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  • Genes, molecules and patients--emerging topics to guide clinical pain research.

    3 July 2018

    This review selectively explores some areas of pain research that, until recently, have been poorly understood. We have chosen four topics that relate to clinical pain and we discuss the underlying mechanisms and related pathophysiologies contributing to these pain states. A key issue in pain medicine involves crucial events and mediators that contribute to normal and abnormal pain signaling, but remain unseen without genetic, biomarker or imaging analysis. Here we consider how the altered genetic make-up of familial pains reveals the human importance of channels discovered by preclinical research, followed by the contribution of receptors as stimulus transducers in cold sensing and cold pain. Finally we review recent data on the neuro-immune interactions in chronic pain and the potential targets for treatment in cancer-induced bone pain.

  • Characterisation and mechanisms of bradykinin-evoked pain in man using iontophoresis.

    3 July 2018

    Bradykinin (BK) is an inflammatory mediator that can evoke oedema and vasodilatation, and is a potent algogen signalling via the B1 and B2 G-protein coupled receptors. In naïve skin, BK is effective via constitutively expressed B2 receptors (B2R), while B1 receptors (B1R) are purported to be upregulated by inflammation. The aim of this investigation was to optimise BK delivery to investigate the algesic effects of BK and how these are modulated by inflammation. BK iontophoresis evoked dose- and temperature-dependent pain and neurogenic erythema, as well as thermal and mechanical hyperalgesia (P < 0.001 vs saline control). To differentiate the direct effects of BK from indirect effects mediated by histamine released from mast cells (MCs), skin was pretreated with compound 4880 to degranulate the MCs prior to BK challenge. The early phase of BK-evoked pain was reduced in degranulated skin (P < 0.001), while thermal and mechanical sensitisation, wheal, and flare were still evident. In contrast to BK, the B1R selective agonist des-Arg9-BK failed to induce pain or sensitise naïve skin. However, following skin inflammation induced by ultraviolet B irradiation, this compound produced a robust pain response. We have optimised a versatile experimental model by which BK and its analogues can be administered to human skin. We have found that there is an early phase of BK-induced pain which partly depends on the release of inflammatory mediators by MCs; however, subsequent hyperalgesia is not dependent on MC degranulation. In naïve skin, B2R signaling predominates, however, cutaneous inflammation results in enhanced B1R responses.

  • Characterisation of a peripheral neuropathic component of the rat monoiodoacetate model of osteoarthritis.

    3 July 2018

    Joint degeneration observed in the rat monoiodoacetate (MIA) model of osteoarthritis shares many histological features with the clinical condition. The accompanying pain phenotype has seen the model widely used to investigate the pathophysiology of osteoarthritis pain, and for preclinical screening of analgesic compounds. We have investigated the pathophysiological sequellae of MIA used at low (1 mg) or high (2 mg) dose. Intra-articular 2 mg MIA induced expression of ATF-3, a sensitive marker for peripheral neuron stress/injury, in small and large diameter DRG cell profiles principally at levels L4 and 5 (levels predominated by neurones innervating the hindpaw) rather than L3. At the 7 day timepoint, ATF-3 signal was significantly smaller in 1 mg MIA treated animals than in the 2 mg treated group. 2 mg, but not 1 mg, intra-articular MIA was also associated with a significant reduction in intra-epidermal nerve fibre density in plantar hindpaw skin, and produced spinal cord dorsal and ventral horn microgliosis. The 2 mg treatment evoked mechanical pain-related hypersensitivity of the hindpaw that was significantly greater than the 1 mg treatment. MIA treatment produced weight bearing asymmetry and cold hypersensitivity which was similar at both doses. Additionally, while pregabalin significantly reduced deep dorsal horn evoked neuronal responses in animals treated with 2 mg MIA, this effect was much reduced or absent in the 1 mg or sham treated groups. These data demonstrate that intra-articular 2 mg MIA not only produces joint degeneration, but also evokes significant axonal injury to DRG cells including those innervating targets outside of the knee joint such as hindpaw skin. This significant neuropathic component needs to be taken into account when interpreting studies using this model, particularly at doses greater than 1 mg MIA.

  • ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data.

    3 July 2018

    BACKGROUND: Measuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR) technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s). RESULTS: We have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.html CONCLUSIONS: These packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things) read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples.

  • NGF but not NT-3 or BDNF prevents the A fiber sprouting into lamina II of the spinal cord that occurs following axotomy.

    3 July 2018

    There is a stereotypical pattern of primary afferent terminations within the mature spinal cord; however, this pattern is not immutable. Peripheral axotomy causes A fibers to sprout into lamina II, a region from which they are normally excluded. We have investigated the role of neurotrophins in this response. Rats which had undergone sciatic axotomy were treated intrathecally with NGF, BDNF, or NT-3. A fibers were visualized using transganglionic labeling with cholera toxin B subunit; small fibers were visualized using CGRP immunostaining. NGF (12 microg/day for 2 weeks), but not NT-3 or BDNF, prevented both the axotomy-induced reduction in CGRP staining within lamina II and the sprouting of A fibers into this region. It is likely that the prevention of A fiber sprouting is a secondary consequence of NGF rescuing small fibers. This effect of NGF on dorsal horn sprouting has implications both for our understanding of the maintenance of CNS connectivity and for the treatment of neuropathic pain states.

  • The use of nerve and muscle biopsy in the diagnosis of vasculitis: a 5 year retrospective study.

    3 July 2018

    INTRODUCTION: Peripheral nerve vasculitis is an important condition which can be diagnostically challenging and is one of the principal current indications for nerve and muscle biopsy. Previous studies have suggested that combined nerve and muscle biopsy (usually of the superficial peroneal nerve and peroneus brevis muscle) produces a higher diagnostic yield than nerve biopsy alone in the investigation of vasculitis. OBJECTIVE: To determine whether in our two centres combined nerve (usually the sural) and muscle (usually the vastus lateralis) biopsy improved diagnostic yield compared with nerve biopsy alone. METHODS: We interrogated our database of all nerve biopsies (usually of the sural nerve) performed at our institutions over 5 years and identified 53 cases of biopsy proven peripheral nerve vasculitis. Clinicopathological and neurophysiological data in these patients were reviewed. RESULTS: The most common clinical presentation was with a painful asymmetric axonal polyneuropathy or mononeuritis multiplex (66% of cases). Nerve biopsy demonstrated definite vasculitis in 36%, probable vasculitis in 62% and no vasculitis in 2% of cases. In 24 patients a muscle biopsy (usually the vastus lateralis) was also performed and vasculitis was demonstrated in 46% of these (in 13% showing definite and 33% probable vasculitis). There was only one patient in whom vasculitis was demonstrated in muscle but not in peripheral nerve. CONCLUSION: Combined nerve (usually sural) and vastus lateralis muscle biopsy did not significantly increase the diagnostic yield compared with nerve biopsy alone. A sensible approach to the diagnosis of peripheral nerve vasculitis is to choose a nerve to biopsy which is clinically affected and amenable to biopsy. If the sural nerve is chosen, the data suggest that it is not routinely worth doing a vastus lateralis biopsy at the same time, whereas if the superficial peroneal nerve is chosen, it seems appropriate to do a combined superficial peroneal nerve and peroneus brevis biopsy. It is still not known if both the sural and superficial peroneal nerves are involved clinically which one gives the higher yield if biopsied.

  • Artemin has potent neurotrophic actions on injured C-fibres.

    3 July 2018

    In this study, we have investigated the effects of artemin (ARTN), one of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors, on C-fibres following nerve injury in the adult rat. GDNF family receptor alpha (GFRalpha) 3, the ligand binding domain of the ARTN receptor, is expressed in 34% of dorsal root ganglion (DRG) cells, predominantly in the peptidergic population of C-fibres and in a proportion of the isolectin B4 (IB4)-binding population. Interestingly, only 30% of GFRalpha3-expressing DRG cells co-expressed RET (the signal transducing domain). In agreement with previous studies, treatment with ARTN prevented many of the nerve injury-induced changes in the histochemistry of both the peptidergic and the IB4-binding populations of small, but not large, diameter DRG cells. In addition, ARTN treatment maintained C-fibre conduction velocity, and C-fibre evoked substance P release within the dorsal horn following nerve injury. ARTN was also protective following capsaicin treatment, which produces selective C-fibre injury. Given the potent neurotrophic actions of ARTN on C-fibres, it may therefore provide potential for the treatment of nerve injury, particularly in the maintenance of small fibre function.

  • Canomad presenting without ophthalmoplegia and responding to intravenous immunoglobulin.

    3 July 2018

    The acronym CANOMAD encompasses chronic ataxic neuropathy combined with ophthalmoplegia, M protein, cold agglutinins, and anti-disialosyl antibodies.Herein we describe 2 patients presenting with progressive ataxic neuropathy who only developed ophthalmoplegia after a significant delay post-presentation, which in 1 case had features indicative of brainstem dysfunction. Both patients were found to have an IgM paraprotein and anti-disialosyl antibodies. They responded to treatment with intravenous immunoglobulin, thus illustrating the importance of diagnosing this condition.