Nerve Injury-induced Neuropathic Pain Research Articles

Penehyclidine hydrochloride suppressed peripheral nerve injury-induced neuropathic pain by inhibiting microglial MAPK/p-p38/IL-1β pathway activation.

BackgroundMillions of people suffered from neuropathic pain, which is related to neuroinflammation in the central nervous system. Penehyclidine hydrochloride is a premedication of general anesthesia, which has been confirmed possessing neuroprotective effects against various neurodegenerative or neuroinflammatory diseases. However, it is not clear that whether penehyclidine hydrochloride could suppress neuropathic pain through its anti-neuroinflammatory effects.MethodsThis study investigated the effects of penehyclidine hydrochloride on rat spinal nerve ligation injury-induced neuropathic pain with behavioral, morphological, and molecular biological methods in animals.ResultsThe results indicated that penehyclidine hydrochloride could attenuate spinal nerve ligation-induced neuropathic pain without any motor impairment and had no effect on sham-operated animals after repeated intraperitoneal administration. Intraperitoneal penehyclidine hydrochloride could suppress spinal nerve ligation-induced ipsilateral spinal dorsal horn microglial activation with downregulation of OX42 expression. Moreover, intraperitoneal penehyclidine hydrochloride inhibited spinal nerve ligation-induced spinal p-p38 mitogen-activated protein kinase expression, which was specially colocalized with the spinal dorsal horn microglia. Furthermore, intraperitoneal penehyclidine hydrochloride could depress spinal neuroinflammation by suppressing spinal nerve ligation-induced interleukin (IL)-1β over-expression.ConclusionThese results indicated that the anti-allodynic effects of penehyclidine hydrochloride on spinal nerve ligation-induced neuropathic pain did not rely on motor impairment. Inhibiting spinal microglial p-p38/IL-1β pathway activation might contribute to the anti-allodynic effect of penehyclidine hydrochloride on nerve injury-induced neuropathic pain.

Dopamine receptor D2, but not D1, mediates descending dopaminergic pathway-produced analgesic effect in a trigeminal neuropathic pain mouse model.

Neuropathic pain represents a challenge to clinicians because it is resistant to commonly prescribed analgesics due to its largely unknown mechanisms. Here, we investigated a descending dopaminergic pathway-mediated modulation of trigeminal neuropathic pain. We performed chronic constriction injury of the infraorbital nerve from the maxillary branch of trigeminal nerve to induce trigeminal neuropathic pain in mice. Our retrograde tracing showed that the descending dopaminergic projection from hypothalamic A11 nucleus to spinal trigeminal nucleus caudalis is bilateral. Optogenetic/chemogenetic manipulation of dopamine receptors D1 and D2 in the spinal trigeminal nucleus caudalis produced opposite effects on the nerve injury-induced trigeminal neuropathic pain. Specific excitation of dopaminergic neurons in the A11 nucleus attenuated the trigeminal neuropathic pain through the activation of D2 receptors in the spinal trigeminal nucleus caudalis. Conversely, specific ablation of the A11 dopaminergic neurons exacerbated such pain. Our results suggest that the descending A11-spinal trigeminal nucleus caudalis dopaminergic projection is critical for the modulation of trigeminal neuropathic pain and could be manipulated to treat such pain.

Riluzole induces LTD of spinal nociceptive signaling via postsynaptic GluR2 receptors

PurposeRiluzole – a major therapeutic medicine for patients with amyotrophic lateral sclerosis – reportedly has anti-nociceptive and anti-allodynic efficacies in neuropathic pain models. However, little is known about its effect on neurotransmission in the spinal superficial dorsal horn (SDH). The present study aims to investigate the effects of riluzole on the synaptic transmission of SDH nociceptive pathways in both physiological and pathological conditions.Materials and methodsSpinal nerve ligation was used to produce a neuropathic pain model. Mechanical allodynia behavior was assessed with Von Frey filaments. Riluzole’s effects on nociceptive synaptic transmission under both physiological and pathological conditions were examined by patch-clamp recordings in rat SDH neurons.ResultsThe principal findings of the present study are three-fold. First, we affirm that riluzole has a remarkable long-lasting analgesic effect on both in vitro and in vivo pathological pain models. Second, the prolonged inhibitory effects of riluzole on spinal nociceptive signaling are mediated by both presynaptic and postsynaptic mechanisms. Finally, endocytosis of post-synaptic GluR2 contributes to the riluzole-induced long-term depression (LTD) of the spinal nociceptive pathway.ConclusionThe present study finds that riluzole induces LTD of nociceptive signaling in the SDH and produces long-lasting anti-allodynia effects in nerve injury-induced neuropathic pain conditions via postsynaptic AMPA receptors associated with the endocytosis of GluR2.

Role of spinal dopamine D2 receptors in analgesic effect of duloxetine in rats with neuropathic pain

Objective To evaluate the involvement of spinal dopamine D2 receptors in the analgesic effect of duloxetine in neuropathic pain rat models. Methods Experiment I, twenty-four male SD rats, weighting 200-250 g, were randomly divided into 4 groups (n=6), respectively receiving intraperitoneal injection of dimethyl sulfoxide (DMSO) (group DS), 3 mg/kg duloxetine (group D1), 10 mg/kg duloxetine (group D2), and 30 mg/kg duloxetine (group D3). These treatments started 7 days after spinal L5 nerve ligation, and the mechanical withdraw threshold (MWT) was determined at 7 time points: before spinal nerve ligation (SNL) surgery (T0), before duloxetine injection (T1), and 15 min (T2), 30 min (T3), 60 min (T4), 120 min (T5), 180 min (T6) after duloxetine injection. Experiment Ⅱ, twenty-four male SD rats, weighting 200-250 g, were subjected to intrathecal catheters insertion, and were randomly divided into 4 groups (n=6). In group DS, DMSO (group DS+DS) or 30 μg sulpride (group Sul+DS) was administered intrathecally 15 min before DMSO intraperitoneal injection. In group Dul, DMSO (group DS+Dul) or 30 μg sulpride (group Sul+Dul) was administered intrathecally 15 min before 30 mg/kg duloxetine intraperitoneal injection. Mechanical paw withdraw threshold was determined before and after duloxetine or DMSO injection. Experiment Ⅲ, twelve male SD rats, weighting 200-250 g, were randomly divided into 2 groups (n=6), respectively receiving intraperitoneal injection of DMSO (group N) and 10 mg/kg duloxetine (group D). Then, the concentrations of dopamine in the spinal cord were measured with microdialysis before and after injection of duloxetine or DMSO injection. Results Compared with group DS, the MWT was significantly increased in group D3 at T2-T6 (P<0.05). Compared with group DS+Dul, the MWT was dramatically decreased in group Sul+Dul at T2-T6 (P<0.05). Compared with group N, the concentrations of dopamine in the spinal cord was dramatically increased in group D at T8-T13 (P<0.05). Conclusions Duloxetine relieved spinal nerve injury-induced neuropathic pain in rats, which may be related to the enhancement of dopamine release and activation of D2 receptors in the spinal cord dorsal horn. Key words: Neuropathic pain; Duloxetine; Spinal cord dorsal horn; Dopamine D2 receptors

Mesenchymal stem cell exosomes as a cell-free therapy for nerve injury-induced pain in rats.

Nerve injury-induced neuropathic pain is difficult to treat. In this study, we used exosomes derived from human umbilical cord mesenchymal stem cell (UCMSC) as a cell-free therapy for nerve injury-induced pain in rats. Isolated UCMSC exosomes range in size from 30 to 160 nm and contain CD63, HSP60, and CD81 exosome markers. After L5/6 spinal nerve ligation surgery, single intrathecal injection of exosomes reversed nerve ligation-induced mechanical and thermal hypersensitivities of right hindpaw of rats at initial and well-developed pain stages. Moreover, continuous intrathecal infusion of exosomes achieved excellent preventive and reversal effects for nerve ligation-induced pain. In immunofluorescent study, lots of Exo-green-labelled exosomes could be found majorly in the ipsilateral L5 spinal dorsal horn, dorsal root ganglion, and peripheral axons, suggesting the homing ability of UCMSC exosomes. They also appeared in the central terminals or cell bodies of IB4, CGRP, and NF200 sensory neurons. In addition, exosome treatment suppressed nerve ligation-induced upregulation of c-Fos, CNPase, GFAP, and Iba1. All these data suggest that the analgesic effects of exosomes may involve their actions on neuron and glial cells. Exosomes also inhibited the level of TNF-α and IL-1β, while enhanced the level of IL-10, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in the ipsilateral L5/6 dorsal root ganglion of nerve-ligated rats, indicating anti-inflammatory and proneurotrophic abilities. Protein analysis revealed the content of vascular endothelial growth factor C, angiopoietin-2, and fibroblast growth factor-2 in the exosomes. In summary, intrathecal infusion of exosomes from UCMSCs may be considered as a novel therapeutic approach for nerve injury-induced pain.

Contribution of CD137L to Sensory Hypersensitivity in a Murine Model of Neuropathic Pain.

CD137L (4-1BBL) is a costimulatory molecule whose signaling can promote monocyte/macrophage functions; however, CD137L-mediated microglial response and its role in neuropathic pain remain unknown. We investigated CD137L following peripheral nerve injury-induced neuropathic pain using a spinal nerve L5 transection (L5Tx) murine model in both sexes. First, C57BL/6_CD137L knock-out (KO) mice displayed decreased mechanical and diminished heat hypersensitivity compared to wild-type (WT) controls, beginning on day 3 to up to day 35 post-L5Tx. Purified anti-mouse CD137L neutralizing monoclonal antibody (0.1 or 0.5 µg) was also used to identify CD137L’s window of action in BALB/c mice. Anti-CD137L antibody was intrathecally administered either from day 0 (before surgery) to day 7 (early treatment), or from day 6 to 13 post-L5Tx (late treatment), and nociceptive thresholds were assessed before surgery to up to day 35 post-surgery. Early treatment with anti-CD137L reduced L5Tx-induced mechanical but not heat hypersensitivity, while later treatment did not alter either sensitivity. Pro- versus anti-inflammatory responses within the lumbar spinal cord following L5Tx were further evaluated via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) in time-course studies. Following L5Tx, female CD137L KO mice did not show increased iNOS mRNA and had reduced numbers of IL-1β+ cells compared to WT. At 21 d post-surgery, CD137L KO mice had higher total numbers of arginase (Arg)-1+ cells and Arg-1+ microglia. Altogether, results indicate that spinal cord CD137L contributes to the development of peripheral nerve injury-induced neuropathic pain, which may be in part mediated through CD137L’s modulation of the pro- and anti-inflammatory balance within the spinal cord.

Involvement of lysophosphatidic acid-induced astrocyte activation underlying the maintenance of partial sciatic nerve injury-induced neuropathic pain.

We have previously demonstrated that lysophosphatidic acid (LPA) plays key roles in the initial mechanisms for neuropathic pain (NeuP) development. Here, we examined whether LPA receptor mechanisms and LPA production are related to the glial activation at a late stage after partial sciatic nerve ligation (pSNL) by use of microglial inhibitor, Mac1-saporin or astrocyte inhibitor, and L-α-aminoadipate (L-AA). Although single intrathecal injection of LPA1/3 antagonist, Ki-16425 did not affect the pain threshold at day 7 after the spinal cord injury, repeated treatments of each compound gradually reversed the basal pain threshold to the control level. The intrathecal administration of a microglia inhibitor, Mac-1-saporin reversed the late hyperalgesia and LPA production at day 14 after the pSNL, whereas L-AA inhibited the hyperalgesia, but had no effect on LPA production. The involvement of LPA receptors in astrocyte activation in vivo was evidenced by the findings that Ki-16425 treatments abolished the upregulation of CXCL1 in activated astrocytes in the spinal dorsal horn of mice at day 14 after the pSNL, and that Ki-16425 reversed the LPA-induced upregulation of several chemokine gene expressions in primary cultured astrocytes. Finally, we found that significant hyperalgesia was observed with intrathecal administration of primary cultured astrocytes, which had been stimulated by LPA in a Ki-16425-reversible manner. All these findings suggest that LPA production and LPA1/3 receptor activation through differential glial mechanisms play key roles in the maintenance as well as initiation mechanisms in NeuP.

Chronic constriction injury-induced microRNA-146a-5p alleviates neuropathic pain through suppression of IRAK1/TRAF6 signaling pathway

BackgroundmicroRNA-146a-5p (miRNA-146a-5p) is a key molecule in the negative regulation pathway of TLRs and IL-1 receptor (TIR) signaling. Our recent study demonstrated that MyD88-dependent signaling pathway of TIR in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) plays a role in peripheral nerve injury-induced neuropathic pain. However, it was not clear whether and how miRNA-146a-5p regulates the TIR pathway of DRG and SDH in the development of neuropathic pain.MethodsThe sciatic nerve chronic constriction injury (CCI) model of rat was used to induce chronic neuropathic pain. The levels and cellular distribution of miRNA-146a-5p were detected with quantitative real-time PCR (qPCR) and fluorescent in situ hybridization (FISH). The RNA level, protein level, and cellular distribution of IRAK1 and TRAF6 that is targeted by miRNA-146a-5p were detected with qPCR, western blot, and immunofluorescent. The pain-related behavioral effect of miRNA-146a-5p was accessed after intrathecal administration. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia.ResultsWe found that the level of miRNA-146a-5p significantly increased in L4-L6 DRGs and SDH after CCI surgery; meanwhile, the protein level of IRAK1 and TRAF6 in DRGs was significantly increased after CCI. Intrathecal injection of miR146a-5p agomir or miRNA-146a-5p antagomir regulates miRNA-146a-5p level of L4-L6 DRGs and SDH. We found that intrathecal injection of miR146a-5p agomir can alleviate mechanical and thermal hyperalgesia in CCI rats and reverse the upregulation of IRAK1 and TRAF6 of L4-L6 DRGs and SDH induced by CCI. We furthermore found that intrathecal injection of miRNA-146a-5p antagomir can exacerbate the mechanical and thermal pain-related behavior of CCI rats and meanwhile increase IRAK1 and TRAF6 of L4-L6 DRGs and SDH expression even further.ConclusionsmiRNA-146a-5p of DRG and SDH can modulate the development of CCI-induced neuropathic pain through inhibition of IRAK1 and TRAF6 in the TIR signaling pathway. Hence, miRNA-146a-5p may serve as a potential therapeutic target for neuropathic pain.

Region-specific deletions of the glutamate transporter GLT1 differentially affect nerve injury-induced neuropathic pain in mice.

Glutamate is a major excitatory neurotransmitter and plays an important role in neuropathic pain, which is frequently caused by nerve damage. According to recent studies, nerve injury induces changes in glutamatergic transmission in the spinal cord and several supraspinal regions, including the periaqueductal gray (PAG). Among glutamate signaling components, accumulating evidence suggests that the glial glutamate transporter GLT1 plays a critical role in neuropathic pain. Indeed, GLT1 expression is reduced in the spinal cord but increased in the PAG after nerve injury, suggesting that the role of GLT1 in neuropathic pain may vary according to the brain region. In this study, we generated PAG-specific and spinal cord-specific GLT1 knockout mice. Nerve injury-induced neuropathic pain was enhanced in spinal cord-specific GLT1 knockout mice but alleviated in PAG-specific GLT1 knockout mice. Thus, nerve injury may enhance glutamatergic neurotransmission from primary sensory neurons to the post-synaptic dorsal horn following downregulation of GLT1 in the spinal cord and result in inadequate descending pain inhibition caused by GLT1 upregulation in the PAG, resulting in neuropathic pain. In addition, ceftriaxone upregulated GLT1 expression in the spinal cord, but not the PAG, of control mice and attenuated tactile hypersensitivity in nerve-injured control mice but not in nerve-injured spinal cord-specific GLT1 knockout mice. Based on these results, the anti-neuropathic pain effect of ceftriaxone is mediated by the upregulation of GLT1 expression in the spinal cord. Thus, selective upregulation of spinal GLT1 and/or downregulation of GLT1 in the PAG represents a potentially novel strategy for the treatment of neuropathic pain.

Neuropathic Pain Causes Pyramidal Neuronal Hyperactivity in the Anterior Cingulate Cortex.

The anterior cingulate cortex (ACC) is thought to be important for acute pain perception as well as the development of chronic pain after peripheral nerve injury. Nevertheless, how ACC neurons respond to sensory stimulation under chronic pain states is not well understood. Here, we used an in vivo two-photon imaging technique to monitor the activity of individual neurons in the ACC of awake, head restrained mice. Calcium imaging in the dorsal ACC revealed robust somatic activity in layer 5 (L5) pyramidal neurons in response to peripheral noxious stimuli, and the degree of evoked activity was correlated with the intensity of noxious stimulation. Furthermore, the activation of ACC neurons occurred bilaterally upon noxious stimulation to either contralateral or ipsilateral hind paws. Notably, with nerve injury-induced neuropathic pain in one limb, L5 pyramidal neurons in both sides of the ACC showed enhanced activity in the absence or presence of pain stimuli. These results reveal hyperactivity of L5 pyramidal neurons in the bilateral ACC during the development of neuropathic pain.

Effective control of neuropathic pain by transient expression of hepatocyte growth factor in a mouse chronic constriction injury model.

Hepatocyte growth factor (HGF) is a multifunctional protein that contains angiogenic and neurotrophic properties. In the current study, we investigated the analgesic effects of HGF by using a plasmid DNA that was designed to express 2 isoforms of human HGF—pCK-HGF-X7 (or VM202)—in a chronic constriction injury (CCI) –induced mouse neuropathic pain model. Intramuscular injection of pCK-HGF-X7 into proximal thigh muscle induced the expression of HGF in the muscle, sciatic nerve, and dorsal root ganglia (DRG). This gene transfer procedure significantly attenuated mechanical allodynia and thermal hyperalgesia after CCI. Injury-induced expression of activating transcription factor 3, calcium channel subunit α2δ1, and CSF1 in the ipsilateral DRG neurons was markedly down-regulated in the pCK-HGF-X7–treated group, which suggested that HGF might exert its analgesic effects by inhibiting pain-mediating genes in the sensory neurons. In addition, suppressed CSF1 expression in DRG neurons by pCK-HGF-X7 treatment was accompanied by a noticeable suppression of the nerve injury–induced glial cell activation in the spinal cord dorsal horn. Taken together, our data show that pCK-HGF-X7 attenuates nerve injury–induced neuropathic pain by inhibiting pain-related factors in DRG neurons and subsequent spinal cord glial activation, which suggests its therapeutic efficacy in the treatment of neuropathic pain.—Nho, B., Lee, J., Lee, J., Ko, K. R., Lee, S. J., Kim, S. Effective control of neuropathic pain by transient expression of hepatocyte growth factor in a mouse chronic constriction injury model.

The HDAC1/c-JUN complex is essential in the promotion of nerve injury-induced neuropathic pain through JNK signaling

Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. Administration of a selective HDAC1 inhibitor (LG325) in SNI-subjected mice significantly attenuated behavior related to injury-induced pain. Understanding the HDAC1 pathway in epigenetic regulation of pathological pain is of great medical relevance. Spared nerve injury (SNI) mice showed a significant increase in the HDAC1 protein levels within spinal cord in coincidence with the nociceptive phenotype at 1 and 3 weeks after nerve injury. No variation in HDAC3, DNMT3a, AcH3, MBD3 and MeCP2 levels was detected. Increased expression of HDAC1 is accompanied by activation of the JNK-c-Jun signaling pathway. A robust spinal JNK-1 overphosphorylation was observed post nerve-injury along with a selective JNK-dependent increase in p-c-Jun and HDAC1 protein levels. Co-immunoprecipitation experiments showed the presence of a heterodimeric complex between HDAC1 and c-Jun in SNI mice indicating that these transcription factors can act together to regulate transcription through heterodimerization. Stimulation of c-Jun phosphorylation was prevented by the selective HDAC1 inhibitor LG325. We found that HDAC1 was associated with c-Jun in nuclei of spinal dorsal horn astrocytes expressing JNK. On the other hand, the presence of HDAC1 and c-Jun interaction was not detected in control mice. These findings provide new insights into the mechanisms underlying the anti-nociceptive activity of HDAC inhibitors. Taken together, these data support a role for histone deacetylase in the emergence of neuropathic pain.

LPA5 signaling is involved in multiple sclerosis-mediated neuropathic pain in the cuprizone mouse model

Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain.

Anti-nociceptive Role of CXCL1 in a Murine Model of Peripheral Nerve Injury-induced Neuropathic Pain

Both spinal cord infiltrating CD4+ T lymphocytes and microglial CD40 contribute to the maintenance of neuropathic pain-like behaviors induced by spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we sought to investigate the involvement of multiple chemokines in microglial CD40-mediated and CD4+ T lymphocytes-mediated L5Tx-induced sensory hypersensitivity. Spinal cord chemokine expression in CD4 knockout (KO), CD40 KO, and wild type (WT) BALB/c mice was determined at the protein level via multiplex assays and at the RNA level via quantitative real-time PCR. In WT mice, L5Tx induced significant increases in CCL2, CCL3, and CCL5 expression (protein and RNA) up to day 21 post-L5Tx, while CD4 KO mice displayed blunted, predominantly non-significant, responses in these chemokines at protein levels post-L5Tx. L5Tx also induced increased expression of these chemokines in CD40 KO mice; however, the overall protein levels of these chemokines were significantly lower than those in WT mice. Further, L5Tx induced a significant increase in CXCL1 at the protein level and in CXCR2 at RNA level only in CD40 KO mice. Intrathecal administration of CXCL1 in WT mice significantly reduced L5Tx-induced mechanical hypersensitivity. CD40 KO mice also displayed higher levels of Ly6G (neutrophil marker) RNA expression in the lumbar spinal cord post-L5Tx. Altogether, our data suggest that CD4+ T lymphocytes and microglial CD40 mediate their pro-nociceptive effects in part by promoting selected chemokine responses, and more importantly, CXCL1 can play an anti-nociceptive role in peripheral nerve injury-induced neuropathic pain, which is possibly mediated by infiltrating neutrophils.

Dehydrocorydaline attenuates bone cancer pain by shifting microglial M1/M2 polarization toward the M2 phenotype.

Bone cancer pain remains a major challenge in patients with primary or metastatic bone cancer due to a lack of understanding the mechanisms. Previous studies have revealed the two distinct functional polarization states of microglia (classically activated M1 and alternatively activated M2) in the spinal cord in nerve injury–induced neuropathic pain. However, whether microglia in the spinal cord polarize to M1 and M2 phenotypes and contribute to the development of bone cancer pain remains unclear. In this study, we used a mouse model with bone cancer to characterize the M1/M2 polarization of microglia in the spinal cord during the development of bone cancer pain, and investigated the antinociceptive effects of dehydrocorydaline, an alkaloidal component isolated from Rhizoma corydalis on bone cancer pain. Our results show that microglia in the spinal cord presented increased M1 polarization and decreased M2 polarization, while overproduction of IL-1β and inhibited expression of IL-10 was detected during bone cancer pain development. Intraperitoneal administration of dehydrocorydaline (10 mg/kg) had significant antinociceptive effects on day 14 after osteosarcoma cell implantation, accompanied by suppressed M1 phenotype and upregulated M2 phenotype of microglia in the spinal cord, while alleviated inflammatory response was observed then. These results suggest that the imbalanced polarization of microglia toward the M1 phenotype in the spinal cord may contribute to the development of bone cancer pain, while dehydrocorydaline helps to attenuate bone cancer pain, with microglial polarization shifting toward the M2 phenotype in the spinal cord.

Differential sensory fiber specificity and LPA receptor-involvement between partial sciatic nerve injury-induced and high-fat diet-induced neuropathic pain

Differential sensory fiber specificity and LPA receptor-involvement between partial sciatic nerve injury-induced and high-fat diet-induced neuropathic pain

ATF2, but not ATF3, participates in the maintenance of nerve injury-induced tactile allodynia and thermal hyperalgesia.

Transcription factors are proteins that modulate the transcriptional rate of target genes in the nucleus in response to extracellular or cytoplasmic signals. Activating transcription factors 2 (ATF2) and 3 (ATF3) respond to environmental signals and maintain cellular homeostasis. There is evidence that inflammation and nerve injury modulate ATF2 and ATF3 expression. However, the function of these transcription factors in pain is unknown. The purpose of this study was to investigate the contribution of ATF2 and ATF3 to nerve injury-induced neuropathic pain. L5/6 spinal nerve ligation induced tactile allodynia and thermal hyperalgesia. Moreover, nerve damage enhanced ATF2 and ATF3 protein expression in injured L5/6 dorsal root ganglia and spinal cord but not in uninjured L4 dorsal root ganglia. Nerve damage also enhanced ATF2 immunoreactivity in dorsal root ganglia and spinal cord 7 to 21 days post-injury. Repeated intrathecal post-treatment with a small-interfering RNA targeted against ATF2 (ATF2 siRNA) or anti-ATF2 antibody partially reversed tactile allodynia and thermal hyperalgesia. In contrast, ATF3 siRNA or anti-ATF3 antibody did not modify nociceptive behaviors. ATF2 immunoreactivity was found in dorsal root ganglia and spinal cord co-labeling with NeuN mainly in non-peptidergic (IB4+) but also in peptidergic (CGRP+) neurons. ATF2 was found mainly in small- and medium-sized neurons. These results suggest that ATF2, but not ATF3, is found in strategic sites related to spinal nociceptive processing and participates in the maintenance of neuropathic pain in rats.

Presynaptic inhibition of nociceptive neurotransmission by somatosensory neuron-secreted suppressors.

Noxious stimuli cause pain by activating cutaneous nociceptors. The Aδ- and C-fibers of dorsal root ganglion (DRG) neurons convey the nociceptive signals to the laminae I-II of spinal cord. In the dorsal horn of spinal cord, the excitatory afferent synaptic transmission is regulated by the inhibitory neurotransmitter γ-aminobutyric acid and modulators such as opioid peptides released from the spinal interneurons, and by serotonin, norepinepherine and dopamine from the descending inhibitory system. In contrast to the accumulated evidence for these central inhibitors and their neural circuits in the dorsal spinal cord, the knowledge about the endogenous suppressive mechanisms in nociceptive DRG neurons remains very limited. In this review, we summarize our recent findings of the presynaptic suppressive mechanisms in nociceptive neurons, the BNP/NPR-A/PKG/BKCa channel pathway, the FSTL1/α1Na+-K+ ATPase pathway and the activin C/ERK pathway. These endogenous suppressive systems in the mechanoheat nociceptors may also contribute differentially to the mechanisms of nerve injury-induced neuropathic pain or inflammation-induced pain.

Sex-Dependent Glial Signaling in Pathological Pain: Distinct Roles of Spinal Microglia and Astrocytes.

Increasing evidence suggests that spinal microglia regulate pathological pain in males. In this study, we investigated the effects of several microglial and astroglial modulators on inflammatory and neuropathic pain following intrathecal injection in male and female mice. These modulators were the microglial inhibitors minocycline and ZVEID (a caspase-6 inhibitor) and the astroglial inhibitors L-α-aminoadipate (L-AA, an astroglial toxin) and carbenoxolone (a connexin 43 inhibitor), as well as U0126 (an ERK kinase inhibitor) and D-JNKI-1 (a c-Jun N-terminal kinase inhibitor). We found that spinal administration of minocycline or ZVEID, or Caspase6 deletion, reduced formalin-induced inflammatory and nerve injury-induced neuropathic pain primarily in male mice. In contrast, intrathecal L-AA reduced neuropathic pain but not inflammatory pain in both sexes. Intrathecal U0126 and D-JNKI-1 reduced neuropathic pain in both sexes. Nerve injury caused spinal upregulation of the astroglial markers GFAP and Connexin 43 in both sexes. Collectively, our data confirmed male-dominant microglial signaling but also revealed sex-independent astroglial signaling in the spinal cord in inflammatory and neuropathic pain.

DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons

Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG.