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1.  G protein-gated inwardly rectifying potassium channel subunits 1 and 2 are down-regulated in rat dorsal root ganglion neurons and spinal cord after peripheral axotomy 
Molecular Pain  2015;11:44.
Increased nociceptive neuronal excitability underlies chronic pain conditions. Various ion channels, including sodium, calcium and potassium channels have pivotal roles in the control of neuronal excitability. The members of the family of G protein-gated inwardly rectifying potassium (GIRK) channels, GIRK1–4, have been implicated in modulating excitability. Here, we investigated the expression and distribution of GIRK1 and GIRK2 in normal and injured dorsal root ganglia (DRGs) and spinal cord of rats.
We found that ~70% of the DRG neurons expressed GIRK1, while only <10% expressed GIRK2. The neurochemical profiles of GIRK1- and GIRK2-immunoreactive neurons were characterized using the neuronal markers calcitonin gene-related peptide, isolectin-B4 and neurofilament-200, and the calcium-binding proteins calbindin D28k, calretinin, parvalbumin and secretagogin. Both GIRK subunits were expressed in DRG neurons with nociceptive characteristics. However, while GIRK1 was widely expressed in several sensory neuronal subtypes, GIRK2 was detected mainly in a group of small C-fiber neurons. In the spinal dorsal horn, GIRK1- and -2-positive cell bodies and processes were mainly observed in lamina II, but also in superficial and deeper layers. Abundant GIRK1-, but not GIRK2-like immunoreactivity, was found in the ventral horn (laminae VI–X). Fourteen days after axotomy, GIRK1 and GIRK2 were down-regulated in DRG neurons at the mRNA and protein levels. Both after axotomy and rhizotomy there was a reduction of GIRK1- and -2-positive processes in the dorsal horn, suggesting a presynaptic localization of these potassium channels. Furthermore, nerve ligation caused accumulation of both subunits on both sides of the lesion, providing evidence for anterograde and retrograde fast axonal transport.
Our data support the hypothesis that reduced GIRK function is associated with increased neuronal excitability and causes sensory disturbances in post-injury conditions, including neuropathic pain.
Electronic supplementary material
The online version of this article (doi:10.1186/s12990-015-0044-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4511542  PMID: 26199148
Axonal transport; Ca2+-binding proteins; CGRP; Galanin; GIRK channel; Nerve injury; Neuropathic pain; Neuropeptides; NPY; Nociceptor; Somatostatin
2.  Identification of lncRNA expression profile in the spinal cord of mice following spinal nerve ligation-induced neuropathic pain 
Molecular Pain  2015;11:43.
Neuropathic pain that caused by lesion or dysfunction of the nervous system is associated with gene expression changes in the sensory pathway. Long noncoding RNAs (lncRNAs) have been reported to be able to regulate gene expression. Identifying lncRNA expression patterns in the spinal cord under normal and neuropathic pain conditions is essential for understanding the genetic mechanisms behind the pathogenesis of neuropathic pain.
Spinal nerve ligation (SNL) induced rapid and persistent pain hypersensitivity, characterized by mechanical allodynia and heat hyperalgesia. Meanwhile, astrocytes and microglia were dramatically activated in the ipsilateral spinal cord dorsal horn at 10 days after SNL. Further lncRNA microarray and mRNA microarray analysis showed that the expression profiles of lncRNA and mRNA between SNL and sham-operated mice were greatly changed at 10 days. The 511 differentially expressed (>2 fold) lncRNAs (366 up-regulated, 145 down-regulated) and 493 mRNAs (363 up-regulated, 122 down-regulated) were finally identified. The expression patterns of several lncRNAs and mRNAs were further confirmed by qPCR. Functional analysis of differentially expressed (DE) mRNAs showed that the most significant enriched biological processes of up-regulated genes in SNL include immune response, defense response, and inflammation response, which are important pathogenic mechanisms underlying neuropathic pain. 35 DE lncRNAs have neighboring or overlapping DE mRNAs in genome, which is related to Toll-like receptor signaling, cytokine–cytokine receptor interaction, and peroxisome proliferator-activated receptor signaling pathway.
Our findings uncovered the expression pattern of lncRNAs and mRNAs in the mice spinal cord under neuropathic pain condition. These lncRNAs and mRNAs may represent new therapeutic targets for the treatment of neuropathic pain.
PMCID: PMC4504460  PMID: 26184882
LncRNA; Spinal cord; Spinal nerve ligation; Neuropathic pain
3.  Distribution of raphespinal fibers in the mouse spinal cord 
Molecular Pain  2015;11:42.
Serotonergic raphespinal neurons and their fibers have been mapped in large mammals, but the non-serotonergic ones have not been studied, especially in the mouse. The present study aimed to investigate the termination pattern of fibers arising from the hindbrain raphe and reticular nuclei which also have serotonergic neurons by injecting the anterograde tracer BDA into them.
We found that raphespinal fibers terminate in both the dorsal and ventral horns in addition to lamina 10. There is a shift of the fibers in the ventral horn towards the dorsal and lateral part of the gray matter. Considerable variation in the termination pattern also exists between raphe nuclei with raphe magnus having more fibers terminating in the dorsal horn. Fibers from the adjacent gigantocellular reticular nucleus show similar termination pattern as those from the raphe nuclei with slight difference. Immunofluorescence staining showed that raphespinal fibers were heterogeneous and serotoninergic fibers were present in all laminae but mainly in laminae 1, 2, medial lamina 8, laminae 9 and 10. Surprisingly, immunofluorescence staining on clarified spinal cord tissue revealed that serotoninergic fibers formed bundles regularly in a short distance along the rostrocaudal axis in the medial part of the ventral horn and they extended towards the lateral motor neuron column area.
Serotonergic and non-serotonergic fibers arising from the hindbrain raphe and reticular nuclei had similar termination pattern in the mouse spinal cord with subtle difference. The present study provides anatomical foundation for the multiple roles raphe and adjacent reticular nuclei play.
Electronic supplementary material
The online version of this article (doi:10.1186/s12990-015-0046-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4502924  PMID: 26173454
Hindbrain; Raphe nuclei; Reticular nuclei; Serotonin; Spinal cord; Raphespinal tract; CLARITY; Anterograde tracing
4.  Role of anoctamin-1 and bestrophin-1 in spinal nerve ligation-induced neuropathic pain in rats 
Molecular Pain  2015;11:41.
Calcium-activated chloride channels (CaCCs) activation induces membrane depolarization by increasing chloride efflux in primary sensory neurons that can facilitate action potential generation. Previous studies suggest that CaCCs family members bestrophin-1 and anoctamin-1 are involved in inflammatory pain. However, their role in neuropathic pain is unclear. In this investigation we assessed the involvement of these CaCCs family members in rats subjected to the L5/L6 spinal nerve ligation. In addition, anoctamin-1 and bestrophin-1 mRNA and protein expression in dorsal root ganglion (DRG) and spinal cord was also determined in the presence and absence of selective inhibitors.
L5/L6 spinal nerve ligation induced mechanical tactile allodynia. Intrathecal administration of non-selective CaCCs inhibitors (NPPB, 9-AC and NFA) dose-dependently reduced tactile allodynia. Intrathecal administration of selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) also dose-dependently diminished tactile allodynia and thermal hyperalgesia. Anoctamin-1 and bestrophin-1 mRNA and protein were expressed in the dorsal spinal cord and DRG of naïve, sham and neuropathic rats. L5/L6 spinal nerve ligation rose mRNA and protein expression of anoctamin-1, but not bestrophin-1, in the dorsal spinal cord and DRG from day 1 to day 14 after nerve ligation. In addition, repeated administration of CaCCs inhibitors (T16Ainh-A01, CaCCinh-A01 or NFA) or anti-anoctamin-1 antibody prevented spinal nerve ligation-induced rises in anoctamin-1 mRNA and protein expression. Following spinal nerve ligation, the compound action potential generation of putative C fibers increased while selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) attenuated such increase.
There is functional anoctamin-1 and bestrophin-1 expression in rats at sites related to nociceptive processing. Blockade of these CaCCs suppresses compound action potential generation in putative C fibers and lessens established tactile allodynia. As CaCCs activity contributes to neuropathic pain maintenance, selective inhibition of their activity may function as a tool to generate analgesia in nerve injury pain states.
Electronic supplementary material
The online version of this article (doi:10.1186/s12990-015-0042-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4487556  PMID: 26130088
Allodynia; Anoctamin-1; Bestrophin-1; Calcium-activated chloride channels; Neuropathic pain; Spinal nerve ligation
5.  Temporal dynamics of anxiety phenotypes in a dental pulp injury model 
Molecular Pain  2015;11:40.
Accumulating clinical and preclinical evidence indicates that chronic pain is often comorbid with persistent low mood and anxiety. However, the mechanisms underlying pain-induced anxiety, such as its causality, temporal progression, and relevant neural networks are poorly understood, impeding the development of efficacious therapeutic approaches.
Here, we have identified the sequential emergence of anxiety phenotypes in mice subjected to dental pulp injury (DPI), a prototypical model of orofacial pain that correlates with human toothache. Compared with sham controls, mice subjected to DPI by mechanically exposing the pulp to the oral environment exhibited significant signs of anxiogenic effects, specifically, altered behaviors on the elevated plus maze (EPM), novelty-suppressed feeding (NSF) tests at 1 but not 3 days after the surgery. Notably, at 7 and 14 days, the DPI mice again avoided the open arm, center area, and novelty environment in the EPM, open field, and NSF tests, respectively. In particular, DPI-induced social phobia and increased repetitive grooming did not occur until 14 days after surgery, suggesting that DPI-induced social anxiety requires a long time. Moreover, oral administration of an anti-inflammatory drug, ibuprofen, or an analgesic agent, ProTx-II, which is a selective inhibitor of NaV1.7 sodium channels, both significantly alleviated DPI-induced avoidance in mice. Finally, to investigate the underlying central mechanisms, we pharmacologically blocked a popular form of synaptic plasticity with a GluA2-derived peptide, long-term depression, as that treatment significantly prevented the development of anxiety phenotype upon DPI.
Together, these results suggest a temporally progressive causal relationship between orofacial pain and anxiety, calling for more in-depth mechanistic studies on concomitant pain and anxiety disorders.
PMCID: PMC4487070  PMID: 26122003
Dental pulp injury; Pain; Anxiety; Social phobia; Synaptic plasticity
6.  Function and postnatal changes of dural afferent fibers expressing TRPM8 channels 
Molecular Pain  2015;11:37.
Genome-wide association studies have identified TRPM8 (transient receptor potential melastatin 8) as one of the susceptibility genes for common migraine. Here, we investigated the postnatal changes of TRPM8-expressing dural afferent fibers as well as the function of dural TRPM8 channels in mice.
First, we quantified the density and the number of axonal branches of TRPM8-expressing fibers in the dura of mice expressing farnesylated enhanced green fluorescent protein (EGFPf) from one TRPM8 allele between postnatal day 2 (P2) to adulthood. The number of axonal branches on individual dural EGFP-positive fibers was decreased by 30% between P2 and P11. The density of dural EGFP-positive fibers was subsequently reduced by 50% between P16 and P21. Conversely, the density and the number of branches of axons expressing calcitonin gene-related peptide remained stable in postnatal mouse dura. The density of TRPM8-expressing fibers innervating the mouse cornea epithelium was significantly increased from P2 to adulthood. Next, we tested the function of dural TRPM8 channels in adult mice and found that TRPM8 agonist menthol effectively inhibited the nocifensive behavior evoked by dural application of inflammatory mediators.
Our results indicate that the TRPM8-expressing dural afferent fibers undergo cell- and target tissue-specific axonal pruning during postnatal development. Activation of dural TRPM8 channels decreases meningeal irritation-evoked nocifensive behavior in adult mice. This provides a framework to further explore the role of postnatal changes of TRPM8-expressing dural afferents in the pathophysiology of pediatric and adult migraine.
Electronic supplementary material
The online version of this article (doi:10.1186/s12990-015-0043-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4480580  PMID: 26111800
Migraine; Headache; TRPM8; CGRP; Dural afferent fibers
7.  Expression changes of microRNA-1 and its targets Connexin 43 and brain-derived neurotrophic factor in the peripheral nervous system of chronic neuropathic rats 
Molecular Pain  2015;11:39.
MicroRNAs (miRNAs) are involved in the neuroplastic changes which induce and maintain neuropathic pain. However, it is unknown whether nerve injury leads to altered miRNA expression and modulation of pain relevant target gene expression within peripheral nerves. In the present study, expression profiles of miR-1 and the pain-relevant targets, brain derived neurotrophic factor (BDNF) and Connexin 43 (Cx43), were studied in peripheral neuropathic pain, which was induced by chronic constriction injury (CCI) of the sciatic nerve in rats. The expression of miR-1 was investigated in the sciatic nerve, dorsal root ganglion (DRG) and the ipsilateral spinal cord by qPCR. Changes of BDNF and Cx43 expression patterns were studied using qPCR, Western blot analysis, ELISA and immunohistochemistry.
In sciatic nerves of naïve rats, expression levels of miR-1 were more than twice as high as in DRG and spinal cord. In neuropathic rats, CCI lead to a time-dependent downregulation of miR-1 in the sciatic nerve but not in DRG and spinal cord. Likewise, protein expression of the miR-1 targets BDNF and Cx43 was upregulated in the sciatic nerve and DRG after CCI. Immunohistochemical staining revealed an endoneural abundancy of Cx43 in injured sciatic nerves which was absent after Sham operation.
This study demonstrates that CCI leads to a regulation of miRNAs (miR-1) in the peripheral nervous system. This regulation is associated with alterations in the expression and localization of the miR-1 dependent pain-relevant proteins BDNF and Cx43. Further studies will have to explore the function of miRNAs in the context of neuropathic pain in the peripheral nervous system.
PMCID: PMC4482165  PMID: 26111928
microRNA; miR-1; Connexin 43 (Cx43); BDNF; Neuropathic pain; Chronic constriction injury (CCI)
8.  Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT2R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies 
Molecular Pain  2015;11:38.
The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherally-restricted analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging.
AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine.
The major AT2R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.
PMCID: PMC4482278  PMID: 26111701
Angiotensin II; AngIII; Ang-(1-7); AT2R; Antagonist; EMA401; Human DRG neurons; Peripheral nerve injury; Calcium imaging; Hypersensitivity; Neuropathic pain; Neurites
9.  Spinal cord stimulation modulates supraspinal centers of the descending antinociceptive system in rats with unilateral spinal nerve injury 
Molecular Pain  2015;11:36.
The descending antinociceptive system (DAS) is thought to play crucial roles in the antinociceptive effect of spinal cord stimulation (SCS), especially through its serotonergic pathway. The nucleus raphe magnus (NRM) in the rostral ventromedial medulla is a major source of serotonin [5-hydroxytryptamine (5-HT)] to the DAS, but the role of the dorsal raphe nucleus (DRN) in the ventral periaqueductal gray matter is still unclear. Moreover, the influence of the noradrenergic pathway is largely unknown. In this study, we evaluated the involvement of these serotonergic and noradrenergic pathways in SCS-induced antinociception by behavioral analysis of spinal nerve-ligated (SNL) rats. We also investigated immunohistochemical changes in the DRN and locus coeruleus (LC), regarded as the adrenergic center of the DAS, and expression changes of synthetic enzymes of 5-HT [tryptophan hydroxylase (TPH)] and norepinephrine [dopamine β-hydroxylase (DβH)] in the spinal dorsal horn.
Intrathecally administered methysergide, a 5-HT1- and 5-HT2-receptor antagonist, and idazoxan, an α2-adrenergic receptor antagonist, equally abolished the antinociceptive effect of SCS. The numbers of TPH-positive serotonergic and phosphorylated cyclic AMP response element binding protein (pCREB)-positive neurons and percentage of pCREB-positive serotonergic neurons in the DRN significantly increased after 3-h SCS. Further, the ipsilateral-to-contralateral immunoreactivity ratio of DβH increased in the LC of SNL rats and reached the level seen in naïve rats, even though the number of pCREB-positive neurons in the LC was unchanged by SNL and SCS. Moreover, 3-h SCS did not increase the expression levels of TPH and DβH in the spinal dorsal horn.
The serotonergic and noradrenergic pathways of the DAS are involved in the antinociceptive effect of SCS, but activation of the DRN might primarily be responsible for this effect, and the LC may have a smaller contribution. SCS does not potentiate the synthetic enzymes of 5HT and norepinephrine in the neuropathic spinal cord.
PMCID: PMC4479321  PMID: 26104415
Descending antinociceptive system; Dorsal raphe nucleus; Locus coeruleus; Neuropathic pain; Noradrenergic pathway; Serotonergic pathway; Spinal cord; Spinal cord stimulation
10.  PI3K mediated activation of GSK-3β reduces at-level primary afferent growth responses associated with excitotoxic spinal cord injury dysesthesias 
Molecular Pain  2015;11:35.
Neuropathic pain and sensory abnormalities are a debilitating secondary consequence of spinal cord injury (SCI). Maladaptive structural plasticity is gaining recognition for its role in contributing to the development of post SCI pain syndromes. We previously demonstrated that excitotoxic induced SCI dysesthesias are associated with enhanced dorsal root ganglia (DRG) neuronal outgrowth. Although glycogen synthase kinase-3β (GSK-3β) is a known intracellular regulator neuronal growth, the potential contribution to primary afferent growth responses following SCI are undefined. We hypothesized that SCI triggers inhibition of GSK-3β signaling resulting in enhanced DRG growth responses, and that PI3K mediated activation of GSK-3β can prevent this growth and the development of at-level pain syndromes.
Excitotoxic SCI using intraspinal quisqualic acid (QUIS) resulted in inhibition of GSK-3β in the superficial spinal cord dorsal horn and adjacent DRG. Double immunofluorescent staining showed that GSK-3βP was expressed in DRG neurons, especially small nociceptive, CGRP and IB4-positive neurons. Intrathecal administration of a potent PI3-kinase inhibitor (LY294002), a known GSK-3β activator, significantly decreased GSK-3βP expression levels in the dorsal horn. QUIS injection resulted in early (3 days) and sustained (14 days) DRG neurite outgrowth of small and subsequently large fibers that was reduced with short term (3 days) administration of LY294002. Furthermore, LY294002 treatment initiated on the date of injury, prevented the development of overgrooming, a spontaneous at-level pain related dysesthesia.
QUIS induced SCI resulted in inhibition of GSK-3β in primary afferents and enhanced at-level DRG intrinsic growth (neurite elongation and initiation). Early PI3K mediated activation of GSK-3β attenuated QUIS-induced DRG neurite outgrowth and prevented the development of at-level dysesthesias.
PMCID: PMC4475622  PMID: 26093674
GSK-3β; Spinal cord injury (SCI); Pain; Dysesthesias; Dorsal root ganglia (DRG); Neurite outgrowth
11.  Tolerance to the antinociceptive effects of chronic morphine requires c-Jun N-terminal kinase 
Molecular Pain  2015;11:34.
Morphine and fentanyl are opioid analgesics in wide clinical use that act through the μ-opioid receptor (MOR). However, one limitation of their long-term effectiveness is the development of tolerance. Receptor desensitization has been proposed as a putative mechanism driving tolerance to G protein-coupled receptor (GPCR) agonists. Recent studies have found that tolerance to morphine is mediated by the c-Jun N-terminal Kinase (JNK) signaling pathway. The goal of the present study was to test the hypotheses that: 1) JNK inhibition will be antinociceptive on its own; 2) JNK inhibition will augment morphine antinociception and; 3) JNK mediates chronic tolerance for the antinociceptive effects of morphine using acute (hotplate and tail-flick), inflammatory (10 μl of formalin 2.5 %) and chemotherapy (cisplatin 5 mg/kg ip once weekly)-induced neuropathic pain assays.
We found that JNK inhibition by SP600125 (3 mg/kg) produces a greater antinociceptive effect than morphine (6 mg/kg) alone in the formalin test. Moreover, co-administration of morphine (6 mg/kg) with SP600125 (3 mg/kg) produced a sub-additive antinociceptive effect in the formalin test. We also show that pre-treatment with SP600125 (3 or 10 mg/kg), attenuates tolerance to the antinociceptive effects of morphine (10 mg/kg), but not fentanyl (0.3 mg/kg), in the tail-flick and hotplate tests. Pre-treatment with SP600125 also attenuates tolerance to the hypothermic effects of both morphine and fentanyl. We also examined the role of JNK in morphine tolerance in a cisplatin-induced model of neuropathic pain. Interestingly, treatment with SP600125 (3 mg/kg) alone attenuated mechanical and cold allodynia in a chemotherapy-induced pain model using cisplatin. Strikingly, SP600125 (3 mg/kg) pre-treatment prolonged the anti-allodynic effect of morphine by several days (5 and 7 days for mechanical and cold, respectively).
These results demonstrate that JNK signaling plays a crucial role in mediating antinociception as well as chronic tolerance to the antinociceptive effects of morphine in acute, inflammatory, and neuropathic pain states. Thus, inhibition of JNK signaling pathway, via SP600125, represents an efficacious pharmacological approach to delay tolerance to the antinociceptive effects of chronic morphine in diverse pain models.
PMCID: PMC4465461  PMID: 26065412
Morphine; Fentanyl; JNK; GPCR; GRK; Cisplatin; Desensitization; Tolerance; Mu opioid receptor; Chemotherapy
12.  The opioid peptide dynorphin A induces leukocyte responses via integrin Mac-1 (αMβ2, CD11b/CD18) 
Molecular Pain  2015;11:33.
Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (αMβ2, CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.
In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages.
Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12990-015-0027-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4481117  PMID: 26036990
Dynorphin A; Integrin Mac-1 (CD11b/CD18); Leukocytes; Opioid peptides; Adhesion molecules
13.  mTOR, a new potential target for chronic pain and opioid-induced tolerance and hyperalgesia 
Molecular Pain  2015;11:32.
Chronic pain is a major public health problem with limited treatment options. Opioids remain a routine treatment for chronic pain, but extended exposure to opioid therapy can produce opioid tolerance and hyperalgesia. Although the mechanisms underlying chronic pain, opioid-induced tolerance, and opioid-induced hyperalgesia remain to be uncovered, mammalian target of rapamycin (mTOR) is involved in these disorders. The mTOR complex 1 and its triggered protein translation are required for the initiation and maintenance of chronic pain (including cancer pain) and opioid-induced tolerance/hyperalgesia. Given that mTOR inhibitors are FDA-approved drugs and an mTOR inhibitor is approved for the treatment of several cancers, these findings suggest that mTOR inhibitors will likely have multiple clinical benefits, including anticancer, antinociception/anti-cancer pain, and antitolerance/hyperalgesia. This paper compares the role of mTOR complex 1 in chronic pain, opioid-induced tolerance, and opioid-induced hyperalgesia.
PMCID: PMC4455918  PMID: 26024835
Pain; NMTOR; Opioid; Hyperalgesia; Tolerance
14.  Novel expression pattern of neuropeptide Y immunoreactivity in the peripheral nervous system in a rat model of neuropathic pain 
Molecular Pain  2015;11:31.
Neuropeptide Y (NPY) has been implicated in the modulation of pain. Under normal conditions, NPY is found in interneurons in the dorsal horn of the spinal cord and in sympathetic postganglionic neurons but is absent from the cell bodies of sensory neurons. Following peripheral nerve injury NPY is dramatically upregulated in the sensory ganglia. How NPY expression is altered in the peripheral nervous system, distal to a site of nerve lesion, remains unknown. To address this question, NPY expression was investigated using immunohistochemistry at the level of the trigeminal ganglion, the mental nerve and in the skin of the lower lip in relation to markers of sensory and sympathetic fibers in a rat model of trigeminal neuropathic pain.
At 2 and 6 weeks after chronic constriction injury (CCI) of the mental nerve, de novo expression of NPY was seen in the trigeminal ganglia, in axons in the mental nerve, and in fibers in the upper dermis of the skin. In lesioned animals, NPY immunoreactivity was expressed primarily by large diameter mental nerve sensory neurons retrogradely labelled with Fluorogold. Many axons transported this de novo NPY to the periphery as NPY-immunoreactive (IR) fibers were seen in the mental nerve both proximal and distal to the CCI. Some of these NPY-IR axons co-expressed Neurofilament 200 (NF200), a marker for myelinated sensory fibers, and occasionally colocalization was seen in their terminals in the skin. Peptidergic and non-peptidergic C fibers expressing calcitonin gene-related peptide (CGRP) or binding isolectin B4 (IB4), respectively, never expressed NPY. CCI caused a significant de novo sprouting of sympathetic fibers into the upper dermis of the skin, and most, but not all of these fibers, expressed NPY.
This is the first study to provide a comprehensive description of changes in NPY expression in the periphery after nerve injury. Novel expression of NPY in the skin comes mostly from sprouted sympathetic fibers. This information is fundamental in order to understand where endogenous NPY is expressed, and how it might be acting to modulate pain in the periphery.
PMCID: PMC4449610  PMID: 26012590
Neuropeptide tyrosine; NPY; Innervation; NF200; CGRP; IB4; Sympathetic nervous system; Skin; Mental nerve; Trigeminal; Pain
15.  Released lipids regulate Transient Receptor Potential Channel (TRP)-dependent oral cancer pain 
Molecular Pain  2015;11:30.
Pain in the head neck area is an early symptom in oral cancer, supporting the hypothesis that cancer cells control the activities of surrounding nociceptors at the site of the tumor. Several reports implicate TRPV1 and TRPA1 in cancer pain, although there is a large gap in knowledge since the mechanisms for tumor-induced activation of these TRP receptors are unknown. Interestingly, TRP-active lipids such as linoleic acid, arachidonic acid, hydroxyoctadecadienoic acid and hydroxyeicosatetraenoic acid are significantly elevated in the saliva of oral cancer patients compared to normal patients, supporting a possible linkage between these lipids and oral cancer pain. We therefore hypothesize that oral squamous cell carcinomas release certain lipids that activate TRPV1 and/or TRPA1 on sensory neurons, contributing to the development of oral cancer pain.
Lipid extracts were made from conditioned media of three human oral squamous cell carcinoma (OSCC) cell lines as well as one normal human oral keratinocytes cell line. These were then injected intraplantarly into rat hindpaws to measure spontaneous nocifensive behavior, as well as thermal and mechanical allodynia. For interventional experiments, the animals were pretreated with AMG517 (TRPV1 antagonist) or HC030031 (TRPA1 antagonist) prior to extract injection.
These studies demonstrate that lipids released from the three OSCC cell lines, but not the normal cell line, were capable of producing significant spontaneous nocifensive behaviors, as well as thermal and mechanical allodynia. Notably each of the cell lines produced a different magnitude of response for each of three behavioral assays. Importantly, pre-treatment with a TRPVI antagonist blocked lipid-mediated nocifensive and thermal hypersensitivity, but not mechanical hypersensitivity. In addition, pre-treatment with a TRPA1 antagonist only reversed thermal hypersensitivity without affecting lipid-induced nocifensive behavior or mechanical allodynia.
These data reveal a novel mechanism for cancer pain and provide strong direction for future studies evaluating the cellular mechanism regulating the TRP-active lipids by OSCC tumors.
PMCID: PMC4456056  PMID: 26007300
Lipids; TRPV1; TRPA1; Oral squamous cell carcinoma; Cancer pain
16.  Upregulation of T-type Ca2+ channels in long-term diabetes determines increased excitability of a specific type of capsaicin-insensitive DRG neurons 
Molecular Pain  2015;11:29.
Previous studies have shown that increased excitability of capsaicin-sensitive DRG neurons and thermal hyperalgesia in rats with short-term (2–4 weeks) streptozotocin-induced diabetes is mediated by upregulation of T-type Ca2+ current. In longer–term diabetes (after the 8th week) thermal hyperalgesia is changed to hypoalgesia that is accompanied by downregulation of T-type current in capsaicin-sensitive small-sized nociceptors. At the same time pain symptoms of diabetic neuropathy other than thermal persist in STZ-diabetic animals and patients during progression of diabetes into later stages suggesting that other types of DRG neurons may be sensitized and contribute to pain. In this study, we examined functional expression of T-type Ca2+ channels in capsaicin-insensitive DRG neurons and excitability of these neurons in longer-term diabetic rats and in thermally hypoalgesic diabetic rats.
Here we have demonstrated that in STZ-diabetes T-type current was upregulated in capsaicin-insensitive low-pH-sensitive small-sized nociceptive DRG neurons of longer-term diabetic rats and thermally hypoalgesic diabetic rats. This upregulation was not accompanied by significant changes in biophysical properties of T-type channels suggesting that a density of functionally active channels was increased. Sensitivity of T-type current to amiloride (1 mM) and low concentration of Ni2+ (50 μM) implicates prevalence of Cav3.2 subtype of T-type channels in the capsaicin-insensitive low-pH-sensitive neurons of both naïve and diabetic rats. The upregulation of T-type channels resulted in the increased neuronal excitability of these nociceptive neurons revealed by a lower threshold for action potential initiation, prominent afterdepolarizing potentials and burst firing. Sodium current was not significantly changed in these neurons during long-term diabetes and could not contribute to the diabetes-induced increase of neuronal excitability.
Capsaicin-insensitive low-pH-sensitive type of DRG neurons shows diabetes-induced upregulation of Cav3.2 subtype of T-type channels. This upregulation results in the increased excitability of these neurons and may contribute to nonthermal nociception at a later-stage diabetes.
PMCID: PMC4490764  PMID: 25986602
Diabetes; Pain; T-type calcium channels; DRG neurons; Excitability; Capsaicin-insensitive
17.  Pain perception in acute model mice of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 
Molecular Pain  2015;11:28.
Pain is the most prominent non-motor symptom observed in patients with Parkinson’s disease (PD). However, the mechanisms underlying the generation of pain in PD have not been well studied. We used a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD to analyze the relationship between pain sensory abnormalities and the degeneration of dopaminergic neurons.
The latency to fall off the rotarod and the total distance traveled in round chamber were significantly reduced in MPTP-induced PD mice, consistent with motor dysfunction. MPTP-treated mice also showed remarkably shorter nociceptive response latencies compared to saline-treated mice and the subcutaneous injection of L-3,4-dihydroxyphenylalanine (L-DOPA) partially reversed pain hypersensitivity induced by MPTP treatment. We found that degeneration of cell bodies and fibers in the substantia nigra pars compacta and the striatum of MPTP-treated mice. In addition, astrocytic and microglial activation was seen in the subthalamic nucleus and neuronal activity was significantly increased in the striatum and globus pallidus. However, we did not observe any changes in neurons, astrocytes, and microglia of both the dorsal and ventral horns in the spinal cord after MPTP treatment.
These results suggest that the dopaminergic nigrostriatal pathway may have a role in inhibiting noxious stimuli, and that abnormal inflammatory responses and neural activity in basal ganglia is correlated to pain processing in PD induced by MPTP treatment.
PMCID: PMC4448854  PMID: 25981600
Dopaminergic pathway; Subthalamic nucleus; Inflammation; Astrogliosis
18.  Current gene therapy using viral vectors for chronic pain 
Molecular Pain  2015;11:27.
The complexity of chronic pain and the challenges of pharmacotherapy highlight the importance of development of new approaches to pain management. Gene therapy approaches may be complementary to pharmacotherapy for several advantages. Gene therapy strategies may target specific chronic pain mechanisms in a tissue-specific manner. The present collection of articles features distinct gene therapy approaches targeting specific mechanisms identified as important in the specific pain conditions. Dr. Fairbanks group describes commonly used gene therapeutics (herpes simplex viral vector (HSV) and adeno-associated viral vector (AAV)), and addresses biodistribution and potential neurotoxicity in pre-clinical models of vector delivery. Dr. Tao group addresses that downregulation of a voltage-gated potassium channel (Kv1.2) contributes to the maintenance of neuropathic pain. Alleviation of chronic pain through restoring Kv1.2 expression in sensory neurons is presented in this review. Drs Goins and Kinchington group describes a strategy to use the replication defective HSV vector to deliver two different gene products (enkephalin and TNF soluble receptor) for the treatment of post-herpetic neuralgia. Dr. Hao group addresses the observation that the pro-inflammatory cytokines are an important shared mechanism underlying both neuropathic pain and the development of opioid analgesic tolerance and withdrawal. The use of gene therapy strategies to enhance expression of the anti-pro-inflammatory cytokines is summarized. Development of multiple gene therapy strategies may have the benefit of targeting specific pathologies associated with distinct chronic pain conditions (by Guest Editors, Drs. C. Fairbanks and S. Hao).
PMCID: PMC4446851  PMID: 25962909
Pain; Viral vectors; Gene therapy
19.  Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin 
Molecular Pain  2015;11:26.
The skin is a morphologically complex organ that serves multiple complementary functions, including an important role in thermoregulation, which is mediated by a rich vasculature that is innervated by sympathetic and sensory endings. Two autosomal dominant disorders characterized by episodes of severe pain, inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD) have been directly linked to mutations that enhance the function of sodium channel Nav1.7. Pain attacks are accompanied by reddening of the skin in both disorders. Nav1.7 is known to be expressed at relatively high levels within both dorsal root ganglion (DRG) and sympathetic ganglion neurons, and mutations that enhance the activity of Nav1.7 have been shown to have profound effects on the excitability of both cell-types, suggesting that dysfunction of sympathetic and/or sensory fibers, which release vasoactive peptides at skin vasculature, may contribute to skin reddening in IEM and PEPD.
In the present study, we demonstrate that smooth muscle cells of cutaneous arterioles and arteriole-venule shunts (AVS) in the skin express sodium channel Nav1.7. Moreover, Nav1.7 is expressed by endothelial cells lining the arterioles and AVS and by sensory and sympathetic fibers innervating these vascular elements.
These observations suggest that the activity of mutant Nav1.7 channels in smooth muscle cells of skin vasculature and innervating sensory and sympathetic fibers contribute to the skin reddening and/or pain in IEM and PEPD.
PMCID: PMC4447014  PMID: 25957174
Arteriole-venule shunt; Cutaneous arterioles; Dermis; Smooth muscle cells; Sodium channels; Vascular myocytes
20.  Polysulfide evokes acute pain through the activation of nociceptive TRPA1 in mouse sensory neurons 
Molecular Pain  2015;11:24.
Hydrogen sulfide (H2S) is oxidized to polysulfide. Recent reports show that this sulfur compound modulates various biological functions. We have reported that H2S is involved in inflammatory pain in mice. On the other hand, little is known about the functional role of polysulfide in sensory neurons. Here we show that polysulfide selectively stimulates nociceptive TRPA1 and evokes acute pain, using TRPA1-gene deficient mice (TRPA1(−/−)), a heterologous expression system and a TRPA1-expressing cell line.
In wild-type mouse sensory neurons, polysulfide elevated the intracellular Ca concentration ([Ca2+]i) in a dose-dependent manner. The half maximal effective concentration (EC50) of polysulfide was less than one-tenth that of H2S. The [Ca2+]i responses to polysulfide were observed in neurons responsive to TRPA1 agonist and were inhibited by blockers of TRPA1 but not of TRPV1. Polysulfide failed to evoke [Ca2+]i increases in neurons from TRPA1(−/−) mice. In RIN-14B cells, constitutively expressing rat TRPA1, polysulfide evoked [Ca2+]i increases with the same EC50 value as in sensory neurons. Heterologously expressed mouse TRPA1 was activated by polysulfide and that was suppressed by dithiothreitol. Analyses of the TRPA1 mutant channel revealed that cysteine residues located in the internal domain were related to the sensitivity to polysulfide. Intraplantar injection of polysulfide into the mouse hind paw induced acute pain and edema which were significantly less than in TRPA1(−/−) mice.
The present data suggest that polysulfide functions as pronociceptive substance through the activation of TRPA1 in sensory neurons. Since the potency of polysulfide is higher than parental H2S and this sulfur compound is generated under pathophysiological conditions, it is suggested that polysulfide acts as endogenous ligand for TRPA1. Therefore, TRPA1 may be a promising therapeutic target for endogenous sulfur compound-related algesic action.
PMCID: PMC4428232  PMID: 25934637
Transient Receptor Potential Channels (TRP Channels); Calcium imaging; Dorsal root ganglia; Heterologous expression
21.  Minocycline does not affect long-term potentiation in the anterior cingulate cortex of normal adult mice 
Molecular Pain  2015;11:25.
It has been reported that activated microglia plays important roles in chronic pain-related sensory signaling at the spinal cord dorsal horn. Less is known about the possible contribution of microglia to cortical plasticity that has been found to be important for chronic pain. In the present study, we used a 64-channel multi-electrode array recording system to investigate the role of microglia in cortical plasticity of the anterior cingulate cortex (ACC) in normal adult mice. We found that bath application of minocycline, an inhibitor of microglial activation, had no effect on postsynaptic LTP (post-LTP) induced by theta burst stimulation in the ACC. Furthermore, presynaptic LTP (pre-LTP) induced by the combination of low-frequency stimulation with a GluK1-containing kainate receptor agonist was also not affected. The spatial distribution of post-LTP or pre-LTP among the cingulate network is also unaltered by minocycline. Our results suggest that minocycline does not affect cingulate plasticity and neurons are the major player in pain-related cortical plasticity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12990-015-0025-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4464617  PMID: 25933605
Minocycline; Multi-electrode array; Presynaptic long-term potentiation; Postsynaptic long-term potentiation; Anterior cingulate cortex
22.  In situ patch-clamp recordings from Merkel cells in rat whisker hair follicles, an experimental protocol for studying tactile transduction in tactile-end organs 
Molecular Pain  2015;11:23.
Mammals use tactile end-organs to perform sensory tasks such as environmental exploration, social interaction, and tactile discrimination. However, cellular and molecular mechanisms underlying tactile transduction in tactile end-organs remain poorly understood. The patch-clamp recording technique may be the most valuable approach for detecting and studying tactile transduction in tactile end-organs, but it is technically challenging because tactile transduction elements in an end-organ are normally inaccessible by patch-clamp recording electrodes. Here we describe an in situ patch-clamp recording protocol for the study of tactile transduction in Merkel cells of rat whisker hair follicles, one of the most sensitive tactile end-organs in mammals. This technique offers an opportunity to explore the identities and properties of ion channels that are involved in tactile transduction in whisker hair follicles, and it may also lend a useful tool for researchers to study other tactile end-organs. The experimental protocol describes procedures for 1) tissue dissection and whisker hair follicle preparation, 2) device setup and steps for performing patch-clamp recordings from Merkel cells in a whisker hair follicle, 3) methods of delivering mechanical stimuli, and 4) intra-follicle microinjection for receptor knockdown in whisker hair follicles. The main procedures in this protocol, from tissue preparation to whole-cell patch-clamp recordings, can be completed in a few hours.
PMCID: PMC4437660  PMID: 25907165
Piezo2 channel; Touch; Mechanotransduction; Tactile discrimination; Merkel cells; Whisker hair follicles
23.  Experimental evidence for alleviating nociceptive hypersensitivity by single application of capsaicin 
Molecular Pain  2015;11:22.
The single application of high-concentration of capsaicin has been used as an analgesic therapy of persistent pain. However, its effectiveness and underlying mechanisms remain to be further evaluated with experimental approaches. The present study provided evidence showing that the single application of capsaicin dose-dependently alleviated nociceptive hypersensitivity, and reduced the action potential firing in small-diameter neurons of the dorsal root ganglia (DRG) in rats and mice. Pre-treatment with capsaicin reduced formalin-induced acute nocifensive behavior after a brief hyperalgesia in rats and mice. The inhibitory effects of capsaicin were calcium-dependent, and mediated by the capsaicin receptor (transient receptor potential vanilloid type-1). We further found that capsaicin exerted inhibitory effects on the persistent nociceptive hypersensitivity induced by peripheral inflammation and nerve injury. Thus, these results support the long-lasting and inhibitory effects of topical capsaicin on persistent pain, and the clinic use of capsaicin as a pain therapy.
PMCID: PMC4422461  PMID: 25896608
24.  Chronic dry eye symptoms after LASIK: parallels and lessons to be learned from other persistent post-operative pain disorders 
Molecular Pain  2015;11:21.
Laser in-situ keratomileusis (LASIK) is a commonly performed surgical procedure used to correct refractive error. LASIK surgery involves cutting a corneal flap and ablating the stroma underneath, with known damage to corneal nerves. Despite this, the epidemiology of persistent pain and other long-term outcomes after LASIK surgery are not well understood. Available data suggest that approximately 20-55% of patients report persistent eye symptoms (generally regarded as at least 6 months post-operation) after LASIK surgery. While it was initially believed that these symptoms were caused by ocular surface dryness, and referred to as “dry eye,” it is now increasingly understood that corneal nerve damage produced by LASIK surgery resembles the pathologic neuroplasticity associated with other forms of persistent post-operative pain. In susceptible patients, these neuropathological changes, including peripheral sensitization, central sensitization, and altered descending modulation, may underlie certain persistent dry eye symptoms after LASIK surgery. This review will focus on the known epidemiology of symptoms after LASIK and discuss mechanisms of persistent post-op pain due to nerve injury that may be relevant to these patients. Potential preventative and treatment options based on approaches used for other forms of persistent post-op pain and their application to LASIK patients are also discussed. Finally, the concept of genetic susceptibility to post-LASIK ocular surface pain is presented.
PMCID: PMC4411662  PMID: 25896684
LASIK; Photorefractive keratectomy; Dry eye; Chronic pain; Neuropathic pain; Persistent post-operative pain; Photoallodynia; Peripheral sensitization; Central sensitization
25.  In vivo patch-clamp analysis of the antinociceptive actions of TRPA1 activation in the spinal dorsal horn 
Molecular Pain  2015;11:20.
Transient receptor potential (TRP) channels are nonselective cation channels expressed in a variety of sensory structures, and are important molecular mediators of thermal, mechanical, cellular and chemical signals. We investigated the function of one key member of the TRP superfamily, TRPA1, in the spinal dorsal horn using in vivo patch-clamp recordings.
The application of allyl isothiocyanate (AITC), a TRPA1 agonist, significantly increased the frequency and amplitude of inhibitory postsynaptic currents (IPSCs; holding potential (VH) = 0 mV) as well as excitatory postsynaptic currents (EPSCs; VH = −70 mV) in substantia gelatinosa (SG) neurons. The AITC-induced increases in EPSC frequency and amplitude were resistant to the Na+ channel blocker tetrodotoxin (TTX). In the presence of the glutamate receptor antagonists CNQX and AP5, AITC did not generate any synaptic activity. The AITC-induced increases in IPSC frequency and amplitude were abolished by TTX or glutamate receptor antagonists. Moreover, the duration of IPSCs enhanced by TRPA1 activation were significantly longer than those of EPSCs enhanced by activation of this channel in the spinal dorsal horn. AITC induced hyperpolarization of the membrane potential of SG neurons in the spinal cord but depolarized the membrane potential in the presence of TTX. Furthermore, we examined the effects of mechanical stimuli to the skin during TRPA1 activation in the spinal dorsal horn in normal rats in both voltage-clamp and current-clamp modes. In the peripheral tissue stimuli test, AITC significantly suppressed EPSCs evoked by pinch or air puff stimulation of the skin. In current-clamp mode, AITC significantly suppressed excitatory postsynaptic potentials (EPSPs) evoked by pinch stimuli.
TRPA1 appears to be localized not only at presynaptic terminals on SG neurons, enhancing glutamate release, but also in the terminals of primary afferents innervating spinal inhibitory interneurons, which have synaptic interactions with SG neurons. This study offers further insight into the mechanisms underlying the possible antinociceptive actions of TRPA1 activation in the spinal dorsal horn. Our findings suggest that pharmacological activation of spinal TRPA1 channels may have therapeutic potential for the treatment of pain.
PMCID: PMC4422151  PMID: 25896791
TRPA1; In vivo patch-clamp; Allyl isothiocyanate; Antinociceptive action

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