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First paragraph (this article has no abstract) Persistent stimulation of nociceptors results in sensitization of nociceptive sensory neurons, which is associated with hyperalgesia and allodynia. The release of NO and subsequent synthesis of cGMP in the spinal cord are involved in this process. cGMP-dependent protein kinase I (PKG-I) has been suggested to act as a downstream target of cGMP, but its exact role in nociception hadn't been characterized yet. To further evaluate the NO/cGMP/PKG-I pathway in nociception we assessed the effects of PKG-I inhibiton and activaton in the rat formalin assay and analyzed the nociceptive behavior of PKG-I-/- mice. Open access article.
Oral presentation from 4th International Conference of cGMP Generators, Effectors and Therapeutic Implications ; Regensburg, Germany. 19–21 June 2009 Background: An exaggerated pain sensitivity is the dominant feature of inflammatory and neuropathic pain both in the clinical setting and in experimental animal models. It manifests as pain in response to normally innocuous stimuli (allodynia), increased response to noxious stimuli (hyperalgesia) or spontaneous pain, and can persist long after the initial injury is resolved. Research over the last decades has revealed that several signaling pathways in the spinal cord essentially contribute to the pain sensitization. To test the contribution of cGMP produced by NO-sensitive guanylyl cyclase (NO-GC) to pain sensitization, we investigated the localization of NO-GC in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). Results: We show that NO-GC (β1 subunit) is distinctly expressed in neurons of the mouse spinal cord, while its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by spinal administration of drugs releasing NO. Surprisingly, during spinal nociceptive processing cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), while cGKI can be activated by natriuretic peptide receptor-B (NPR-B) dependent cGMP production. Conclusion: Taken together, our results provide evidence that NO-GC has a dominant role in the development of exaggerated pain sensitivity during inflammatory and neuropathic pain. Furthermore, beside the NO-mediated cGMP synthesis, cGMP produced by NPR-B contributes to pain sensitization by activation of cGKI.
Background: R-flurbiprofen, one of the enantiomers of flurbiprofen racemate, is inactive with respect to cyclooxygenase inhibition, but shows analgesic properties without relevant toxicity. Its mode of action is still unclear. Methodology/Principal Findings: We show that R-flurbiprofen reduces glutamate release in the dorsal horn of the spinal cord evoked by sciatic nerve injury and thereby alleviates pain in sciatic nerve injury models of neuropathic pain in rats and mice. This is mediated by restoring the balance of endocannabinoids (eCB), which is disturbed following peripheral nerve injury in the DRGs, spinal cord and forebrain. The imbalance results from transcriptional adaptations of fatty acid amide hydrolase (FAAH) and NAPE-phospholipase D, i.e. the major enzymes involved in anandamide metabolism and synthesis, respectively. R-flurbiprofen inhibits FAAH activity and normalizes NAPE-PLD expression. As a consequence, R-Flurbiprofen improves endogenous cannabinoid mediated effects, indicated by the reduction of glutamate release, increased activity of the anti-inflammatory transcription factor PPAR gamma and attenuation of microglia activation. Antinociceptive effects are lost by combined inhibition of CB1 and CB2 receptors and partially abolished in CB1 receptor deficient mice. R-flurbiprofen does however not cause changes of core body temperature which is a typical indicator of central effects of cannabinoid-1 receptor agonists. Conclusion: Our results suggest that R-flurbiprofen improves the endogenous mechanisms to regain stability after axonal injury and to fend off chronic neuropathic pain by modulating the endocannabinoid system and thus constitutes an attractive, novel therapeutic agent in the treatment of chronic, intractable pain.
Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.
R-flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S-flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E2 (PGE2) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R-flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R-flurbiprofen traps PGE2 inside of the cells by inhibiting multidrug resistance–associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R-flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R-flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.
Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis
(2017)
Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the ‘symptom-free’ intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach.
Monoclonal antibodies (mAb) are promising therapeutics in multiple sclerosis and multiple new candidates have been developed, hence increasing the need for some agreement for preclinical mAb studies. We systematically analyzed publications of experimental autoimmune encephalomyelitis (EAE) studies showing effects of monoclonal antibodies. A PubMed search retrieved 570 records, out of which 122 studies with 253 experiments were eligible based on experimental design, number of animals and presentation of time courses of EAE scores. Analysis of EAE models, treatment schedules, single and total doses, routes of administration, and onset of treatment from pre-immunization up to 35 days after immunization revealed high heterogeneity. Total doses ranged from 0.1 to 360 mg/kg for observation times of up to 35 days after immunization. About half of experiments (142/253) used total doses of 10–70 mg/kg. Employing this range, we tested anti-Itga4 as a reference mAb at varying schedules and got no, mild or substantial EAE-score reductions, depending on the mouse strain and onset of the treatment. The result agrees with the range of outcomes achieved in 10 reported anti-Itga4 experiments. Studies comparing low and high doses of various mAbs or early vs. late onset of treatment did not reveal dose-effect or timing-effect associations, with a tendency towards better outcomes with preventive treatments starting within the first week after immunization. The systematic comparison allows for extraction of some “common” design characteristics, which may be helpful to further assess the efficacy of mAbs and role of specific targets in preclinical models of multiple sclerosis.
GTP cyclohydrolase (GCH1) governs de novo synthesis of the enzyme cofactor, tetrahydrobiopterin (BH4), which is essential for biogenic amine production, bioactive lipid metabolism and redox coupling of nitric oxide synthases. Overproduction of BH4 via upregulation of GCH1 in sensory neurons is associated with nociceptive hypersensitivity in rodents, and neuron‐specific GCH1 deletion normalizes nociception. The translational relevance is revealed by protective polymorphisms of GCH1 in humans, which are associated with a reduced chronic pain. Because myeloid cells constitute a major non‐neuronal source of BH4 that may contribute to BH4‐dependent phenotypes, we studied here the contribution of myeloid‐derived BH4 to pain and itch in lysozyme M Cre‐mediated GCH1 knockout (LysM‐GCH1−/−) and overexpressing mice (LysM‐GCH1‐HA). Unexpectedly, knockout or overexpression in myeloid cells had no effect on nociceptive behaviour, but LysM‐driven GCH1 knockout reduced, and its overexpression increased the scratching response in Compound 48/80 and hydroxychloroquine‐evoked itch models, which involve histamine and non‐histamine dependent signalling pathways. Mechanistically, GCH1 overexpression increased BH4, nitric oxide and hydrogen peroxide, and these changes were associated with increased release of histamine and serotonin and degranulation of mast cells. LysM‐driven GCH1 knockout had opposite effects, and pharmacologic inhibition of GCH1 provided even stronger itch suppression. Inversely, intradermal BH4 provoked scratching behaviour in vivo and BH4 evoked an influx of calcium in sensory neurons. Together, these loss‐ and gain‐of‐function experiments suggest that itch in mice is contributed by BH4 release plus BH4‐driven mediator release from myeloid immune cells, which leads to activation of itch‐responsive sensory neurons.
Lysophosphatidic acid (LPA) is a synaptic phospholipid, which regulates cortical excitation/inhibition (E/I) balance and controls sensory information processing in mice and man. Altered synaptic LPA signaling was shown to be associated with psychiatric disorders. Here, we show that the LPA-synthesizing enzyme autotaxin (ATX) is expressed in the astrocytic compartment of excitatory synapses and modulates glutamatergic transmission. In astrocytes, ATX is sorted toward fine astrocytic processes and transported to excitatory but not inhibitory synapses. This ATX sorting, as well as the enzymatic activity of astrocyte-derived ATX are dynamically regulated by neuronal activity via astrocytic glutamate receptors. Pharmacological and genetic ATX inhibition both rescued schizophrenia-related hyperexcitability syndromes caused by altered bioactive lipid signaling in two genetic mouse models for psychiatric disorders. Interestingly, ATX inhibition did not affect naive animals. However, as our data suggested that pharmacological ATX inhibition is a general method to reverse cortical excitability, we applied ATX inhibition in a ketamine model of schizophrenia and rescued thereby the electrophysiological and behavioral schizophrenia-like phenotype. Our data show that astrocytic ATX is a novel modulator of glutamatergic transmission and that targeting ATX might be a versatile strategy for a novel drug therapy to treat cortical hyperexcitability in psychiatric disorders.
Based on increasing evidence suggesting that MS pathology involves alterations in bioactive lipid metabolism, the present analysis was aimed at generating a complex serum lipid-biomarker. Using unsupervised machine-learning, implemented as emergent self-organizing maps of neuronal networks, swarm intelligence and Minimum Curvilinear Embedding, a cluster structure was found in the input data space comprising serum concentrations of d = 43 different lipid-markers of various classes. The structure coincided largely with the clinical diagnosis, indicating that the data provide a basis for the creation of a biomarker (classifier). This was subsequently assessed using supervised machine-learning, implemented as random forests and computed ABC analysis-based feature selection. Bayesian statistics-based biomarker creation was used to map the diagnostic classes of either MS patients (n = 102) or healthy subjects (n = 301). Eight lipid-markers passed the feature selection and comprised GluCerC16, LPA20:4, HETE15S, LacCerC24:1, C16Sphinganine, biopterin and the endocannabinoids PEA and OEA. A complex classifier or biomarker was developed that predicted MS at a sensitivity, specificity and accuracy of approximately 95% in training and test data sets, respectively. The present successful application of serum lipid marker concentrations to MS data is encouraging for further efforts to establish an MS biomarker based on serum lipidomics.