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1.  Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury 
eLife  null;5:e12661.
Neuropathic pain following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons, which begins to normalise over time. We investigated the composition and distribution of shaker-type-potassium channels (Kv1 channels) within the nodal complex of myelinated axons following injury. At the neuroma that forms after damage, expression of Kv1.1 and 1.2 (normally localised to the juxtaparanode) was markedly decreased. In contrast Kv1.4 and 1.6, which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in rats and humans. Within the dorsal root (a site remote from injury) we noted a redistribution of Kv1-channels towards the paranode. Blockade of Kv1 channels with α-DTX after injury reinstated hyperexcitability of A-fibre axons and enhanced mechanosensitivity. Changes in the molecular composition and distribution of axonal Kv1 channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury.
DOI: http://dx.doi.org/10.7554/eLife.12661.001
eLife digest
Around 20% of the world’s population experiences long-lasting “chronic” pain, which often results in poor sleep, depression and anxiety. One of the most disabling forms of chronic pain is called neuropathic pain, which results from injuries to sensory nerves. Pain or discomfort is felt in response to touches that are not normally painful. Neuropathic pain is difficult to treat as we do not fully understand the molecular mechanisms that cause it.
Stimulating a nerve causes it to produce action potentials. At a molecular level, these action potentials are generated by ions moving into and out of the neuron through proteins called ion channels. The movement of sodium ions into a neuron triggers an action potential, and the movement of potassium ions out of the neuron returns it to a resting state.
After a sensory nerve is cut or otherwise damaged it becomes hyperactive and produces spontaneous electrical activity that the brain interprets as pain signals. However, it is not fully understood how cutting a nerve affects the ion channels in a way that generates this hyperactivity.
Different types of ion channel are found in different regions of the nerve cell; for example, type 1 potassium channels are normally found in a region called the juxtaparanode at the axon of the neuron. Calvo et al. have now tracked what happens to type 1 potassium channels after nerve injury in rats. Soon after nerve damage occurred, nearly all of these ion channels disappeared from the juxtaparanode. At the same time, electrical activity in the cut nerve increased, and the recovering animals responded in ways that suggested they were hypersensitive to the nerve being touched.
Three weeks after the injury, most rats lost their hypersensitivity and the electrical activity in the cut nerve returned to near-normal levels. Calvo et al. found that the recovering nerves contained new subtypes of type 1 potassium channels. These potassium channels did not just appear in the juxtaparanode: they also invaded the ‘fence’ region that normally separates potassium channels from sodium channels. The same was observed to happen in the nerves of patients that suffer from neuropathic pain due to a nerve injury.
At this late time point after nerve injury, blocking the activity of potassium channels produced the same abnormal increase in the nerve’s electrical activity as seen immediately after the nerve had been cut. The rats’ hypersensitivity to touch also returned. This suggests that the appearance of the new potassium channel subtypes might be a protective mechanism that reduces the activity of a damaged nerve to decrease pain.
These findings suggest new ways of treating neuropathic pain. Further studies are now needed to investigate whether drugs that can activate the new potassium channel subtypes could stop pain from an injured nerve becoming a long-term problem.
DOI: http://dx.doi.org/10.7554/eLife.12661.002
doi:10.7554/eLife.12661
PMCID: PMC4841771  PMID: 27033551
neuropathic pain; neuropathy; shaker type potassium channels; juxtaparanode; hypersensitivity; Human; Rat
2.  The Expression of Inflammatory Mediators in Bladder Pain Syndrome 
European Urology  2016;70(2):283-290.
Background
Bladder pain syndrome (BPS) pathology is poorly understood. Treatment strategies are empirical, with limited efficacy, and affected patients have diminished quality of life.
Objective
We examined the hypothesis that inflammatory mediators within the bladder contribute to BPS pathology.
Design, setting, and participants
Fifteen women with BPS and 15 women with stress urinary incontinence without bladder pain were recruited from Cork University Maternity Hospital from October 2011 to October 2012. During cystoscopy, 5-mm bladder biopsies were taken and processed for gene expression analysis. The effect of the identified genes was tested in laboratory animals.
Outcome measures and statistical analysis
We studied the expression of 96 inflammation-related genes in diseased and healthy bladders. We measured the correlation between genes and patient clinical profiles using the Pearson correlation coefficient.
Results and limitations
Analysis revealed 15 differentially expressed genes, confirmed in a replication study. FGF7 and CCL21 correlated significantly with clinical outcomes. Intravesical CCL21 instillation in rats caused increased bladder excitability and increased c-fos activity in spinal cord neurons. CCL21 atypical receptor knockout mice showed significantly more c-fos upon bladder stimulation with CCL21 than wild-type littermates. There was no change in FGF7-treated animals. The variability in patient samples presented as the main limitation. We used principal component analysis to identify similarities within the patient group.
Conclusions
Our study identified two biologically relevant inflammatory mediators in BPS and demonstrated an increase in nociceptive signalling with CCL21. Manipulation of this ligand is a potential new therapeutic strategy for BPS.
Patient summary
We compared gene expression in bladder biopsies of patients with bladder pain syndrome (BPS) and controls without pain and identified two genes that were increased in BPS patients and correlated with clinical profiles. We tested the effect of these genes in laboratory animals, confirming their role in bladder pain. Manipulating these genes in BPS is a potential treatment strategy.
Take Home Message
Bladder pain syndrome (BPS) pathology remains poorly understood, and its treatment is difficult. We identified mediators that, from our study of their action in the bladders of laboratory animals, may be implicated in disease pathology. These mediators are unexploited therapeutic targets for BPS treatment.
doi:10.1016/j.eururo.2016.02.058
PMCID: PMC4926725  PMID: 26965559
Animal model; Bladder pain syndrome; CCL21; Clinical correlation; FGF7; Gene expression analysis; Interstitial cystitis; Pain behaviour
3.  Ultraviolet Radiation on the Skin: A Painful Experience? 
CNS Neuroscience & Therapeutics  2015;22(2):118-126.
Summary
Excessive exposure of skin to ultraviolet radiation (UVR) has dramatic clinical effects in humans, and it is a significant public health concern. Discomfort and sensory changes caused by skin sunburn are the main common features experienced by many of us, a phenomena triggered by the combination of long and short wavelengths radiation (UVA and UVB, respectively). Although the biological processes underlying UVR exposure are not fully understood, in the last few years many studies have made significant progress in characterizing sunburn at the cellular and molecular levels, making use of both humans and laboratory animal models. Here we review and reason that UVR can be used as an excellent model of sensitization and inflammation for pain research. UVR, particularly UVB, produces a controllable and sterile inflammation that causes a robust dose‐dependent hypersensitivity with minimal confounding effects. Importantly, we show that UVR animal models precisely recapitulate the sensory, cellular, and molecular changes observed in human skin, giving it great confidence as a translational model. Furthermore, in this article, we give an overview of the pharmacology underlying UVB inflammation, the latest advances in the field, and potential new targets for inflammatory pain.
doi:10.1111/cns.12444
PMCID: PMC4833175  PMID: 26331607
Inflammation; Pain; Pharmacology; UVB; UVR
4.  Ensuring transparency and minimization of methodologic bias in preclinical pain research: PPRECISE considerations 
Pain  2015;157(4):901-909.
Improved reporting standards, and adoption of experimental protocols that emphasize control of experimental bias, are likely to improve understanding and confidence in pain biology.
Abstract
There is growing concern about lack of scientific rigor and transparent reporting across many preclinical fields of biological research. Poor experimental design and lack of transparent reporting can result in conscious or unconscious experimental bias, producing results that are not replicable. The Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION) public–private partnership with the U.S. Food and Drug Administration sponsored a consensus meeting of the Preclinical Pain Research Consortium for Investigating Safety and Efficacy (PPRECISE) Working Group. International participants from universities, funding agencies, government agencies, industry, and a patient advocacy organization attended. Reduction of publication bias, increasing the ability of others to faithfully repeat experimental methods, and increased transparency of data reporting were specifically discussed. Parameters deemed essential to increase confidence in the published literature were clear, specific reporting of an a priori hypothesis and definition of primary outcome measure. Power calculations and whether measurement of minimal meaningful effect size to determine these should be a core component of the preclinical research effort provoked considerable discussion, with many but not all agreeing. Greater transparency of reporting should be driven by scientists, journal editors, reviewers, and grant funders. The conduct of high-quality science that is fully reported should not preclude novelty and innovation in preclinical pain research, and indeed, any efforts that curtail such innovation would be misguided. We believe that to achieve the goal of finding effective new treatments for patients with pain, the pain field needs to deal with these challenging issues.
doi:10.1097/j.pain.0000000000000458
PMCID: PMC4794131  PMID: 26683237
Transparent reporting; Consensus; Bias; Internal validity
5.  Dimethylarginine dimethylaminohydrolase 1 is involved in spinal nociceptive plasticity 
Pain  2015;156(10):2052-2060.
Supplemental Digital Content is Available in the Text.
Inhibition of dimethylarginine dimethylaminohydrolase 1 attenuates pain-related behavior and hyperexcitability in pain conditions associated with excessive nitric oxide production, representing a novel therapeutic target.
Abstract
Activation of neuronal nitric oxide synthase, and consequent production of nitric oxide (NO), contributes to spinal hyperexcitability and enhanced pain sensation. All NOS isoforms are inhibited endogenously by asymmetric dimethylarginine, which itself is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Inhibition of DDAH can indirectly attenuate NO production by elevating asymmetric dimethylarginine concentrations. Here, we show that the DDAH-1 isoform is constitutively active in the nervous system, specifically in the spinal dorsal horn. DDAH-1 was found to be expressed in sensory neurons within both the dorsal root ganglia and spinal dorsal horn; L-291 (NG–[2-Methoxyethyl]-l-arginine methyl ester), a DDAH-1 inhibitor, reduced NO synthesis in cultured dorsal root ganglia neurons. Spinal application of L-291 decreased N-methyl-d-aspartate–dependent postdischarge and windup of dorsal horn sensory neurons—2 measures of spinal hyperexcitability. Finally, spinal application of L-291 reduced both neuronal and behavioral measures of formalin-induced central sensitization. Thus, DDAH-1 may be a potential therapeutic target in neuronal disorders, such as chronic pain, where elevated NO is a contributing factor.
doi:10.1097/j.pain.0000000000000269
PMCID: PMC4770343  PMID: 26098438
Spinal hyperexcitability; Nitric oxide; Neuronal nitric oxide synthase; Asymmetric dimethylargenine; Dimethylarginine dimethylaminohydrolase inhibition
6.  The Role of G-Protein Receptor 84 in Experimental Neuropathic Pain 
The Journal of Neuroscience  2015;35(23):8959-8969.
G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiological function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mechanical or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter molecule 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array analysis revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addition, the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1β, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger associated with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired.
doi:10.1523/JNEUROSCI.3558-14.2015
PMCID: PMC4461694  PMID: 26063927
cytokine; G-protein linked receptors; inflammation; macrophage; microglia; neuropathic pain
7.  Tamoxifen induces cellular stress in the nervous system by inhibiting cholesterol synthesis 
Background
Tamoxifen (TAM) is an important cancer therapeutic and an experimental tool for effecting genetic recombination using the inducible Cre-Lox technique. Despite its widespread use in the clinic and laboratory, we know little about its effects on the nervous system. This is of significant concern because TAM, via unknown mechanisms, induces cognitive impairment in humans. A hallmark of cellular stress is induction of Activating Transcription Factor 3 (Atf3), and so to determine whether TAM induces cellular stress in the adult nervous system, we generated a knock-in mouse in which Atf3 promoter activity drives transcription of TAM-dependent Cre recombinase (Cre-ERT2); when crossed with tdtomato reporter mice, Atf3 induction results in robust and permanent genetic labeling of cells in which it is up-regulated even transiently.
Results
We found that granular neurons of the olfactory bulb and dentate gyrus, vascular cells and ependymal cells throughout the brain, and peripheral sensory neurons expressed tdtomato in response to TAM treatment. We also show that TAM induced Atf3 up-regulation through inhibition of cholesterol epoxide hydrolase (ChEH): reporter expression was mitigated by delivery in vitamin E-rich wheat germ oil (vitamin E depletes ChEH substrates), and was partially mimicked by a ChEH-specific inhibitor.
Conclusions
This work demonstrates that TAM stresses cells of the adult central and peripheral nervous systems and highlights concerns about clinical and experimental use of TAM. We propose TAM administration in vitamin E-rich vehicles such as wheat germ oil as a simple remedy.
doi:10.1186/s40478-015-0255-6
PMCID: PMC4660723  PMID: 26610346
Dentate gyrus; Olfactory bulb; Activating transcription factor 3; Cholesterol biosynthesis; Sensory neurons
8.  Short-term effect of acute and repeated urinary bladder inflammation on thigmotactic behaviour in the laboratory rat 
F1000Research  2015;4:109.
Understanding the non-sensory components of the pain experience is crucial to developing effective treatments for pain conditions. Chronic pain is associated with increased incidence of anxio-depressive disorders, and patients often report feelings of vulnerability which can decrease quality of life. In animal models of pain, observation of behaviours such as thigmotaxis can be used to detect such affective disturbances by exploiting the influence of nociceptive stimuli on the innate behavioural conflict between exploration of a novel space and predator avoidance behaviour. This study investigates whether acute and repeated bladder inflammation in adult female Wistar rats increases thigmotactic behaviour in the open field paradigm, and aims to determine whether this correlates with activation in the central amygdala, as measured by c-Fos immunoreactivity. Additionally, up-regulation of inflammatory mediators in the urinary bladder was measured using RT-qPCR array featuring 92 transcripts to examine how local mediators change under experimental conditions. We found acute but not repeated turpentine inflammation of the bladder increased thigmotactic behaviour (decreased frequency of entry to the inner zone) in the open field paradigm, a result that was also observed in the catheter-only instrumentation group. Decreases in locomotor activity were also observed in both models in turpentine and instrumentation groups. No differences were observed in c-Fos activation, although a general increased in activation along the rostro-caudal axis was seen. Inflammatory mediator up-regulation was greatest following acute inflammation, with CCL12, CCL7, and IL-1β significantly up-regulated in both conditions when compared to naïve tissue. These results suggest that acute catheterisation, with or without turpentine inflammation, induces affective alterations detectable in the open field paradigm accompanied by up-regulation of multiple inflammatory mediators.
doi:10.12688/f1000research.6255.1
PMCID: PMC4850861  PMID: 27158443
Pain; Inflammation; Open Field; Cytokines; Amygdala; Behaviour; c-Fos
9.  HDAC4 is required for inflammation-associated thermal hypersensitivity 
The FASEB Journal  2015;29(8):3370-3378.
Transcriptional alterations are characteristic of persistent pain states, but the key regulators remain elusive. HDAC4 is a transcriptional corepressor that has been linked to synaptic plasticity and neuronal excitability, mechanisms that may be involved in peripheral and central sensitization. Using a conditional knockout (cKO) strategy in mice, we sought to determine whether the loss of HDAC4 would have implications for sensory neuron transcription and nociception. HDAC4 was found to be largely unnecessary for transcriptional regulation of naïve sensory neurons but was essential for appropriate transcriptional responses after injury, with Calca and Trpv1 expression consistently down-regulated in HDAC4 cKO compared to levels in the littermate controls (0.2–0.44-fold change, n = 4 in 2 separate experiments). This down-regulation corresponded to reduced sensitivity to 100 nM capsaicin in vitro (IC50 = 230 ± 20 nM, 76 ± 4.4% wild-type capsaicin responders vs. 56.9 ± 4.7% HDAC4 cKO responders) and to reduced thermal hypersensitivity in the complete Freund’s adjuvant (CFA) model of inflammatory pain (1.3–1.4-fold improvement over wild-type controls; n = 5–12, in 2 separate experiments). These data indicate that HDAC4 is a novel inflammatory pain mediator and may be a good therapeutic target, capable of orchestrating the regulation of multiple downstream effectors.—Crow, M., Khovanov, N., Kelleher, J. H., Sharma, S., Grant, A. D., Bogdanov, Y., Wood, J. N., McMahon, S. B., Denk, F. HDAC4 is required for inflammation-associated thermal hypersensitivity.
doi:10.1096/fj.14-264440
PMCID: PMC4511203  PMID: 25903105
epigenetics; pain; nerve growth factor; transcription
10.  Sensory Neuron Downregulation of the Kv9.1 Potassium Channel Subunit Mediates Neuropathic Pain following Nerve Injury 
Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.
doi:10.1523/JNEUROSCI.3561-12.2012
PMCID: PMC3713313  PMID: 23197740
11.  PainNetworks: A web-based resource for the visualisation of pain-related genes in the context of their network associations 
Pain  2013;154(12):2586.e1-2586.e12.
Hundreds of genes are proposed to contribute to nociception and pain perception. Historically, most studies of pain-related genes have examined them in isolation or alongside a handful of other genes. More recently the use of systems biology techniques has enabled us to study genes in the context of the biological pathways and networks in which they operate.
Here we describe a Web-based resource, available at http://www.PainNetworks.org. It integrates interaction data from various public databases with information on known pain genes taken from several sources (eg, The Pain Genes Database) and allows the user to examine a gene (or set of genes) of interest alongside known interaction partners. This information is displayed by the resource in the form of a network.
The user can enrich these networks by using data from pain-focused gene expression studies to highlight genes that change expression in a given experiment or pairs of genes showing correlated expression patterns across different experiments. Genes in the networks are annotated in several ways including biological function and drug binding.
The Web site can be used to find out more about a gene of interest by looking at the function of its interaction partners. It can also be used to interpret the results of a functional genomics experiment by revealing putative novel pain-related genes that have similar expression patterns to known pain-related genes and by ranking genes according to their network connections with known pain genes.
We expect this resource to grow over time and become a valuable asset to the pain community.
doi:10.1016/j.pain.2013.09.003
PMCID: PMC3863956  PMID: 24036287
Systems biology; Protein–protein interactions; Protein interaction network; Web-based resource; Pain genes; Microarrays
13.  Chronic Pain: Emerging Evidence for the Involvement of Epigenetics 
Neuron  2012;73(3):435-444.
Epigenetic processes, such as histone modifications and DNA methylation, have been associated with many neural functions including synaptic plasticity, learning, and memory. Here, we critically examine emerging evidence linking epigenetic mechanisms to the development or maintenance of chronic pain states. Although in its infancy, research in this area potentially unifies several pathophysiological processes underpinning abnormal pain processing and opens up a different avenue for the development of novel analgesics.
doi:10.1016/j.neuron.2012.01.012
PMCID: PMC3996727  PMID: 22325197
14.  Genome-Wide Transcriptional Profiling of Skin and Dorsal Root Ganglia after Ultraviolet-B-Induced Inflammation 
PLoS ONE  2014;9(4):e93338.
Ultraviolet-B (UVB)-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24), chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5), the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (R = 0.8022). In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain.
doi:10.1371/journal.pone.0093338
PMCID: PMC3986071  PMID: 24732968
15.  Molecular Mechanisms Underlying the Enhanced Analgesic Effect of Oxycodone Compared to Morphine in Chemotherapy-Induced Neuropathic Pain 
PLoS ONE  2014;9(3):e91297.
Oxycodone is a μ-opioid receptor agonist, used for the treatment of a large variety of painful disorders. Several studies have reported that oxycodone is a more potent pain reliever than morphine, and that it improves the quality of life of patients. However, the neurobiological mechanisms underlying the therapeutic action of these two opioids are only partially understood. The aim of this study was to define the molecular changes underlying the long-lasting analgesic effects of oxycodone and morphine in an animal model of peripheral neuropathy induced by a chemotherapic agent, vincristine. Using a behavioural approach, we show that oxycodone maintains an optimal analgesic effect after chronic treatment, whereas the effect of morphine dies down. In addition, using DNA microarray technology on dorsal root ganglia, we provide evidence that the long-term analgesic effect of oxycodone is due to an up-regulation in GABAB receptor expression in sensory neurons. These receptors are transported to their central terminals within the dorsal horn, and subsequently reinforce a presynaptic inhibition, since only the long-lasting (and not acute) anti-hyperalgesic effect of oxycodone was abolished by intrathecal administration of a GABAB receptor antagonist; in contrast, the morphine effect was unaffected. Our study demonstrates that the GABAB receptor is functionally required for the alleviating effect of oxycodone in neuropathic pain condition, thus providing new insight into the molecular mechanisms underlying the sustained analgesic action of oxycodone.
doi:10.1371/journal.pone.0091297
PMCID: PMC3949760  PMID: 24618941
16.  Chondroitinase ABC-Mediated Plasticity of Spinal Sensory Function 
The Journal of Neuroscience  2008;28(46):11998-12009.
Experimental therapeutics designed to enhance recovery from spinal cord injury (SCI) primarily focus on augmenting the growth of damaged axons by elevating their intrinsic growth potential and/or by nullifying the influence of inhibitory proteins present in the mature CNS. However, these strategies may also influence the wiring of intact pathways. The direct contribution of such effects to functional restoration after injury has been mooted, but as yet not been described. Here, we provide evidence to support the hypothesis that reorganization of intact spinal circuitry enhances function after SCI. Adult rats that underwent unilateral cervical spared-root lesion (rhizotomy of C5, C6, C8, and T1, sparing C7) exhibited profound sensory deficits for 4 weeks after injury. Delivery of a focal intraspinal injection of the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC (ChABC) was sufficient to restore sensory function after lesion. In vivo electrophysiological recordings confirm that behavioral recovery observed in ChABC-treated rats was consequent on reorganization of intact C7 primary afferent terminals and not regeneration of rhizotomized afferents back into the spinal cord within adjacent segments. These data confirm that intact spinal circuits have a profound influence on functional restoration after SCI. Furthermore, comprehensive understanding of these targets may lead to therapeutic interventions that can be spatially tailored to specific circuitry, thereby reducing unwanted maladaptive axon growth of distal pathways.
doi:10.1523/JNEUROSCI.3877-08.2008
PMCID: PMC3844838  PMID: 19005065
chondroitin sulfate; plasticity; spinal cord injury; traumatic injury; dorsal rhizotomy; proteoglycan
17.  Opening paths to novel analgesics: the role of potassium channels in chronic pain 
Trends in Neurosciences  2014;37(3):146-158.
Highlights
•Potassium (K+) channels are crucial determinants of neuronal excitability.•Nerve injury or inflammation alters K+ channel activity in neurons of the pain pathway.•These changes can render neurons hyperexcitable and cause chronic pain.•Therapies targeting K+ channels may provide improved pain relief in these states.
Chronic pain is associated with abnormal excitability of the somatosensory system and remains poorly treated in the clinic. Potassium (K+) channels are crucial determinants of neuronal activity throughout the nervous system. Opening of these channels facilitates a hyperpolarizing K+ efflux across the plasma membrane that counteracts inward ion conductance and therefore limits neuronal excitability. Accumulating research has highlighted a prominent involvement of K+ channels in nociceptive processing, particularly in determining peripheral hyperexcitability. We review salient findings from expression, pharmacological, and genetic studies that have untangled a hitherto undervalued contribution of K+ channels in maladaptive pain signaling. These emerging data provide a framework to explain enigmatic pain syndromes and to design novel pharmacological treatments for these debilitating states.
doi:10.1016/j.tins.2013.12.002
PMCID: PMC3945816  PMID: 24461875
potassium channel; pain; dorsal root ganglia; pharmacotherapy
18.  Genes and epigenetic processes as prospective pain targets 
Genome Medicine  2013;5(2):12.
Chronic pain affects approximately one in five adults, resulting in a greatly reduced quality of life and a higher risk of developing co-morbidities such as depression. Available treatments often provide inadequate pain relief, but it is hoped that through deeper understanding of the molecular mechanisms underlying chronic pain states we can discover new and improved therapies. Although genetic research has flourished over the past decade and has identified many key genes in pain processing, the budding field of epigenetics promises to provide new insights and a more dynamic view of pain regulation. This review gives an overview of basic mechanisms and current therapies to treat pain, and discusses the clinical and preclinical evidence for the contribution of genetic and epigenetic factors, with a focus on how this knowledge can affect drug development.
doi:10.1186/gm416
PMCID: PMC3706821  PMID: 23409739
19.  A comparison of RNA-seq and exon arrays for whole genome transcription profiling of the L5 spinal nerve transection model of neuropathic pain in the rat 
Molecular Pain  2014;10:7.
Background
The past decade has seen an abundance of transcriptional profiling studies of preclinical models of persistent pain, predominantly employing microarray technology. In this study we directly compare exon microarrays to RNA-seq and investigate the ability of both platforms to detect differentially expressed genes following nerve injury using the L5 spinal nerve transection model of neuropathic pain. We also investigate the effects of increasing RNA-seq sequencing depth. Finally we take advantage of the “agnostic” approach of RNA-seq to discover areas of expression outside of annotated exons that show marked changes in expression following nerve injury.
Results
RNA-seq and microarrays largely agree in terms of the genes called as differentially expressed. However, RNA-seq is able to interrogate a much larger proportion of the genome. It can also detect a greater number of differentially expressed genes than microarrays, across a wider range of fold changes and is able to assign a larger range of expression values to the genes it measures. The number of differentially expressed genes detected increases with sequencing depth. RNA-seq also allows the discovery of a number of genes displaying unusual and interesting patterns of non-exonic expression following nerve injury, an effect that cannot be detected using microarrays.
Conclusion
We recommend the use of RNA-seq for future high-throughput transcriptomic experiments in pain studies. RNA-seq allowed the identification of a larger number of putative candidate pain genes than microarrays and can also detect a wider range of expression values in a neuropathic pain model. In addition, RNA-seq can interrogate the whole genome regardless of prior annotations, being able to detect transcription from areas of the genome not currently annotated as exons. Some of these areas are differentially expressed following nerve injury, and may represent novel genes or isoforms. We also recommend the use of a high sequencing depth in order to detect differential expression for genes with low levels of expression.
doi:10.1186/1744-8069-10-7
PMCID: PMC4021616  PMID: 24472155
Whole-genome transcription profiling; Exon arrays; Microarrays; RNA-Sequencing; RNA-seq; Next generation sequencing; Spinal nerve transection; Nerve injury; Neuropathic pain; Differential gene expression
20.  Defining the nociceptor transcriptome 
Unbiased “omics” techniques, such as next generation RNA-sequencing, can provide entirely novel insights into biological systems. However, cellular heterogeneity presents a significant barrier to analysis and interpretation of these datasets. The neurons of the dorsal root ganglia (DRG) are an important model for studies of neuronal injury, regeneration and pain. The majority of investigators utilize a dissociated preparation of whole ganglia when studying cellular and molecular function. We demonstrate that the standard methods for producing these preparations gives a 10%-neuronal mixture of cells, with the remainder of cells constituting satellite glia and other non-neuronal cell types. Using a novel application of magnetic purification, we consistently obtain over 95% pure, viable neurons from adult tissue, significantly enriched for small diameter nociceptors expressing the voltage gated ion channel Nav1.8. Using genome-wide RNA-sequencing we compare the currently used (10% neuronal) and pure (95% nociceptor) preparations and find 920 genes enriched. This gives an unprecedented insight into the molecular composition of small nociceptive neurons in the DRG, potentially altering the interpretation of previous studies performed at the tissue level, and indicating a number of novel markers of this widely-studied population of cells. We anticipate that the ease of use, affordability and speed of this technique will see it become widely adopted, delivering a greatly improved capacity to study the roles of nociceptors in health and disease.
doi:10.3389/fnmol.2014.00087
PMCID: PMC4227287  PMID: 25426020
pain; nociceptors; nociception; somatosensation; dorsal root ganglion; RNA-sequencing; peripheral nervous system; regeneration
21.  Kv2 dysfunction after peripheral axotomy enhances sensory neuron responsiveness to sustained input☆ 
Experimental Neurology  2014;251(100):115-126.
Peripheral nerve injuries caused by trauma are associated with increased sensory neuron excitability and debilitating chronic pain symptoms. Axotomy-induced alterations in the function of ion channels are thought to largely underlie the pathophysiology of these phenotypes. Here, we characterise the mRNA distribution of Kv2 family members in rat dorsal root ganglia (DRG) and describe a link between Kv2 function and modulation of sensory neuron excitability. Kv2.1 and Kv2.2 were amply expressed in cells of all sizes, being particularly abundant in medium-large neurons also immunoreactive for neurofilament-200. Peripheral axotomy led to a rapid, robust and long-lasting transcriptional Kv2 downregulation in the DRG, correlated with the onset of mechanical and thermal hypersensitivity. The consequences of Kv2 loss-of-function were subsequently investigated in myelinated neurons using intracellular recordings on ex vivo DRG preparations. In naïve neurons, pharmacological Kv2.1/Kv2.2 inhibition by stromatoxin-1 (ScTx) resulted in shortening of action potential (AP) after-hyperpolarization (AHP). In contrast, ScTx application on axotomized neurons did not alter AHP duration, consistent with the injury-induced Kv2 downregulation. In accordance with a shortened AHP, ScTx treatment also reduced the refractory period and improved AP conduction to the cell soma during high frequency stimulation. These results suggest that Kv2 downregulation following traumatic nerve lesion facilitates greater fidelity of repetitive firing during prolonged input and thus normal Kv2 function is postulated to limit neuronal excitability. In summary, we have profiled Kv2 expression in sensory neurons and provide evidence for the contribution of Kv2 dysfunction in the generation of hyperexcitable phenotypes encountered in chronic pain states.
Highlights
•Kv2.1 and Kv2.2 are expressed in rat dorsal root ganglion neurons.•Kv2 subunits are most abundant in myelinated sensory neurons.•Kv2.1 and Kv.2 subunits are downregulated in a traumatic nerve injury pain model.•Kv2 inhibition ex vivo allows higher firing rates during sustained stimulation.•We conclude that Kv2 channels contribute to limiting peripheral neuron excitability.
doi:10.1016/j.expneurol.2013.11.011
PMCID: PMC3898477  PMID: 24252178
AP, action potential; APD50, AP half width; AHPD50, after-hyperpolarization half width; ATF3, activating transcription factor 3; CGRP, calcitonin gene-related peptide; CNS, central nervous system; DRG, dorsal root ganglion; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IB4, isolectin B4; IHC, immunohistochemistry; IR, input resistance; ISH, in situ hybridization; Kv channel, voltage-gated potassium channel; NF200, neurofilament 200; RP, refractory period; ScTx, stromatoxin-1; SNT, spinal nerve transection; Neuropathic pain; Potassium channel; Dorsal root ganglia
22.  Synthesis of Lipid Mediators during UVB-Induced Inflammatory Hyperalgesia in Rats and Mice 
PLoS ONE  2013;8(12):e81228.
Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.
doi:10.1371/journal.pone.0081228
PMCID: PMC3857181  PMID: 24349046
23.  Probing Functional Properties of Nociceptive Axons Using a Microfluidic Culture System 
PLoS ONE  2013;8(11):e80722.
Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. Microfluidic chambers (MFCs) can provide unique insight into the axonal compartment independent of the soma. Here we demonstrate how an MFC based cell culture system can be readily adapted for the study of axonal function in vitro. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized DRG axons using calcium imaging at the soma for pharmacological screening or patch-clamp electrophysiology for detailed biophysical characterisation. To demonstrate the adaptability of the system, we report by way of example functional changes in nociceptor axons following sensitization by neurotrophins and axotomy in vitro. We show that NGF can locally sensitize axonal responses to capsaicin, independent of the soma. Axotomizing neurons in MFC results in a significant increase in the proportion of neurons that respond to axonal stimulation, and interestingly leads to accumulation of Nav1.8 channels in regenerating axons. Axotomy also augmented AP amplitude following axotomy and altered activation thresholds in a subpopulation of regenerating axons. We further show how the system can readily be used to study modulation of axonal function by non-neuronal cells such as keratinocytes. Hence we describe a novel in vitro platform for the study of axonal function and a surrogate model for nerve injury and sensitization.
doi:10.1371/journal.pone.0080722
PMCID: PMC3835735  PMID: 24278311
24.  Chemokine expression in peripheral tissues from the Monosodium Iodoacetate model of chronic joint pain 
Molecular Pain  2013;9:57.
Background
Chronic pain arising from degenerative diseases of the joint such as osteoarthritis (OA) has a strong peripheral component which is likely to be mediator driven. Current treatments which reduce the production of such mediators i.e. non-steroidal anti-inflammatory drugs (NSAIDs), can help to lessen pain in OA patients. However, this is not always the case and complete pain relief is rarely achieved, suggesting that additional unidentified mediators play a role. Here we have investigated the notion that chemokines might act as such pain mediators in OA.
Results
Using the monosodium iodoacetate (MIA) model of chronic joint pain the expression of over 90 different inflammatory mediators, mainly cytokines and chemokines, were measured in tissues taken from the femorotibial joint (cartilage, subchondral bone, fat pad) using custom-made quantitative real-time polymerase chain reaction (qPCR) array cards. At both the day 3 and 14 time points, numerous inflammatory mediators were significantly up-regulated in these tissues, although it was clear that the largest transcriptional dysregulation occurred in the cartilage. Using individual qPCR to measure immune cell markers, a significant infiltration of macrophages was measured in the cartilage and fat pad at day 3. Neutrophil infiltration was also measured in the fat pad at the same time point, but no infiltration was observed at day 14. Combination of mRNA expression data from different time points and tissues identified the chemokines, CCL2, 7 and 9 as being consistently up-regulated. The overall increase in CCL2 expression was also measured at the protein level.
Conclusion
Chemokines in general and CCL2, 7 and 9 in particular, represent promising targets for further studies into the identification of new pain mediators in chronic joint pain.
doi:10.1186/1744-8069-9-57
PMCID: PMC3835139  PMID: 24206615
Pain; Chemokine; Osteoarthritis; Monosodium iodoacetate; Macrophages; Neutrophils
25.  HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain 
Pain  2013;154(9):1668-1679.
Summary
Intrathecal delivery of histone deacetylase inhibitors ameliorates hypersensitivity in models of neuropathic pain. This effect may be mediated at the level of the spinal cord through inhibition of HDAC1 function.
Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug–induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.
doi:10.1016/j.pain.2013.05.021
PMCID: PMC3763368  PMID: 23693161
Histone deacetylase; Histone deacetylase inhibitors; Neuropathic pain

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