those subjected to spinal nerve ligation, using sequential application of semiselective channel blockers (nisoldipine for L-type, SNX-111 or ω-conotoxin GVIA for N-type, agatoxin IVA or ω-conotoxin MVIIC for P/Q-type, and SNX-482 for a component of R-type) during either square wave depolarizations or action potential waveform voltage commands. Using sequential administration of multiple blockers, proportions of total Ca2+ current attributable to different subtypes and the effect of injury depended on the sequence of blocker administration and type of depolarization command. Overall, however, N-type and L-type currents comprised the dominant components of ICa in sensory neurons under control conditions, and these subtypes showed the greatest loss of current following injury (L-type 26-71% loss, N-type 0-51% loss). Further exploration of N-type current identified by its sensitivity to ω-conotoxin GVIA applied alone showed that injury reduced the peak N-type current during step depolarization by 68% and decreased the total charge entry during action potential waveform stimulation by 44%. Isolation of N-type current by blockade of all other subtypes demonstrated a 50% loss with injury, and also revealed an injury-related rightward shift in the activation curve. Non-stationary noise analyses of N-type current in injured neurons revealed unitary channel current and number of channels that were not different from control, which indicates that injury-induced loss of current is due to a decrease in channel open probability. Our findings suggest that diminished Ca2+ influx through N-type and L-type channels may contribute to sensory neuron dysfunction and pain after nerve injury., (Published by Elsevier Ltd.)

Painful peripheral neuropathies produced by nerve trauma are accompanied by substantial axonal degeneration and by a response in spinal cord microglia that is characterized by hypertrophy and increased expression of several intracellular and cell-surface markers, including ionizing calcium-binding adapter molecule 1 (Iba1) and Cd11b (a complement receptor 3 antigen recognized by the OX42 antibody). The microglia response has been hypothesized to be essential for the pathogenesis of the neuropathic pain state. In contrast, the painful peripheral neuropathies produced by low doses of cancer chemotherapeutics do not produce degeneration of axons in the peripheral nerve, although they do cause partial degeneration of the sensory axons' distal-most tips, that is the intraepidermal nerve fibers that form the axons' terminal receptor arbors. The question thus arises as to whether the relatively minor and distal axonal injury characterizing the chemotherapy-evoked neuropathies is sufficient to evoke the microglial response that is seen after traumatic nerve injury. We examined the lumbar spinal cord of rats with painful peripheral neuropathies due to the anti-neoplastic agents, paclitaxel, vincristine, and oxaliplatin, and the anti-retroviral agent, 2',3'-dideoxycytidine (ddC), and compared them to rats with a complete sciatic nerve transection and the partial sciatic nerve injury produced in the chronic constriction injury model (CCI). As expected, microglia hypertrophy and increased expression of Iba1 were pronounced in the nerve transection and CCI animals. However, there was no microglia hypertrophy or increased Iba1 staining in the animals treated with paclitaxel, vincristine, oxaliplatin, or ddC. These results suggest that the mechanisms that produce neuropathic pain after exposure to chemotherapeutics may be fundamentally different than those operating after nerve trauma., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuropathic pain conditions for which treatment is sought are characterized by complex behavioural disturbances, as well as "pain." Recent studies using chronic constriction injury of the sciatic nerve have shown that rats develop three distinct patterns of disability characterized by changes in social-interactions and sleep-wake cycle behaviours post-injury: (i) Persistent Disability, (ii) Transient Disability and (iii) No Disability. These patterns occur despite all rats showing identical levels of allodynia and hyperalgesia (i.e., pain). In rats, social-interactions and sleep-wake cycle behaviours are regulated in part, by neural networks, which converge on the periaqueductal grey (PAG). We sought therefore to identify neural adaptations in the PAG, 6 days following chronic constriction injury (CCI), the time at which rats in which disabilities persist are first distinguished from those without disabilities (i.e., No Disability and Transient Disability). GeneChips, RT-PCR and Western blotting revealed the select up-regulation in translation and transcription of glial fibrillary acidic protein (GFAP) and Vimentin in rats with Persistent Disability. Significant increases in GFAP immunoreactivity were localized histologically to the lateral and caudal ventrolateral columns of the PAG. This anatomically specific pattern of increased GFAP suggests activation of astrocytes by select neural pathways, which likely include afferents of both spinal and nucleus of the solitary tract (NTS) origin. The PAG columns in which astrocytes are activated play significant roles in modulating both social-interactions and the sleep-wake cycle. It is possible therefore that the persistent disabilities seen in a subgroup of CCI rats are in part a functional consequence of this specific pattern of astrocyte activation., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

Peripheral nerve injury causes a variety of alterations in pain-related gene expression in primary afferent, which underlie the neuronal plasticity in neuropathic pain. One of the characteristic alterations is a long-lasting downregulation of voltage-gated potassium (K(v)) channel, including K(v)4.3, in the dorsal root ganglion (DRG). The present study showed that nerve injury reduces the messenger RNA (mRNA) expression level of K(v)4.3 gene, which contains a conserved neuron-restrictive silencer element (NRSE), a binding site for neuron-restrictive silencer factor (NRSF). Moreover, we found that injury causes an increase in direct NRSF binding to K(v)4.3-NRSE in the DRG, using chromatin immunoprecipitation (ChIP) assay. ChIP assay further revealed that acetylation of histone H4, but not H3, at K(v)4.3-NRSE is markedly reduced at day 7 post-injury. Finally, the injury-induced K(v)4.3 downregulation was significantly blocked by antisense-knockdown of NRSF. Taken together, these data suggest that nerve injury causes an epigenetic silencing of K(v)4.3 gene mediated through transcriptional suppressor NRSF in the DRG., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Satellite glial cells in the dorsal root ganglion (DRG), like the better-studied glia cells in the spinal cord, react to peripheral nerve injury or inflammation by activation, proliferation, and release of messengers that contribute importantly to pathological pain. It is not known how information about nerve injury or peripheral inflammation is conveyed to the satellite glial cells. Abnormal spontaneous activity of sensory neurons, observed in the very early phase of many pain models, is one plausible mechanism by which injured sensory neurons could activate neighboring satellite glial cells. We tested effects of locally inhibiting sensory neuron activity with sodium channel blockers on satellite glial cell activation in a rat spinal nerve ligation (SNL) model. SNL caused extensive satellite glial cell activation (as defined by glial fibrillary acidic protein [GFAP] immunoreactivity) which peaked on day 1 and was still observed on day 10. Perfusion of the axotomized DRG with the Na channel blocker tetrodotoxin (TTX) significantly reduced this activation at all time points. Similar findings were made with a more distal injury (spared nerve injury model), using a different sodium channel blocker (bupivacaine depot) at the injury site. Local DRG perfusion with TTX also reduced levels of nerve growth factor (NGF) in the SNL model on day 3 (when activated glia are an important source of NGF), without affecting the initial drop of NGF on day 1 (which has been attributed to loss of transport from target tissues). Local perfusion in the SNL model also significantly reduced microglia activation (OX-42 immunoreactivity) on day 3 and astrocyte activation (GFAP immunoreactivity) on day 10 in the corresponding dorsal spinal cord. The results indicate that early spontaneous activity in injured sensory neurons may play important roles in glia activation and pathological pain.

Chronic neuropathic pain remains an unmet clinical problem because it is often resistant to conventional analgesics. Metabotropic glutamate receptors (mGluRs) are involved in nociceptive processing at the spinal level, but their functions in neuropathic pain are not fully known. In this study, we investigated the role of group III mGluRs in the control of spinal excitatory and inhibitory synaptic transmission in a rat model of neuropathic pain induced by L5/L6 spinal nerve ligation. Whole-cell recording of lamina II neurons was performed in spinal cord slices from control and nerve-ligated rats. The baseline amplitude of glutamatergic EPSCs evoked from primary afferents was significantly larger in nerve-injured rats than in control rats. However, the baseline frequency of GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) was much lower in nerve-injured rats than in control rats. The group III mGluR agonist l(+)-2-amino-4-phosphonbutyric acid (l-AP4) produced a greater inhibition of the amplitude of monosynaptic and polysynaptic evoked EPSCs in nerve-injured rats than in control rats. l-AP4 inhibited the frequency of miniature EPSCs in 66.7% of neurons in control rats but its inhibitory effect was observed in all neurons tested in nerve-injured rats. Furthermore, l-AP4 similarly inhibited the frequency of GABAergic and glycinergic IPSCs in control and nerve-injured rats. Our study suggests that spinal nerve injury augments glutamatergic input from primary afferents but decreases GABAergic and glycinergic input to spinal dorsal horn neurons. Activation of group III mGluRs attenuates glutamatergic input from primary afferents in nerve-injured rats, which could explain the antinociceptive effect of group III mGluR agonists on neuropathic pain.

Repetitive strain injuries (RSI), which include several musculoskeletal disorders and nerve compression injuries, are associated with performance of repetitive and forceful tasks. In this study, we examined in young, adult Sprague-Dawley rats, the effects of performing a voluntary, moderate repetition, high force (MRHF; nine reaches/min; 60% maximum pulling force) task for 12 weeks on motor behavior and nerve function, inflammatory responses in forearm musculoskeletal and nerve tissues and serum, and neurochemical immunoexpression in cervical spinal cord dorsal horns. We observed no change in reach rate, but reduced voluntary participation and grip strength in week 12, and increased cutaneous sensitivity in weeks 6 and 12, the latter indicative of mechanical allodynia. Nerve conduction velocity (NCV) decreased 15% in the median nerve in week 12, indicative of low-grade nerve compression. ED-1 cells increased in distal radius and ulna in week 12, and in the median nerve and forearm muscles and tendons in weeks 6 and 12. Cytokines IL-1alpha, IL-1beta, TNF-alpha, and IL-10 increased in distal forearm bones in week 12, while IL-6 increased in tendon in week 12. However, serum analysis revealed only increased TNF-alpha in week 6 and macrophage inflammatory protein 3a (MIP3a) in weeks 6 and 12. Lastly, Substance P and neurokinin-1 were both increased in weeks 6 and 12 in the dorsal horns of cervical spinal cord segments. These results show that a high force, but moderate repetition task, induced declines in motor and nerve function as well as peripheral and systemic inflammatory responses (albeit the latter was mild). The peripheral inflammatory responses were associated with signs of central sensitization (mechanical allodynia and increased neurochemicals in spinal cord dorsal horns).

The N-methyl-d-aspartate receptor (NMDAR) has been implicated in the etiology of chronic pain. In this regard, this study sought to characterize the localization and expression pattern for the NMDAR-2D subunit in a rat model of neuropathic pain. To this end, one group of rats, 3 weeks post-dorsal root rhizotomy (DRR) and a second group, 3 weeks post-spinal nerve ligation (SNL) and sham surgery, were generated. Dorsal root ganglia (DRG) and/or lumbar spinal cord were excised from DRR, naïve, SNL and sham rats. Both immunohistochemical and real-time PCR analysis confirmed discrete NMDAR-2D subunit expression within the DRG and dorsal horn. However, no overt differences in staining intensity or expression were noted between DRG and spinal cord sections obtained from the different surgical groups. Results also demonstrated that the NMDAR-2D subunit was present within Neu N+ cells in the spinal cord and DRG, but excluded from cells labeled with the astrocytic marker, GFAP, and the microglial maker, OX-42. Lastly, the NMDAR-2D subunit was not co-expressed within neurokinin-1 (NK-1)+ or neurofilament-52 (N-52)+ neurons, but the antibody did co-label a number of isolectin B4+ (IB4) DRG cells. Together, these findings seem to suggest that the NMDAR-2D receptor subunit is present within the cell body region of a population of small diameter sensory afferents and post-synaptically within second order dorsal horn neurons. Although these data suggest that the NMDAR-2D subunit is well poised anatomically to modulate pain neurotransmission, the expression pattern for this subunit is not altered in rats demonstrating the presence of neuropathic-like pain behavior.

Neuropathic pain (NPP) due to sensory nerve injury is, in part, the result of peripheral sensitization leading to a long-lasting increase in synaptic plasticity in the spinal dorsal horn. Thus, activation of GABA-mediated inhibitory inputs from sensory neurons could be beneficial in the alleviation of NPP symptoms. Dorsal root ganglia (DRG) conduct painful stimulation from the periphery to the spinal cord. Long-lasting down-regulation in GABA tone or sensitivity in DRG neurons has been reported in animals with neuropathy. To determine the function of GABA in DRG in the development of NPP, we examined how the acute pharmacological GABA(A)-receptor modulation of L5 DRG in vivo affects the development of NPP in rats with crush injury to the sciatic nerve. Direct application of muscimol and gaboxadol, GABA(A) agonists, to L5 DRG immediately after injury induced dose-dependent alleviation, whereas bicuculline and picrotoxin, GABA(A) antagonists, worsened NPP postaxonal injury. The pain-alleviating effects of muscimol and gaboxadol were blocked by bicuculline. Muscimol, applied at the time of injury, caused complete and long-lasting abolishment of NPP development. However, when muscimol was applied after NPP had already developed, its pain-alleviating effect, although significant, was short-lived. Using a fluorescent tracer, sodium fluorescein, we confirmed that local DRG application results in minimal spread into the corresponding dorsal horn of the ipsilateral spinal cord. GABA(A) receptors in DRG are important in the development of NPP after peripheral nerve injury, making timely exogenous GABAergic manipulation at the DRG level a potentially useful therapeutic modality.

Small-fiber painful peripheral neuropathy, a complication of chronic ethanol ingestion, is more severe in women. In the present study, we have replicated this clinical finding in the rat and evaluated for a role of estrogen and second messenger signaling pathways. The alcohol diet (6.5% ethanol volume:volume in Lieber-DeCarli formula) induced hyperalgesia with more rapid onset and severity in females. Following ovariectomy, alcohol failed to induce hyperalgesia in female rats, well past its time to onset in gonad intact males and females. Estrogen replacement reinstated alcohol neuropathy in the female rat. The protein kinase A (PKA) inhibitor (Walsh inhibitor peptide, WIPTIDE) only attenuated alcohol-induced hyperalgesia in female rats. Inhibitors of protein kinase Cepsilon (PKCepsilon-I) and extracellular-signal related kinase (ERK) 1/2 (2'-amino-3'-methoxyflavone (PD98059) and 1,4-diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio) butadiene (U0126)) attenuated hyperalgesia in males and females, however the degree of attenuation produced by PKCepsilon-I was much greater in females. In conclusion, estrogen plays an important role in the expression of pain associated with alcohol neuropathy in the female rat. In contrast to inflammatory hyperalgesia, in which only the contribution of PKCepsilon signaling is sexually dimorphic, in alcohol neuropathy PKA as well as PKCepsilon signaling is highly sexually dimorphic.

Chronic ethanol consumption produces a painful peripheral neuropathy. The aim of this study was then to investigate the mechanism underlying the neuropathic pain-like state induced by chronic ethanol treatment in rats. Mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal, and it lasted for, at least, 14 weeks. At 24 days after ethanol withdrawal, antinociception of morphine was significantly suppressed and the increased guanosine-5'-o-(3-thio) triphosphate ([(35)S]GTPgammaS) binding to membranes of the spinal cord induced by the selective mu-opioid receptor (MOR) agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO), was significantly decreased under the ethanol-dependent neuropathic pain-like state, whereas the increased [(35)S]GTPgammaS binding to membranes of the spinal cord induced by either the selective delta-opioid receptor (DOR) agonist or kappa-opioid receptor (KOR) agonist was not changed under the ethanol-dependent neuropathic pain-like state. Furthermore, total-MOR immunoreactivity was not changed in the spinal cord of ethanol-fed rats. Under these conditions, immunoblotting showed a robust increase in phosphorylated-cPKC immunoreactivity (p-cPKC-IR) in the spinal cord from chronic ethanol fed-rats, whereas phosphorylated-protein kinase A (PKA), dynamin II and G protein-coupled receptor kinase 2 (GRK2) were not affected in the spinal cord of ethanol-fed rats. These findings suggest that the dysfunction of MOR, but not DOR and KOR, linked to cPKC activation in the spinal cord may be, at least in part, involved in the reduced sensitivity to antinociception induced by morphine under the ethanol-dependent neuropathic pain-like state.

Chemotherapeutics in the taxane and vinca-alkaloid classes sometimes produce a painful peripheral neuropathy for which there is no validated treatment. Experiments with rat models of paclitaxel- and vincristine-evoked pain suggest that these conditions may not respond to all of the analgesics that have efficacy in other models of painful peripheral neuropathy. We tested gabapentin as a potential analgesic for paclitaxel- and vincristine-evoked pain. We used a repeated dosing paradigm because there are precedents showing that repeated drug exposure may be necessary to demonstrate analgesia in neuropathic pain models. Gabapentin is believed to work via binding to voltage-gated calcium channels that contain the alpha-2-delta type-1 (alpha(2)delta-1) subunit, and the expression of this subunit is known to be increased in some painful peripheral neuropathy models. Thus we also examined whether the paclitaxel-evoked pain syndrome was accompanied by an alpha(2)delta-1 increase, and whether gabapentin had any effect on subunit expression. We found that the paclitaxel- and vincristine-evoked mechano-allodynia and mechano-hyperalgesia were significantly reduced by gabapentin, but only with repeated dosing. Paclitaxel-evoked painful peripheral neuropathy was associated with an increased expression of the alpha(2)delta-1 subunit in the spinal dorsal horn, but not in the dorsal root ganglia. The spinal cord increase was normalized by repeated gabapentin injections. Together, these findings suggest that repeated dosing with gabapentin may be beneficial in patients with chemotherapy-evoked painful peripheral neuropathy and that gabapentin's mechanisms of action may include normalization of the nerve injury-evoked increase in calcium channel alpha(2)delta-1 subunit expression.

Spinal cord stimulation (SCS) is an established treatment for chronic neuropathic pain. However, in recent studies conflicting results regarding the effect of SCS were noted in a selected group of patients suffering from complex regional pain syndrome and mechanical allodynia. In the present study we investigated the pain relieving effect of SCS in a rat experimental model of neuropathic pain as related to the severity of mechanical allodynia. Adult male rats (n=45) were submitted to a unilateral sciatic nerve ligation. The level of allodynia was tested using the withdrawal response to tactile stimuli with the von Frey test. A portion of these rats developed marked tactile hypersensitivity in the nerve-lesioned paw (von Frey test), similar to "tactile allodynia" observed after nerve injury in humans. Prior to SCS treatment the rats were subdivided into three groups based on the level of allodynia: mild, moderate and severe. All allodynic rats were treated with SCS (n=27) for 30 min (f=50 Hz; pulse width 0.2 ms and stimulation at 2/3 of motor threshold) at 16 days post-injury. Our data demonstrate a differential effect of SCS related to the severity of the mechanical allodynia. SCS leads to a faster and better pain relief in mildly allodynic rats as compared with the more severely allodynic rats. Thus, we suggest that the selection and subdivision of patient groups similar to those defined in our experimental setting (mild, moderate and severe allodynic) may provide better pre-treatment prediction of possible therapeutic benefits of SCS.

S100beta is a calcium-binding peptide produced mainly by astrocytes that exerts paracrine and autocrine effects on neurons and glia. We have previously shown that S100beta is markedly elevated at the mRNA level in the spinal cord following peripheral inflammation, intraplantar administration of complete Freund's adjuvant in the rat. The purpose of the present study was to further investigate the role of astrocytic S100beta in mediating behavioral hypersensitivity in rodent models of persistent pain. First, we assessed the lumbar spinal cord expression of S100beta at the mRNA and protein level using real-time RT-PCR, Western blot and immunohistochemistry analysis following L5 spinal nerve transection in rats, a rodent model of neuropathic pain. Second, we assessed behavioral hypersensitivity (mechanical allodynia) in wild type and genetically modified mice lacking or overexpressing S100beta following L5 spinal nerve transection. Third, we assessed the expression level of S100beta protein in the CD1 wild type mice after nerve injury. We report that lumbar spinal S100beta mRNA steadily increased from days 4-28 after nerve injury. S100beta protein in the lumbar spinal cord was significantly increased in both rats and mice at day 14 following nerve injury as compared with sham control groups. S100beta genetically deficient mice displayed significantly increased tactile thresholds (reduced response to non-noxious stimuli) after nerve injury as compared with the wild type group. S100beta overexpressing mice displayed significantly decreased tactile threshold responses (enhanced response to non-noxious stimuli). Together, these results from both series of experiments using a peripheral nerve injury model in two different species implicate the involvement of glial-derived S100beta in the pathophysiology of neuropathic pain.

Loss of GABA-mediated inhibition in the spinal cord is thought to mediate allodynia and spontaneous pain after nerve injury. Despite extensive investigation of GABA itself, relatively little is known about how nerve injury alters the receptors at which GABA acts. This study examined levels of GABA(B) receptor protein in the spinal cord dorsal horn, and in the L4 and L5 (lumbar designations) dorsal root ganglia one to 18 weeks after L5 spinal nerve ligation. Mechanical allodynia was maximal by 1 week and persisted at blunted levels for at least 18 weeks after injury. Spontaneous pain behaviors were evident for 6 weeks. Western blotting of dorsal horn detected two isoforms of the GABA(B(1)) subunit and a single GABA(B(2)) subunit. High levels of GABA(B(1a)) and low levels of GABA(B(1b)) protein were present in the dorsal root ganglia. However, GABA(B(2)) protein was not detected in the dorsal root ganglia, consistent with the proposed existence of an atypical receptor composed of GABA(B(1)) homodimers. The levels of GABA(B(1a)), GABA(B(1b)), and GABA(B(2)) protein in the ipsilateral dorsal horn were unchanged at any time after injury. Immunohistochemical staining also did not detect a change in GABA(B(1)) or GABA(B(2)) subunits in dorsal horn segments having a robust loss of isolectin B4 staining. The levels of GABA(B(1a)) protein were also unchanged in the L4 or L5 dorsal root ganglia at any time after spinal nerve ligation. Levels of GABA(B(2)) remained undetectable. Finally, baclofen-stimulated binding of guanosine-5'-(gamma-O-thio)triphosphate in dorsal horn did not differ between sham and ligated rats. Collectively, these results argue that a loss of GABA(B) receptor-mediated inhibition, particularly of central terminals of primary afferents, is unlikely to mediate the development or maintenance of allodynia or spontaneous pain behaviors after spinal nerve injury.

Neuropeptide W-23 and neuropeptide B are each an endogenous ligand of GPR7. GPR7 mRNA has been detected in regions of the cortex, the hippocampus, the hypothalamus and the spinal cord in the rat. GPR7 receptor has structural features in common with both opioid and somatostatin receptors. In the present study, the effects of intrathecal and i.c.v. application of neuropeptide W-23 and neuropeptide B on the level of mechanical allodynia induced by partial sciatic nerve ligation were tested in rats. The level of mechanical allodynia was measured using von Frey filaments. Intrathecal injection of either neuropeptide W-23 or neuropeptide B attenuated the level of mechanical allodynia in a dose dependent manner at a dose between 0.1 and 10 microg, but i.c.v. injection of either neuropeptide W-23 or neuropeptide B had no effect on the level of mechanical allodynia at a dose between 3 and 30 microg. The effect of intrathecal administration of either 10 microg of neuropeptide W-23 or 10 microg of neuropeptide B was not antagonized by i.p. injection of 1 mg/kg of naloxone. Immunohistochemical examination revealed that neuropeptide W-23 was expressed mainly in the small- to medium-sized neuronal profiles in the dorsal root ganglion and that partial sciatic nerve injury decreased the percentage of neuropeptide W-23-like immunoreactivity positive neuronal profiles that were labeled by IB4. These data suggest that neuropeptide W-23 is involved in the nociceptive transmission in spinal cord and that both spinally-applied neuropeptide W-23 and spinally-applied neuropeptide B produce anti-allodynic effects in the partial sciatic nerve ligation model in rat.

Previous studies have indicated that thalamic nucleus submedius is involved in opioid-mediated antinociception in tail flick test and formalin test. The current study examined the effects of opioids microinjected into the thalamic nucleus submedius on the allodynia developed in neuropathic pain model rats, and determined the roles of different subtypes of opioid receptors in the thalamic nucleus submedius opioid-evoked antiallodynia. The allodynic behaviors induced by L5/L6 spinal nerve ligation were assessed by mechanical (von Frey filaments) and cold (4 degrees C plate) stimuli. Morphine (1.0, 2.5, and 5.0 microg) microinjected into the thalamic nucleus submedius contralateral to the nerve injury paw produced a dose-dependent inhibition of the mechanical and cold allodynia, and these effects were reversed by microinjection of the non-selective opioid receptor antagonist naloxone (1.0 microg) into the same site. Microinjection of endomorphin-1 (5.0 microg), a highly selective mu-opioid receptor agonist, and [D-Ala2, D-Leu5]-enkephalin (10 microg), a delta-/mu-opioid receptor agonist, also inhibited the allodynic behaviors, and these effects were blocked by selective mu-opioid receptor antagonist beta-funaltrexamine hydrochloride (3.75 microg). However, the [D-Ala2, D-Leu5]-enkephalin-evoked antiallodynic effects were not influenced by the selective delta-opioid receptor antagonist naltrindole (5.0 microg). Microinjection of the selective kappa-receptor agonist spiradoline mesylate salt (100 microg) into the thalamic nucleus submedius failed to alter the allodynia induced by spinal nerve ligation. These results suggest that the thalamic nucleus submedius is involved in opioid-evoked antiallodynia which is mediated by mu- but not delta- and kappa-opioid receptor in the neuropathic pain model rats.

A transcription factor known as cyclic AMP response element-binding protein has been shown to be involved in the central sensitization in neuropathic pain and inflammation pain. The present study examined the roles of cyclic AMP response element-binding protein and of the phosphorylated cyclic AMP response element-binding protein in the maintenance of mechanical and cold allodynia induced by a neuropathic pain model, "spared nerve injury," in rats. First, the results of immunohistochemical study showed that phosphorylated cyclic AMP response element-binding protein, but not cyclic AMP response element-binding protein, increased bilaterally in the spinal dorsal horn 14 days following spared nerve injury, indicating a possible contribution of phosphorylated cyclic AMP response element-binding protein in spared nerve injury. Second, chronic intrathecal application of cyclic AMP response element-binding protein antisense oligodeoxynucleotide with three doses (10 microg/day, 20 microg/day and 40 microg/day) for 5 days demonstrated that the higher doses (20 and 40 microg) significantly attenuated both mechanical (bilaterally) and cold (ipsilaterally) allodynia, compared with sense oligodeoxynucleotide and the lower dose (10 microg). Western blot results showed that the alleviation in intensity of behavioral performance was accompanied by a significant reduction of total cyclic AMP response element-binding protein and phosphorylated cyclic AMP response element-binding protein in the spinal dorsal horn. Moreover, there were no differences in cyclic AMP response element-binding protein and phosphorylated cyclic AMP response element-binding protein between ipsilateral and contralateral dorsal horns. Our data demonstrate a close association between the expression of behavioral hypersensitivity and cyclic AMP response element-binding protein activation in the spinal dorsal horn following spared nerve injury, supporting the notion that phosphorylated cyclic AMP response element-binding protein may play an important role in the maintenance of chronic neuropathic pain.

Neurofibromatosis type I is a common autosomal dominant disease characterized by formation of multiple benign and malignant tumors. People with this disorder also experience chronic pain, which can be disabling. Neurofibrinomin, the protein product of the NF1 gene (neurofibromin gene (human)), is a guanosine triphosphate activating protein for p21(ras). Loss of NF1 results in an increase in activity of the p21(ras) transduction cascade. Because of the growing evidence suggesting involvement of downstream components of the p21(ras) transduction cascade in the sensitization of nociceptive sensory neurons, we examined the stimulus-evoked release of the neuropeptides, substance P and calcitonin gene-related peptide, from primary sensory neurons of mice with a mutation of the Nf1 gene (neurofibromin gene (mouse)) (Nf1+/-). Measuring immunoreactive substance P and immunoreactive calcitonin gene-related peptide by radioimmunoassay, we demonstrated that capsaicin-stimulated release of neuropeptides is three to five-fold higher in spinal cord slices from Nf1+/- mice than from wildtype mouse tissue. In addition, the potassium and capsaicin-stimulated release of immunoreactive calcitonin gene-related peptide from cultures of sensory neurons isolated from Nf1+/- mice was more than double that from cultures of wildtype neurons. Treatment of wildtype sensory neurons with nerve growth factor for 5-7 days mimicked the enhanced stimulus-evoked release observed from the Nf1+/- neurons. When nerve growth factor was removed 48 h before conducting release experiments, nerve growth factor-induced augmentation of immunoreactive calcitonin gene-related peptide release from Nf1+/- neurons was more pronounced than in Nf1+/- sensory neurons that were treated with nerve growth factor continuously for 5-7 days. Thus, sensory neurons from mice with a heterozygous mutation of the Nf1 gene that is analogous to the human disease neurofibromatosis type I, exhibit increased sensitivity to chemical stimulation. This augmented responsiveness may explain the abnormal pain sensations experienced by people with neurofibromatosis type I and suggests an important role for guanosine triphosphate activating proteins, in the regulation of nociceptive sensory neuron sensitization.