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1.  G9a inhibits CREB-triggered expression of mu opioid receptor in primary sensory neurons following peripheral nerve injury 
Molecular Pain  2016;12:1744806916682242.
Neuropathic pain, a distressing and debilitating disorder, is still poorly managed in clinic. Opioids, like morphine, remain the mainstay of prescribed medications in the treatment of this disorder, but their analgesic effects are highly unsatisfactory in part due to nerve injury-induced reduction of opioid receptors in the first-order sensory neurons of dorsal root ganglia. G9a is a repressor of gene expression. We found that nerve injury-induced increases in G9a and its catalyzed repressive marker H3K9m2 are responsible for epigenetic silencing of Oprm1, Oprk1, and Oprd1 genes in the injured dorsal root ganglia. Blocking these increases rescued dorsal root ganglia Oprm1, Oprk1, and Oprd1 gene expression and morphine or loperamide analgesia and prevented the development of morphine or loperamide-induced analgesic tolerance under neuropathic pain conditions. Conversely, mimicking these increases reduced the expression of three opioid receptors and promoted the mu opioid receptor-gated release of primary afferent neurotransmitters. Mechanistically, nerve injury-induced increases in the binding activity of G9a and H3K9me2 to the Oprm1 gene were associated with the reduced binding of cyclic AMP response element binding protein to the Oprm1 gene. These findings suggest that G9a participates in the nerve injury-induced reduction of the Oprm1 gene likely through G9a-triggered blockage in the access of cyclic AMP response element binding protein to this gene.
doi:10.1177/1744806916682242
PMCID: PMC5153028  PMID: 27927796
G9a; opioid receptor; dorsal root ganglion; nerve injury
2.  Exploring novel paths towards protein signatures of chronic pain 
Molecular Pain  2016;12:1744806916679658.
Pain is a major symptom of many medical conditions and the worldwide number one reason for people to seek medical assistance. It affects the quality of life of patients and poses a heavy financial burden on society with high costs of treatment and lost productivity. Furthermore, the treatment of chronic pain presents a big challenge as pain therapeutics often lack efficacy and exhibit minimal safety profiles. The latter can be largely attributed to the fact that current therapies target molecules with key physiological functions throughout the body. In light of these difficulties, the identification of proteins specifically involved in chronic pain states is of paramount importance for designing selective interventions. Several profiling efforts have been employed with the aim to dissect the molecular underpinnings of chronic pain, both on the level of the transcriptome and proteome. However, generated results are often inconsistent and non-overlapping, which is largely due to inherent technical constraints. A potential solution may be offered by emerging strategies capable of performing standardized and reproducible proteome analysis, such as data-independent acquisition-mass spectrometry (DIA-MS). We have recently demonstrated the applicability of DIA-MS to interrogate chronic pain-related proteome alterations in mice. Based on our results, we aim to provide an overview on DIA-MS and its potential to contribute to the comprehensive characterization of molecular signatures underlying pain pathologies.
doi:10.1177/1744806916679658
PMCID: PMC5153021  PMID: 27920228
Chronic pain; proteomics; data-independent acquisition-mass spectrometry; mass spectrometry; protein signature; mouse models of chronic pain
3.  Involvement of mesolimbic dopaminergic network in neuropathic pain relief by treadmill exercise 
Molecular Pain  2016;12:1744806916681567.
Background
Exercise alleviates pain and it is a central component of treatment strategy for chronic pain in clinical setting. However, little is known about mechanism of this exercise-induced hypoalgesia. The mesolimbic dopaminergic network plays a role in positive emotions to rewards including motivation and pleasure. Pain negatively modulates these emotions, but appropriate exercise is considered to activate the dopaminergic network. We investigated possible involvement of this network as a mechanism of exercise-induced hypoalgesia.
Methods
In the present study, we developed a protocol of treadmill exercise, which was able to recover pain threshold under partial sciatic nerve ligation in mice, and investigated involvement of the dopaminergic reward network in exercise-induced hypoalgesia. To temporally suppress a neural activation during exercise, a genetically modified inhibitory G-protein-coupled receptor, hM4Di, was specifically expressed on dopaminergic pathway from the ventral tegmental area to the nucleus accumbens.
Results
The chemogenetic-specific neural suppression by Gi-DREADD system dramatically offset the effect of exercise-induced hypoalgesia in transgenic mice with hM4Di expressed on the ventral tegmental area dopamine neurons. Additionally, anti-exercise-induced hypoalgesia effect was significantly observed under the suppression of neurons projecting out of the ventral tegmental area to the nucleus accumbens as well.
Conclusion
Our findings suggest that the dopaminergic pathway from the ventral tegmental area to the nucleus accumbens is involved in the anti-nociception under low-intensity exercise under a neuropathic pain-like state.
doi:10.1177/1744806916681567
PMCID: PMC5140073  PMID: 27909152
Chronic pain; mesolimbic dopaminergic network; exercise-induced hypoalgesia; treadmill exercise; DREADD; brain reward system; ventral tegmental area; nucleus accumbens
4.  Selective antagonism of TRPA1 produces limited efficacy in models of inflammatory- and neuropathic-induced mechanical hypersensitivity in rats 
Molecular Pain  2016;12:1744806916677761.
The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in pathophysiological processes that include asthma, cough, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and rodents, and TRPA1 antagonists have been reported to be effective in rodent models of pain. In our pursuit of TRPA1 antagonists as potential therapeutics, we generated AMG0902, a potent (IC90 of 300 nM against rat TRPA1), selective, brain penetrant (brain to plasma ratio of 0.2), and orally bioavailable small molecule TRPA1 antagonist. AMG0902 reduced mechanically evoked C-fiber action potential firing in a skin-nerve preparation from mice previously injected with complete Freund’s adjuvant, supporting the role of TRPA1 in inflammatory mechanosensation. In vivo target coverage of TRPA1 by AMG0902 was demonstrated by the prevention of AITC-induced flinching/licking in rats. However, oral administration of AMG0902 to rats resulted in little to no efficacy in models of inflammatory, mechanically evoked hypersensitivity; and no efficacy was observed in a neuropathic pain model. Unbound plasma concentrations achieved in pain models were about 4-fold higher than the IC90 concentration in the AITC target coverage model, suggesting that either greater target coverage is required for efficacy in the pain models studied or TRPA1 may not contribute significantly to the underlying mechanisms.
doi:10.1177/1744806916677761
PMCID: PMC5131813  PMID: 27899696
TRPA1; inflammatory; neuropathic; pain; AMG0902; rat
5.  Consistent sex-dependent effects of PKMζ gene ablation and pharmacological inhibition on the maintenance of referred pain 
Molecular Pain  2016;12:1744806916675347.
Background
Persistently active PKMζ has been implicated in maintaining spinal nociceptive sensitization that underlies pain hypersensitivity. However, evidence for PKMζ in the maintenance of pain hypersensitivity comes exclusively from short-term studies in males using pharmacological agents of questionable selectivity. The present study examines the contribution of PKMζ to long-lasting allodynia associated with neuropathic, inflammatory, or referred visceral and muscle pain in males and females using pharmacological inhibition or genetic ablation.
Results
Pharmacological inhibition or genetic ablation of PKMζ reduced mild formalin pain and slowly developing contralateral allodynia in nerve-injured rats, but not moderate formalin pain or ipsilateral allodynia in models of neuropathic and inflammatory pain. Pharmacological inhibition or genetic ablation of PKMζ also effectively reduced referred visceral and muscle pain in male, but not in female mice and rats.
Conclusion
We show pharmacological inhibition and genetic ablation of PKMζ consistently attenuate long-lasting pain hypersensitivity. However, differential effects in models of referred versus inflammatory and neuropathic pain, and in males versus females, highlight the roles of afferent input-dependent masking and sex differences in the maintenance of pain hypersensitivity.
doi:10.1177/1744806916675347
PMCID: PMC5131814  PMID: 27899695
Sex differences; protein kinase M-zeta; PKC/Mζ knock-out; muscle pain; visceral pain; tactile allodynia; central sensitization; antinociception; analgesia
6.  Spinal astrocytic activation contributes to mechanical allodynia in a rat model of cyclophosphamide-induced cystitis 
Molecular Pain  2016;12:1744806916674479.
Background
Previous studies have demonstrated that glial cells play an important role in the generation and maintenance of neuropathic pain. Activated glial cells produce numerous mediators such as proinflammatory cytokines that facilitate neuronal activity and synaptic plasticity. Similarly, bladder pain syndrome/interstitial cystitis shares many characteristics of neuropathic pain. However, related report on the involvement of spinal glia in bladder pain syndrome/interstitial cystitis-associated pathological pain and the underlying mechanisms are still lacking. The present study investigated spinal glial activation and underlying molecular mechanisms in a rat model of bladder pain syndrome/interstitial cystitis.
Results
A rat model of bladder pain syndrome/interstitial cystitis was established via systemic injection with cyclophosphamide. Mechanical allodynia was tested with von Frey monofilaments and up-down method. Moreover, Western blots and double immunofluorescence were used to detect the expression and location of glial fibrillary acidic protein, OX42/Iba1, P-P38, NeuN, interleukin (IL)-1β, phosphorylation of N-methyl-D-aspartate receptor 1 (P-NR1), and IL-1 receptor I (IL-1RI) in the L6-S1 spinal cord. We found that glial fibrillary acidic protein rather than OX42/Iba1 or P-P38 was significantly increased in the spinal cord of cyclophosphamide-induced cystitis. L-alpha-aminoadipate but not minocycline markedly attenuated the allodynia. Furthermore, we found that spinal IL-1β was dramatically increased in cyclophosphamide-induced cystitis, and activated astrocytes were the only source of IL-1β release, which contributed to allodynia in cystitis rats. Besides, spinal P-NR1 was statistically increased in cyclophosphamide-induced cystitis and only localized in IL-1RI positive neurons in spinal dorsal horn. Additionally, NR antagonist significantly attenuated the cystitis-induced pain. Interestingly, the time course of the P-NR1 expression paralleled to that of IL-1β or glial fibrillary acidic protein.
Conclusions
Our results demonstrated that astrocytic activation but not microglial activation contributed to the allodynia in cyclophosphamide-induced cystitis and IL-1β released from astrocytes might bind to its endogenous receptor on the neurons inducing the phosphorylation of NR1 subunit, leading to sensory neuronal hyperexcitability and pathological pain.
doi:10.1177/1744806916674479
PMCID: PMC5117243  PMID: 27852964
Bladder pain syndrome; cystitis; glia; cytokines; N-methyl-D-aspartate receptor; pain; spinal cord
7.  A randomized placebo-controlled pilot study of the efficacy and safety of D-cycloserine in people with chronic back pain 
Molecular Pain  2016;12:1744806916678627.
Background
Few effective pharmacological treatment options exist for chronic back pain, the leading cause of disability in the US, and all are associated with significant adverse effects.
Objective
To determine the efficacy and safety of D-cycloserine, a partial agonist to the N-methyl-D-aspartate receptor, in the treatment of chronic low back pain.
Methods
A total of 41 participants with chronic back pain who met all inclusion and exclusion criteria were enrolled in a double-blind, placebo-controlled randomized pilot trial of D-cycloserine. Treatment was administered orally for six weeks at escalating daily doses of 100 mg, 200 mg, and 400 mg, each for two weeks. The primary outcome measure was back pain intensity using the Numeric Rating Scale (0–10). Secondary measures were back pain-related questionnaires: McGill Pain Questionnaire short form, painDETECT, PANAS, and BDI. The pre-specified analysis was a two-way repeated measures analysis of variance.
Results
A treatment difference was observed between groups treated with D-cycloserine and placebo at six weeks of 1.05 ± 3.1 units on the Numeric Rating Scale, with an effect size of 0.4 and p = 0.14. This trend of better chronic back pain relief with D-cycloserine was also observed in the secondary measures. No safety issues were seen.
Conclusion
The difference in mean pain between the D-cycloserine and placebo groups did not reach statistical significance. However, a clinically meaningful effect size in the magnitude of pain relief was observed with a consistent pattern across multiple outcome measures with good safety, supporting further research into the effectiveness of D-cycloserine for chronic back pain.
doi:10.1177/1744806916678627
PMCID: PMC5117251  PMID: 27852965
D-cycloserine; chronic back pain; randomized; clinical trial; N-methyl-D-aspartate agonist
8.  Hippocampal activation of microglia may underlie the shared neurobiology of comorbid posttraumatic stress disorder and chronic pain 
Molecular Pain  2016;12:1744806916679166.
The high comorbidity rates of posttraumatic stress disorder and chronic pain have been widely reported, but the underlying mechanisms remain unclear. Emerging evidence suggested that an excess of inflammatory immune activities in the hippocampus involved in the progression of both posttraumatic stress disorder and chronic pain. Considering that microglia are substrates underlying the initiation and propagation of the neuroimmune response, we hypothesized that stress-induced activation of hippocampal microglia may contribute to the pathogenesis of posttraumatic stress disorder-pain comorbidity. We showed that rats exposed to single prolonged stress, an established posttraumatic stress disorder model, exhibited persistent mechanical allodynia and anxiety-like behavior, which were accompanied by increased activation of microglia and secretion of pro-inflammatory cytokines in the hippocampus. Correlation analyses showed that hippocampal activation of microglia was significantly correlated with mechanical allodynia and anxiety-like behavior. Our data also showed that both intraperitoneal and intra-hippocampal injection of minocycline suppressed single prolonged stress-induced microglia activation and inflammatory cytokines accumulation in the hippocampus, and attenuated both single prolonged stress-induced mechanical allodynia and anxiety-like behavior. Taken together, the present study suggests that stress-induced microglia activation in the hippocampus may serve as a critical mechanistic link in the comorbid relationship between posttraumatic stress disorder and chronic pain. The novel concept introduces the possibility of cotreating chronic pain and posttraumatic stress disorder.
doi:10.1177/1744806916679166
PMCID: PMC5117253  PMID: 27852966
Posttraumatic stress disorder; chronic pain; comorbidity; hippocampus; microglia; stress-induced hyperalgesia
9.  Differential contributions of vasopressin V1A and oxytocin receptors in the amygdala to pain-related behaviors in rats 
Molecular Pain  2016;12:1744806916676491.
Neuroplastic changes in the amygdala account for emotional-affective aspects of pain and involve neuropeptides such as calcitonin gene-related peptide and corticotropin-releasing factor. Another neuropeptide system, central arginine vasopressin, has been implicated in neuropsychiatric disorders, but its role in pain-related emotional expression and neuroplasticity remains to be determined. Here, we tested the hypothesis that arginine vasopressin in the amygdala contributes to pain-related emotional-affective responses, using stereotaxic applications of arginine vasopressin and antagonists for G-protein coupled vasopressin V1A and oxytocin receptors in adult male Sprague-Dawley rats. In normal animals, arginine vasopressin increased audible and ultrasonic vocalizations and anxiety-like behavior (decreased open-arm preference in the elevated plus maze). The facilitatory effects were blocked by a selective V1A antagonist (SR 49059, Relcovaptan) but not by an oxytocin receptor antagonist (L-371,257). L-371,257 had some facilitatory effects on vocalizations. Arginine vasopressin had no effect in arthritic rats (kaolin/carrageenan knee joint pain model). SR 49059 inhibited vocalizations and anxiety-like behavior (elevated plus maze) in arthritic, but not normal, rats and conveyed anxiolytic properties to arginine vasopressin. Arginine vasopressin, SR 49059, and L-371,257 had no significant effects on spinal reflexes. We interpret the data to suggest that arginine vasopressin through V1A in the amygdala contributes to emotional-affective aspects of pain (arthritis model), whereas oxytocin receptors may mediate some inhibitory effects of the vasopressin system.
doi:10.1177/1744806916676491
PMCID: PMC5117246  PMID: 27837170
Vasopressin; oxytocin; pain; anxiety; amygdala
10.  Preventing painful age-related bone fractures 
Molecular Pain  2016;12:1744806916677147.
Age-related bone fractures are usually painful and have highly negative effects on a geriatric patient’s functional status, quality of life, and survival. Currently, there are few analgesic therapies that fully control bone fracture pain in the elderly without significant unwanted side effects. However, another way of controlling age-related fracture pain would be to preemptively administer an osteo-anabolic agent to geriatric patients with high risk of fracture, so as to build new cortical bone and prevent the fracture from occurring. A major question, however, is whether an osteo-anabolic agent can stimulate the proliferation of osteogenic cells and build significant amounts of new cortical bone in light of the decreased number and responsiveness of osteogenic cells in aging bone. To explore this question, geriatric and young mice, 20 and 4 months old, respectively, received either vehicle or a monoclonal antibody that sequesters sclerostin (anti-sclerostin) for 28 days. From days 21 to 28, animals also received sustained administration of the thymidine analog, bromodeoxyuridine (BrdU), which labels the DNA of dividing cells. Animals were then euthanized at day 28 and the femurs were examined for cortical bone formation, bone mineral density, and newly borne BrdU+ cells in the periosteum which is a tissue that is pivotally involved in the formation of new cortical bone. In both the geriatric and young mice, anti-sclerostin induced a significant increase in the thickness of the cortical bone, bone mineral density, and the proliferation of newly borne BrdU+ cells in the periosteum. These results suggest that even in geriatric animals, anti-sclerostin therapy can build new cortical bone and increase the proliferation of osteogenic cells and thus reduce the likelihood of painful age-related bone fractures.
doi:10.1177/1744806916677147
PMCID: PMC5117249  PMID: 27837171
Aging; bone fracture; functional status; quality of life; morbidity; mortality
11.  A painful neuropathy-associated Nav1.7 mutant leads to time-dependent degeneration of small-diameter axons associated with intracellular Ca2+ dysregulation and decrease in ATP levels 
Molecular Pain  2016;12:1744806916674472.
Small fiber neuropathy is a painful sensory nervous system disorder characterized by damage to unmyelinated C- and thinly myelinated Aδ- nerve fibers, clinically manifested by burning pain in the distal extremities and dysautonomia. The clinical onset in adulthood suggests a time-dependent process. The mechanisms that underlie nerve fiber injury in small fiber neuropathy are incompletely understood, although roles for energetic stress have been suggested. In the present study, we report time-dependent degeneration of neurites from dorsal root ganglia neurons in culture expressing small fiber neuropathy-associated G856D mutant Nav1.7 channels and demonstrate a time-dependent increase in intracellular calcium levels [Ca2+]i and reactive oxygen species, together with a decrease in ATP levels. Together with a previous clinical report of burning pain in the feet and hands associated with reduced levels of Na+/K+-ATPase in humans with high altitude sickness, the present results link energetic stress and reactive oxygen species production with the development of a painful neuropathy that preferentially affects small-diameter axons.
doi:10.1177/1744806916674472
PMCID: PMC5102167  PMID: 27821467
ATP; axon degeneration; mitochondria; peripheral neuropathy; reactive oxygen species; sodium-calcium exchanger; voltage-gated sodium channel
12.  Blocking mammalian target of rapamycin alleviates bladder hyperactivity and pain in rats with cystitis 
Molecular Pain  2016;12:1744806916668868.
Background
Bladder disorders associated with interstitial cystitis are frequently characterized by increased contractility and pain. The purposes of this study were to examine (1) the effects of blocking mammalian target of rapamycin (mTOR) on the exaggerated bladder activity and pain evoked by cystitis and (2) the underlying mechanisms responsible for the role of mTOR in regulating cystic sensory activity.
Results
The expression of p-mTOR, mTOR-mediated phosphorylation of p70 ribosomal S6 protein kinase 1 (p-S6K1), 4 E–binding protein 4 (p-4 E-BP1), as well as phosphatidylinositide 3-kinase (p-PI3K) pathway were amplified in cyclophosphamide rats as compared with control rats. Blocking mTOR by intrathecal infusion of rapamycin attenuated bladder hyperactivity and pain. In addition, blocking PI3K signal pathway attenuated activities of mTOR, which was accompanied with decreasing bladder hyperactivity and pain. Inhibition of either mTOR or PI3K blunted the enhanced spinal substance P and calcitonin gene-related peptide in cyclophosphamide rats.
Conclusions
The data for the first time revealed specific signaling pathways leading to cyclophosphamide-induced bladder hyperactivity and pain, including the activation of mTOR and PI3K. Inhibition of these pathways alleviates cystic pain. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of overactive bladder and pain often observed in cystitis.
doi:10.1177/1744806916668868
PMCID: PMC5084610  PMID: 27780878
Cystic pain; mTOR; cystitis; bladder activity; rapamycin
13.  MicroRNA-30b regulates expression of the sodium channel Nav1.7 in nerve injury-induced neuropathic pain in the rat 
Molecular Pain  2016;12:1744806916671523.
Voltage-gated sodium channels, which are involved in pain pathways, have emerged as major targets for therapeutic intervention in pain disorders. Nav1.7, the tetrodotoxin-sensitive voltage-gated sodium channel isoform encoded by SCN9A and predominantly expressed in pain-sensing neurons in the dorsal root ganglion, plays a crucial role in nociception. MicroRNAs are highly conserved, small non-coding RNAs. Through binding to the 3′ untranslated region of their target mRNAs, microRNAs induce the cleavage and/or inhibition of protein translation. Based on bioinformatics analysis using TargetScan software, we determined that miR-30b directly targets SCN9A. To investigate the roles of Nav1.7 and miR-30b in neuropathic pain, we examined changes in the expression of Nav1.7 in the dorsal root ganglion by miR-30b over-expression or knockdown in rats with spared nerve injury. Our results demonstrated that the expression of miR-30b and Nav1.7 was down-regulated and up-regulated, respectively, in the dorsal root ganglion of spared nerve injury rats. MiR-30b over-expression in spared nerve injury rats inhibited SCN9A transcription, resulting in pain relief. In addition, miR-30b knockdown significantly increased hypersensitivity to pain in naive rats. We also observed that miR-30b decreased Nav1.7 expression in PC12 cells. Taken together, our results suggest that miR-30b plays an important role in neuropathic pain by regulating Nav1.7 expression. Therefore, miR-30b may be a promising target for the treatment of chronic neuropathic pain.
doi:10.1177/1744806916671523
PMCID: PMC5081156  PMID: 27765894
Neuropathic pain; sodium channel; microRNA; analgesia
14.  Transcriptome analysis of trigeminal ganglia following masseter muscle inflammation in rats 
Molecular Pain  2016;12:1744806916668526.
Background
Chronic pain in masticatory muscles is a major medical problem. Although mechanisms underlying persistent pain in masticatory muscles are not fully understood, sensitization of nociceptive primary afferents following muscle inflammation or injury contributes to muscle hyperalgesia. It is well known that craniofacial muscle injury or inflammation induces regulation of multiple genes in trigeminal ganglia, which is associated with muscle hyperalgesia. However, overall transcriptional profiles within trigeminal ganglia following masseter inflammation have not yet been determined. In the present study, we performed RNA sequencing assay in rat trigeminal ganglia to identify transcriptome profiles of genes relevant to hyperalgesia following inflammation of the rat masseter muscle.
Results
Masseter inflammation differentially regulated >3500 genes in trigeminal ganglia. Predominant biological pathways were predicted to be related with activation of resident non-neuronal cells within trigeminal ganglia or recruitment of immune cells. To focus our analysis on the genes more relevant to nociceptors, we selected genes implicated in pain mechanisms, genes enriched in small- to medium-sized sensory neurons, and genes enriched in TRPV1-lineage nociceptors. Among the 2320 candidate genes, 622 genes showed differential expression following masseter inflammation. When the analysis was limited to these candidate genes, pathways related with G protein-coupled signaling and synaptic plasticity were predicted to be enriched. Inspection of individual gene expression changes confirmed the transcriptional changes of multiple nociceptor genes associated with masseter hyperalgesia (e.g., Trpv1, Trpa1, P2rx3, Tac1, and Bdnf) and also suggested a number of novel probable contributors (e.g., Piezo2, Tmem100, and Hdac9).
Conclusion
These findings should further advance our understanding of peripheral mechanisms involved in persistent craniofacial muscle pain conditions and provide a rational basis for identifying novel genes or sets of genes that can be potentially targeted for treating such conditions.
doi:10.1177/1744806916668526
PMCID: PMC5066585  PMID: 27702909
Masseter muscle; inflammation; trigeminal ganglia; transcriptome; RNA sequencing
15.  Analgesic effects of adenylyl cyclase inhibitor NB001 on bone cancer pain in a mouse model 
Molecular Pain  2016;12:1744806916652409.
Background
Cancer pain, especially the one caused by metastasis in bones, is a severe type of pain. Pain becomes chronic unless its causes and consequences are resolved. With improvements in cancer detection and survival among patients, pain has been considered as a great challenge because traditional therapies are partially effective in terms of providing relief. Cancer pain mechanisms are more poorly understood than neuropathic and inflammatory pain states. Chronic inflammatory pain and neuropathic pain are influenced by NB001, an adenylyl cyclase 1 (AC1)-specific inhibitor with analgesic effects. In this study, the analgesic effects of NB001 on cancer pain were evaluated.
Results
Pain was induced by injecting osteolytic murine sarcoma cell NCTC 2472 into the intramedullary cavity of the femur of mice. The mice injected with sarcoma cells for four weeks exhibited significant spontaneous pain behavior and mechanical allodynia. The continuous systemic application of NB001 (30 mg/kg, intraperitoneally, twice daily for three days) markedly decreased the number of spontaneous lifting but increased the mechanical paw withdrawal threshold. NB001 decreased the concentrations of cAMP and the levels of GluN2A, GluN2B, p-GluA1 (831), and p-GluA1 (845) in the anterior cingulate cortex, and inhibited the frequency of presynaptic neurotransmitter release in the anterior cingulate cortex of the mouse models.
Conclusions
NB001 may serve as a novel analgesic to treat bone cancer pain. Its analgesic effect is at least partially due to the inhibition of AC1 in anterior cingulate cortex.
doi:10.1177/1744806916652409
PMCID: PMC5019365  PMID: 27612915
NB001; bone cancer pain; adenylyl cyclase 1; anterior cingulate cortex
16.  Downregulation of miR-219 enhances brain-derived neurotrophic factor production in mouse dorsal root ganglia to mediate morphine analgesic tolerance by upregulating CaMKIIγ 
Molecular Pain  2016;12:1744806916666283.
Background
Increasing evidence suggests that microRNAs are functionally involved in the initiation and maintenance of pain hypersensitivity, including chronic morphine analgesic tolerance, through the posttranscriptional regulation of pain-related genes. We have previously demonstrated that miR-219 regulates inflammatory pain in the spinal cord by targeting calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ). However, whether miR-219 regulates CaMKIIγ expression in the dorsal root ganglia to mediate morphine tolerance remains unclear.
Results
MiR-219 expression was downregulated and CaMKIIγ expression was upregulated in mouse dorsal root ganglia following chronic morphine treatment. The changes in miR-219 and CaMKIIγ expression closely correlated with the development of morphine tolerance, which was measured using the reduction of percentage of maximum potential efficiency to thermal stimuli. Morphine tolerance was markedly delayed by upregulating miR-219 expression using miR-219 mimics or downregulating CaMKIIγ expression using CaMKIIγ small interfering RNA. The protein and mRNA expression of brain-derived neurotrophic factor were also induced in dorsal root ganglia by prolonged morphine exposure in a time-dependent manner, which were transcriptionally regulated by miR-219 and CaMKIIγ. Scavenging brain-derived neurotrophic factor via tyrosine receptor kinase B-Fc partially attenuated morphine tolerance. Moreover, functional inhibition of miR-219 via miR-219-sponge in naive mice elicited thermal hyperalgesia and spinal neuronal sensitization, which were both suppressed by CaMKIIγ small interfering RNA or tyrosine receptor kinase B-Fc.
Conclusions
These results demonstrate that miR-219 contributes to the development of chronic tolerance to morphine analgesia in mouse dorsal root ganglia by targeting CaMKIIγ and enhancing CaMKIIγ-dependent brain-derived neurotrophic factor expression.
doi:10.1177/1744806916666283
PMCID: PMC5014090  PMID: 27599867
Morphine tolerance; hyperalgesia; miR-219; CaMKIIγ; brain-derived neurotrophic factor; dorsal root ganglia
17.  Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice 
Molecular Pain  2016;12:1744806916666284.
Background
Small fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown.
Purpose
Determine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal.
Results
We demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro.
Conclusions
These studies establish ganglioside GM3 as a new candidate responsible for neuropathic pain and small fiber neuropathy in diabetes. Moreover, these observations indicate that systemic or topically applied interventions aimed at depleting GM3 may improve both the painful neuropathy and the wound healing impairment in diabetes by protecting against nerve end terminal degeneration, providing a disease-modifying approach to this common, currently intractable medical issue.
doi:10.1177/1744806916666284
PMCID: PMC5011393  PMID: 27590073
Neuropathic pain; small fiber neuropathy; GM3 ganglioside; obesity; glucose intolerance; diabetes
18.  A SCN10A SNP biases human pain sensitivity 
Molecular Pain  2016;12:1744806916666083.
Background:
Nav1.8 sodium channels, encoded by SCN10A, are preferentially expressed in nociceptive neurons and play an important role in human pain. Although rare gain-of-function variants in SCN10A have been identified in individuals with painful peripheral neuropathies, whether more common variants in SCN10A can have an effect at the channel level and at the dorsal root ganglion, neuronal level leading to a pain disorder or an altered normal pain threshold has not been determined.
Results:
Candidate single nucleotide polymorphism association approach together with experimental pain testing in human subjects was used to explore possible common SCN10A missense variants that might affect human pain sensitivity. We demonstrated an association between rs6795970 (G > A; p.Ala1073Val) and higher thresholds for mechanical pain in a discovery cohort (496 subjects) and confirmed it in a larger replication cohort (1005 female subjects). Functional assessments showed that although the minor allele shifts channel activation by −4.3 mV, a proexcitatory attribute, it accelerates inactivation, an antiexcitatory attribute, with the net effect being reduced repetitive firing of dorsal root ganglion neurons, consistent with lower mechanical pain sensitivity.
Conclusions:
At the association and mechanistic levels, the SCN10A single nucleotide polymorphism rs6795970 biases human pain sensitivity.
doi:10.1177/1744806916666083
PMCID: PMC5011395  PMID: 27590072
Nav1.8; dorsal root ganglion; pain; voltage-gated sodium channel
19.  BDNF signaling contributes to oral cancer pain in a preclinical orthotopic rodent model 
Molecular Pain  2016;12:1744806916666841.
The majority of patients with oral cancer report intense pain that is only partially managed by current analgesics. Thus, there is a strong need to study mechanisms as well as develop novel analgesics for oral cancer pain. Current study employed an orthotopic tongue cancer model with molecular and non-reflexive behavioral assays to determine possible mechanisms of oral cancer pain. Human oral squamous cell carcinoma cells line, HSC2, was injected into the tongue of male athymic mice and tumor growth was observed by day 6. Immunohistological analyses revealed a well-differentiated tumor with a localized immune response and pronounced sensory and sympathetic innervation and vascularization. The tumor expressed TMPRSS2, a protein previously reported with oral squamous cell carcinoma. ATF3 expression in trigeminal ganglia was not altered by tumor growth. Molecular characterization of the model demonstrated altered expression of several pain-related genes, out of which up-regulation of BDNF was most striking. Moreover, BDNF protein expression in trigeminal ganglia neurons was increased and inhibition of BDNF signaling with a tyrosine kinase B antagonist, ANA-12, reversed pain-like behaviors induced by the oral tumor. Oral squamous cell carcinoma tumor growth was also associated with a reduction in feeding, mechanical hypersensitivity in the face, as well as spontaneous pain behaviors as measured by the conditioned place preference test, all of which were reversed by analgesics. Interestingly, injection of HSC2 into the hindpaw did not reproduce this spectrum of pain behaviors; nor did injection of a colonic cancer cell line into the tongue. Taken together, this orthotopic oral cancer pain model reproduces the spectrum of pain reported by oral cancer patients, including higher order cognitive changes, and demonstrates that BDNF signaling constitutes a novel mechanism by which oral squamous cell carcinoma induces pain. Identification of the key role of tyrosine kinase B signaling in oral cancer pain may serve as a novel target for drug development.
doi:10.1177/1744806916666841
PMCID: PMC5015823  PMID: 27590070
BDNF; oral cancer pain; orthotopic model; tongue cancer
20.  Repeated activation of delta opioid receptors counteracts nerve injury-induced TNF-α up-regulation in the sciatic nerve of rats with neuropathic pain 
Molecular Pain  2016;12:1744806916667949.
Despite mu opioid receptor agonists are the cornerstones of moderate-to-severe acute pain treatment, their effectiveness in chronic pain conditions is controversial. In contrast to mu opioid receptor agonists, a number of studies have reported the effectiveness of delta opioid receptor agonists on neuropathic pain strengthening the idea that delta opioid receptors gain importance when chronic pain develops. Among other effects, it has been shown that delta opioid receptor activation in optic nerve astrocytes inhibits tumor necrosis factor-α-mediated inflammation in response to severe hypoxia. Considering the involvement of tumor necrosis factor-α in the development and maintenance of neuropathic pain, with this study we sought to correlate the effect of delta opioid receptor agonist on the development of mechanical allodynia to tumor necrosis factor-α expression at the site of nerve injury in rats subjected to chronic constriction injury of the sciatic nerve. To this aim, we measured the levels of tumor necrosis factor-α in the sciatic nerve of rats with neuropathic pain after repeated injections with a delta opioid receptor agonist. Results obtained demonstrated that repeated administrations of the delta opioid receptor agonist SNC80 (10 mg/kg, i.p. for seven consecutive days) significantly inhibited the development of mechanical allodynia in rats with neuropathic pain and that the improvement of neuropathic symptom was timely related to the reduced expression of tumor necrosis factor-α in the rat sciatic nerve. We demonstrated also that when treatment with the delta opioid receptor agonist was suspended both allodynia and tumor necrosis factor-α up-regulation in the sciatic nerve of rats with neuropathic pain were restored. These results show that persistent delta opioid receptor activation significantly attenuates neuropathic pain and negatively regulates sciatic nerve tumor necrosis factor-α expression in chronic constriction injury rats.
doi:10.1177/1744806916667949
PMCID: PMC5024981  PMID: 27590071
Neuropathic pain; delta opioid receptor; tumor necrosis factor-α; mechanical allodynia
21.  Changes in synaptic transmission of substantia gelatinosa neurons after spinal cord hemisection revealed by analysis using in vivo patch-clamp recording 
Molecular Pain  2016;12:1744806916665827.
Background
After spinal cord injury, central neuropathic pain develops in the majority of spinal cord injury patients. Spinal hemisection in rats, which has been developed as an animal model of spinal cord injury in humans, results in hyperexcitation of spinal dorsal horn neurons soon after the hemisection and thereafter. The hyperexcitation is likely caused by permanent elimination of the descending pain systems. We examined the change in synaptic transmission of substantia gelatinosa neurons following acute spinal hemisection by using an in vivo whole-cell patch-clamp technique.
Results
An increased spontaneous action potential firings of substantia gelatinosa neurons was detected in hemisected rats compared with that in control animals. The frequencies and amplitudes of spontaneous excitatory postsynaptic currents and of evoked excitatory postsynaptic currentss in response to non-noxious and noxious stimuli were not different between hemisected and control animals. On the contrary, the amplitude and frequency of spontaneous inhibitory postsynaptic currents of substantia gelatinosa neurons in hemisected animals were significantly smaller and lower, respectively, than those in control animals (P < 0.01). Large amplitude and high-frequency spontaneous inhibitory postsynaptic currents, which could not be elicited by mechanical stimuli, were seen in 44% of substantia gelatinosa neurons in control animals but only in 17% of substantia gelatinosa neurons in hemisected animals. In control animals, such large amplitude spontaneous inhibitory postsynaptic currents were suppressed by spinal application of tetrodotoxin (1 µM). Cervical application of lidocaine (2%, 10 µl) also inhibited such large amplitude of inhibitory postsynaptic currents. The proportion of multi-receptive substantia gelatinosa neurons, which exhibit action potential firing in response to non-noxious and noxious stimuli, was much larger in hemisected animals than in control animals.
Conclusions
These suggest that substantia gelatinosa neurons receive tonic inhibition by spinal inhibitory interneurons which generate persistent action potentials. Spinal hemisection results in hyperexcitation of substantia gelatinosa neurons at least in part by eliminating the tonic descending control of spinal inhibitory interneurons from supraspinal levels.
doi:10.1177/1744806916665827
PMCID: PMC5006296  PMID: 27573517
Substantia gelatinosa; spinal cord injury; hemisection; inhibitory postsynaptic currents; excitatory postsynaptic currents; in vivo; patch-clamp
22.  Transcriptomic and behavioural characterisation of a mouse model of burn pain identify the cholecystokinin 2 receptor as an analgesic target 
Molecular Pain  2016;12:1744806916665366.
Burn injury is a cause of significant mortality and morbidity worldwide and is frequently associated with severe and long-lasting pain that remains difficult to manage throughout recovery. We characterised a mouse model of burn-induced pain using pharmacological and transcriptomic approaches. Mechanical allodynia elicited by burn injury was partially reversed by meloxicam (5 mg/kg), gabapentin (100 mg/kg) and oxycodone (3 and 10 mg/kg), while thermal allodynia and gait abnormalities were only significantly improved by amitriptyline (3 mg/kg) and oxycodone (10 mg/kg). The need for relatively high opioid doses to elicit analgesia suggested a degree of opioid resistance, similar to that shown clinically in burn patients. We thus assessed the gene expression changes in dorsal root ganglion neurons and pathophysiological mechanisms underpinning burn injury-induced pain using a transcriptomic approach. Burn injury was associated with significantly increased expression of genes associated with axon guidance, neuropeptide signalling, behavioural defence response and extracellular signalling, confirming a mixed neuropathic and inflammatory aetiology. Notably, among the pain-related genes that were upregulated post-injury was the cholecystokinin 2 receptor (Cckbr), a G protein-coupled receptor known as a pain target involved in reducing opioid effectiveness. Indeed, the clinically used cholecystokinin receptor antagonist proglumide (30 mg/kg) was effective at reversing mechanical allodynia, with additional analgesia evident in combination with low-dose oxycodone (1 mg/kg), including significant reversal of thermal allodynia. These findings highlight the complex pathophysiological mechanisms underpinning burn injury-induced pain and suggest that cholecystokinin-2 receptor antagonists may be useful clinically as adjuvants to decrease opioid requirements and improve analgesic management.
doi:10.1177/1744806916665366
PMCID: PMC5007901  PMID: 27573516
Burn-induced pain; transcriptome; dorsal root ganglion neurons; animal models; proglumide
23.  Dopaminergic inhibition by G9a/Glp complex on tyrosine hydroxylase in nerve injury-induced hypersensitivity 
Molecular Pain  2016;12:1744806916663731.
The neural balance between facilitation and inhibition determines the final tendency of central sensitization. Nerve injury-induced hypersensitivity was considered as the results from the enhanced ascending facilitation and the diminished descending inhibition. The role of dopaminergic transmission in the descending inhibition has been well documented, but its underlying molecular mechanisms are unclear. Previous studies demonstrated that the lysine dimethyltransferase G9a/G9a-like protein (Glp) complex plays a critical role in cocaine-induced central plasticity, and given cocaine’s role in the nerve system is relied on its function on dopamine system, we herein proposed that the reduced inhibition of dopaminergic transmission was from the downregulation of tyrosine hydroxylase expression by G9a/Glp complex through methylating its gene Th. After approval by the Animal Care and Use Committee, C57BL/6 mice were used for pain behavior using von Frey after spared nerve injury, and Th CpG islands methylation was measured using bisulfite sequencing at different nerve areas. The inhibitor of G9a/Glp, BIX 01294, was administered intraventricularly daily with bolus injection. The protein levels of G9a, Glp, and tyrosine hydroxylase were measured with immunoblotting. Dopamine levels were detected using high-performance liquid chromatography. The expression of G9a but not Glp was upregulated in ventral tegmental area at post-injury day 4 till day 49 (the last day of the behavioral test). Correspondingly, the Th CpG methylation is increased, but the tyrosine hydroxylase expression was downregulated and the dopamine level was decreased. After the intracerebroventriclar injection of BIX 01294 since the post-injury days 7 and 14 for consecutive three days, three weeks, and six weeks, the expression of tyrosine hydroxylase was upregulated with a significant decrease in Th methylation and increase in dopamine level. Moreover, the pain after G9a/Glp inhibitor was attenuated significantly. In sum, methytransferase G9a/Glp complex partially controls dopaminergic transmission by methylating Th in peripheral nerve injury-induced neuropathic pain.
doi:10.1177/1744806916663731
PMCID: PMC5006299  PMID: 27562335
Nerve injury; tyrosine hydroxylase; G9a/Glp; dopamine; desensitization
24.  Diminished supraspinal pain modulation in patients with mild traumatic brain injury 
Molecular Pain  2016;12:1744806916662661.
Background
Chronic pain conditions are highly prevalent in patients with mild traumatic brain injury. Supraspinal diffuse axonal injury is known to dissociate brain functional connectivity in these patients. The effect of this dissociated state on supraspinal pain network is largely unknown. A functional magnetic resonance imaging study was conducted to compare the supraspinal pain network in patients with mild traumatic brain injury to the gender and age-matched healthy controls with the hypothesis that the functional connectivities of the medial prefrontal cortices, a supraspinal pain modulatory region to other pain-related sensory discriminatory and affective regions in the mild traumatic brain injury subjects are significantly reduced in comparison to healthy controls.
Results
The mild traumatic brain injury group (N = 15) demonstrated significantly (P < 0.01, cluster threshold > 150 voxels) less activities in the thalamus, pons, anterior cingulate cortex, insula, dorsolateral prefrontal cortex, and medial prefrontal cortices than the healthy control group (N = 15). Granger Causality Analyses (GCA) indicated while the left medial prefrontal cortices of the healthy control group cast a noticeable degree of outward (to affect) causality inference to multiple pain processing related regions, this outward inference pattern was not observed in the mild traumatic brain injury group. On the other hand, only patients’ bilateral anterior cingulate cortex received multiple inward (to be affected) causality inferences from regions including the primary and secondary somatosensory cortices and the inferior parietal lobe. Resting state functional connectivity analyses indicated that the medial prefrontal cortices of the mild traumatic brain injury group demonstrated a significantly (P < 0.01, F = 3.6, cluster size > 150 voxels) higher degree of functional connectivity to the inferior parietal lobe, premotor and secondary somatosensory cortex than the controls. Conversely, the anterior cingulate cortex of the healthy group demonstrated significantly (P < 0.01, F = 3.84, cluster size > 150 voxels) less degree of functional connectivities to the inferior parietal lobe and secondary somatosensory cortex than their mild traumatic brain injury counterparts.
Conclusions
In short, the current study demonstrates that patients with mild traumatic brain injury and headaches appear to have an altered state of supraspinal modulatory and affective functions related to pain perception.
doi:10.1177/1744806916662661
PMCID: PMC4989585  PMID: 27531671
Traumatic brain injury; chronic posttraumatic headaches; pain; functional magnetic resonance imaging; supraspinal pain processing; mild traumatic brain injury; resting state functional connectivity
25.  High levels of endogenous lipid mediators (N-acylethanolamines) in women with chronic widespread pain during acute tissue trauma 
Molecular Pain  2016;12:1744806916662886.
Although chronic widespread musculoskeletal pain is a significant health problem, the molecular mechanisms involved in developing and maintaining chronic widespread musculoskeletal pain are poorly understood. Central sensitization mechanisms maintained by stimuli from peripheral tissues such as muscle have been suggested. Lipid mediators with anti-inflammatory characteristics such as endogenous ligands of peroxisome proliferator activating receptor-α, oleoylethanolamide, and palmitoylethanolamide are suggested to regulate nociceptive transmission from peripheral locations on route towards the central nervous system. This case–control study investigates the levels of anti-inflammatory lipids in microdialysis samples collected during the first 2 h after microdialysis probe insertion and explores the association of these lipids with different pain characteristics in women with chronic widespread musculoskeletal pain (n = 17) and female healthy controls (n = 19). The levels of oleoylethanolamide, palmitoylethanolamide, and stearoylethanolamide were determined. During sampling of dialysate, pain ratings were conducted using a numeric rating scale. Pain thresholds were registered from upper and lower parts of the body. Oleoylethanolamide and stearoylethanolamide levels were significantly higher (p ≤ 0.05) in chronic widespread musculoskeletal pain at all time points. Numeric rating scale correlated with levels of stearoylethanolamide in chronic widespread musculoskeletal pain. Higher levels of lipid mediators could reflect an altered tissue reactivity in response to microdialysis probe insertion in chronic widespread musculoskeletal pain.
doi:10.1177/1744806916662886
PMCID: PMC5010217  PMID: 27531672
Chronic widespread pain; microdialysis; trapezius muscle; tissue trauma; lipid mediators

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