13 research outputs found

    Acute- and late-phase matrix metalloproteinase (MMP)-9 activity is comparable in female and male rats after peripheral nerve injury.

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    BACKGROUND:In the peripheral nerve, pro-inflammatory matrix metalloproteinase (MMP)-9 performs essential functions in the acute response to injury. Whether MMP-9 activity contributes to late-phase injury or whether MMP-9 expression or activity after nerve injury is sexually dimorphic remains unknown. METHODS:Patterns of MMP-9 expression, activity and excretion were assessed in a model of painful peripheral neuropathy, sciatic nerve chronic constriction injury (CCI), in female and male rats. Real-time Taqman RT-PCR for MMP-9 and its endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) of nerve samples over a 2-month time course of CCI was followed by gelatin zymography of crude nerve extracts and purified MMP-9 from the extracts using gelatin Sepharose-beads. MMP excretion was determined using protease activity assay of urine in female and male rats with CCI. RESULTS:The initial upsurge in nerve MMP-9 expression at day 1 post-CCI was superseded more than 100-fold at day 28 post-CCI. The high level of MMP-9 expression in late-phase nerve injury was accompanied by the reduction in TIMP-1 level. The absence of MMP-9 in the normal nerve and the presence of multiple MMP-9 species (the proenzyme, mature enzyme, homodimers, and heterodimers) was observed at day 1 and day 28 post-CCI. The MMP-9 proenzyme and mature enzyme species dominated in the early- and late-phase nerve injury, consistent with the high and low level of TIMP-1 expression, respectively. The elevated nerve MMP-9 levels corresponded to the elevated urinary MMP excretion post-CCI. All of these findings were comparable in female and male rodents. CONCLUSION:The present study offers the first evidence for the excessive, uninhibited proteolytic MMP-9 activity during late-phase painful peripheral neuropathy and suggests that the pattern of MMP-9 expression, activity, and excretion after peripheral nerve injury is universal in both sexes

    Cholesterol-Dependent LXR Transcription Factor Activity Represses Pronociceptive Effects of Estrogen in Sensory Neurons and Pain Induced by Myelin Basic Protein Fragments

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    BACKGROUND: A bioactive myelin basic protein (MBP) fragment, comprising MBP METHODS: In male and female normal and post-CCI rat sciatic nerves, we assessed: (i) cholesterol precursor and metabolite levels by lipidomics; (ii) MBP RESULTS: CCI regulated LXRα ligand and receptor levels in nerves of both sexes, with cholesterol precursors, desmosterol and 7-DHC, and oxysterol elevated in females relative to males. MBP CONCLUSION: The injury-released bioactive MBP fragments induce pronociceptive changes by selective inactivation of nuclear transcription factors, including LXRα. By Ncoa1 sequestration, bioactive MBP fragments render LXRα function to counteract pronociceptive activity of estrogen/ESR1 in sensory neurons. This effect of MBP fragments is prevalent in females due to high circulating estrogen levels in females relative to males. Restoring LXR activity presents a promising therapeutic strategy in management of neuropathic pain induced by bioactive MBP

    The mechanism of functional up-regulation of P2X3 receptors of trigeminal sensory neurons in a genetic mouse model of Familial Hemiplegic Migraine type 1 (FHM-1)

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    A knock-in (KI) mouse model of FHM-1 expressing the R192Q missense mutation of the Cacna1a gene coding for the \u3b11 subunit of CaV2.1 channels shows, at the level of the trigeminal ganglion, selective functional up-regulation of ATP -gated P2X3 receptors of sensory neurons that convey nociceptive signals to the brainstem. Why P2X3 receptors are constitutively more responsive, however, remains unclear as their membrane expression and TRPV1 nociceptor activity are the same as in wildtype (WT) neurons. Using primary cultures of WT or KI trigeminal ganglia, we investigated whether soluble compounds that may contribute to initiating (or maintaining) migraine attacks, such as TNF\u3b1, CGRP, and BDNF, might be responsible for increasing P2X3 receptor responses. Exogenous application of TNF\u3b1 potentiated P2X3 receptor-mediated currents of WT but not of KI neurons, most of which expressed both the P2X3 receptor and the TNF\u3b1 receptor TNFR2. However, sustained TNF\u3b1 neutralization failed to change WT or KI P2X3 receptor currents. This suggests that endogenous TNF\u3b1 does not regulate P2X3 receptor responses. Nonetheless, on cultures made from both genotypes, exogenous TNF\u3b1 enhanced TRPV1 receptor-mediated currents expressed by a few neurons, suggesting transient amplification of TRPV1 nociceptor responses. CGRP increased P2X3 receptor currents only in WT cultures, although prolonged CGRP receptor antagonism or BDNF neutralization reduced KI currents to WT levels. Our data suggest that, in KI trigeminal ganglion cultures, constitutive up-regulation of P2X3 receptors probably is already maximal and is apparently contributed by basal CGRP and BDNF levels, thereby rendering these neurons more responsive to extracellular ATP. \ua9 2013 Hullugundi et al

    Sensitization of TRPV1 receptors by TNFα.

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    <p>A, Representative traces of currents induced by application of capsaicin (1 µM, 2 s) to WT or R192Q KI neurons in control conditions or after 4 h application of TNFα. B, Histograms show average peak amplitudes of TRPV1-mediated currents (WT control, n = 29; WT TNFα 50 ng/mL n = 19, WT TNFα 100 ng/mL, n = 10; KI control, n = 26; KI TNFα 50 ng/mL, n = 23,, KI TNFα 100 ng/mL n = 9); * = p<0.05, ** = p<0.002, *** = p<0.001.</p

    Number of BDNF expressing neurons in KI culture and effect of BDNF deprivation.

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    <p>A, Examples of β-tubulin positive neurons expressing BDNF in WT or KI cultures. Left panel (green) shows BDNF expression and right panel (red) shows β-tubulin staining of the same neurons. B, Histograms quantifying % of neurons expressing BDNF: KI cultures showed significantly higher number of BDNF positive neurons. N = 4 independent experiments (8 mice), p<0.05. C, Representative traces of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after overnight application of anti-BDNF antibody. D, Histograms show average peak amplitudes of P2X3 receptor-mediated currents (WT control, n = 9; WT anti-BDNF, n = 10; KI control, n = 32; KI anti-BDNF, n = 38); * = p<0.05.</p

    Role of CGRP in KI P2X3 currents.

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    <p>A, Representative traces of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after 2 h CGRP (1 µM) application. B, Histograms show average peak amplitudes of P2X3 receptor-mediated currents (WT control, n = 11; WT CGRP, n = 9; KI control, n = 17; KI CGRP, n = 18), * = p<0.05. C, Representative examples of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after overnight application of the CGRP antagonist CGRP<sub>8–37</sub> (1 µM). D, Histograms show average peak amplitudes of P2X3 receptor-mediated currents (WT control, n = 23; WT CGRP<sub>8–37</sub>, n = 23; KI control, n = 29; KI CGRP<sub>8–37</sub>, n = 30); * = p<0.05.</p

    Effect of TNFα on P2X3 receptor activity and co-expression of its TNFR2 receptors.

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    <p>A, Examples of TNFR2 and P2X3 co-exexpression in (wildtype) WT and R192Q (knockin) KI neurons. Left panel shows P2X3 expression (green), and right panel shows TNFR2 staining (red). B, Histograms quantifying % of cells co-expressing TNFR2 and P2X3: both WT and KI cultures show similar TNFR2 and P2X3 co-expression. N = 3 independent experiments (6 mice). C, Representative traces of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after 4 h TNFα application. D, Histograms show average peak amplitudes of P2X3 receptor-mediated currents: WT control (open bar), n = 30; WT TNFα (stippled bar), n = 38; KI control (grey bar), n = 34; KI TNFα (stippled gray bar), n = 34; ** = p<0.006; * = p<0.05.</p

    Effects of LPS on P2X3 receptors of trigeminal sensory neurons and macrophages from mice expressing the R192Q Cacna1a gene mutation of familial hemiplegic migraine-1

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    A knockin (KI) mouse model with the R192Q missense mutation in the Cacna1a gene commonly detected in familial hemiplegic migraine was used to study whether trigeminal ganglia showed a basal inflammatory profile that could be further enhanced by the lipopolysaccharide (LPS) toxin. Adenosine-5\u2032-triphosphate (ATP)-gated purinergic ionotropic receptor 3 (P2X3) currents expressed by the large majority of trigeminal sensory neurons were taken as functional readout. Cultured R192Q KI trigeminal ganglia showed higher number of active macrophages, basal release of tumor necrosis factor alpha (TNF\u3b1), and larger P2X3 receptor currents with respect to wild type (WT) cells. After 5 h application of LPS in vitro, both WT and R192Q KI cultures demonstrated significant increase in macrophage activation, very large rise in TNF\u3b1 mRNA content, and ambient protein levels together with fall in TNF\u3b1 precursor, suggesting potent release of this inflammatory mediator. Notwithstanding the unchanged expression of P2X3 receptor protein in WT or R192Q KI cultures, LPS evoked a large rise in WT neuronal currents that recovered faster from desensitization. Basal R192Q KI currents were larger than WT ones and could not be further augmented by LPS. These data suggest that KI cultures had a basal neuroinflammatory profile that might facilitate the release of endogenous mediators (including ATP) to activate constitutively hyperfunctional P2X3 receptors and amplify nociceptive signaling by trigeminal sensory neurons. \ua9 2012 Springer Science+Business Media B.V
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