Quantitative and Microstructural Changes of the Blood-Nerve Barrier in Peripheral Neuropathy

Peripheral neuropathy is accompanied by changes in the neuronal environment. The blood-nerve barrier (BNB) is crucial in protecting the neural homeostasis: Tight junctions (TJ) seal paracellular spaces and thus prevent external stimuli from entering. In different models of neuropathic pain, the BNB is impaired, thus contributing to local damage, immune cell invasion and, ultimately, the development of neuropathy with its symptoms. In this study, we examined changes in expression and microstructural localization of two key tight junction proteins (TJP), claudin-1 and the cytoplasmic anchoring ZO-1, in the sciatic nerve of mice subjected to chronic constriction injury (CCI). Via qPCR and analysis of fluorescence immunohistochemistry, a marked downregulation of mRNA as well as decreased fluorescence intensity were observed in the nerve for both proteins. Moreover, a distinct zig-zag structure for both proteins located at cell-cell contacts, indicative of the localization of TJs, was observed in the perineurial compartment of sham-operated animals. This microstructural location in cell-cell-contacts was lost in neuropathy as semiquantified via computational analysis, based on a novel algorithm. In summary, we provide evidence that peripheral neuropathy is not only associated with decrease in relevant TJPs but also exhibits alterations in TJP arrangement and loss in barrier tightness, presumably due to internalization. Specifically, semiquantification of TJP in cell-cell-contacts of microcompartments could be used in the future for routine clinical samples of patients with neuropathy

The role of spinal GABAB receptors in cancer-induced bone pain in rats

Cancer-induced bone pain (CIBP) remains a major challenge in advanced cancer patients due to our lack of understanding of its mechanisms. Previous studies have demonstrated the vital role of GABAB receptors (GABABRs) in regulating nociception and various neuropathic pain models have shown diminished activity of GABABRs. However, the role of spinal GABABRs in CIBP remains largely unknown. In this study, we investigated the specific cellular mechanisms of GABABRs in the development and maintenance of CIBP in rats. Our behavioral results show that both acute and chronic intrathecal treatment with baclofen, a GABABR agonist, significantly attenuated CIBP-induced mechanical allodynia and ambulatory pain. The expression levels of GABABRs were significantly decreased in a time-dependent manner and colocalized mostly with neuron and a minority with astrocyte and microglia. Chronic treatment with baclofen restored the expression of GABABRs and markedly inhibited the activation of cAMP-dependent protein kinase (PKA) and the cAMP-response element-binding protein (CREB) signaling pathway

microRNAs in nociceptive circuits as predictors of future clinical applications

Neuro-immune alterations in the peripheral and central nervous system play a role in the pathophysiology of chronic pain, and non-coding RNAs - and microRNAs (miRNAs) in particular - regulate both immune and neuronal processes. Specifically, miRNAs control macromolecular complexes in neurons, glia and immune cells and regulate signals used for neuro-immune communication in the pain pathway. Therefore, miRNAs may be hypothesized as critically important master switches modulating chronic pain. In particular, understanding the concerted function of miRNA in the regulation of nociception and endogenous analgesia and defining the importance of miRNAs in the circuitries and cognitive, emotional and behavioral components involved in pain is expected to shed new light on the enigmatic pathophysiology of neuropathic pain, migraine and complex regional pain syndrome. Specific miRNAs may evolve as new druggable molecular targets for pain prevention and relief. Furthermore, predisposing miRNA expression patterns and inter-individual variations and polymorphisms in miRNAs and/or their binding sites may serve as biomarkers for pain and help to predict individual risks for certain types of pain and responsiveness to analgesic drugs. miRNA-based diagnostics are expected to develop into hands-on tools that allow better patient stratification, improved mechanism-based treatment, and targeted prevention strategies for high risk individuals. \ua9 2013 Kress, H\ufcttenhofer, Landry, Kuner, Favereaux, Greenberg, Bednarik, Heppenstall, Kronenberg, Malcangio, Rittner, c\ue7eyler, Trajanoski, Mouritzen, Birklein, Sommer and Soreq

Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

<p>Abstract</p> <p>Background</p> <p>Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE) vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS)-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC) on complete Freund's adjuvant-induced inflammatory pain in rats.</p> <p>Results</p> <p>POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin.</p> <p>Conclusion</p> <p>Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.</p

Mycobacteria Attenuate Nociceptive Responses by Formyl Peptide Receptor Triggered Opioid Peptide Release from Neutrophils

In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, β-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia

Die Rolle der Leukozyten bei der Entstehung und Kontrolle von Entzündungsschmerz

Inflammatory pain is modulated by hyperalgesic and analgesic mediators. Hyperalgesic mediators include protons, cytokines, chemokines, and bradykinin produced by leukocytes at the site of inflammation. Simultaneously, leukocytes are known to produce analgesic mediators e.g. opioid peptides including beta- endorphin, met-enkephalin and endomorphins. Opioid peptides are secreted locally following exposure to stress or by local injection of releasing agents like corticotrophin releasing hormone (CRF). Following release, they bind to opioid receptors on sensory nerve terminals and confer peripherally mediated opioid analgesia. In this thesis we examined four aims in inflammatory pain induced by complete Freund’s adjuvant (CFA) in rats: i) role of granulocytes in the generation of inflammatory pain, ii) impact of inflammation on transcription of opioid receptor genes in the dorsal root ganglion (DRG), iii) peripherally mediated opioid analgesia by endomorphins, and iv) signaling pathways of opioid peptide release from granulocytes. The results show that i) granulocytes do not seem to confer pain, because selective recruitment of granulocytes by chemokines does not alter thermal or mechanical nociceptive thresholds. ii) Kappa-opioid receptor mRNA as well as protein in the DRG is upregulated in CFA inflammation mediated by IL-1beta iii) The newly discovered endomorphins can elicit potent analgesia in inflammation. Stress- as well as CRF-induced peripherally mediated analgesia is dependent on the release of classic opioid peptides but also on the release of endomorphins. iv) In granulocytes, opioid peptide release by is CXCR1/2 ligands dependent on Ca2+ from intracellular stores, phosphoinositol-3-kinase (PI3K) and p38 mitogen activated kinase (MAPK). In vivo, intraplantar injection of CXCR2 ligands elicits analgesia in inflammatory pain. Taken together, granulocytes contribute to peripherally mediated opioid analgesia in early inflammation while their role in the generation of inflammatory pain seems to be limited. Interference with granulocyte function using e.g. anti-inflammatory treatments like chemokine receptor antagonists or inhibitors of intracellular signaling pathways might inadvertently impair endogenous peripherally mediated opioid analgesia.Entzündungsschmerz wird durch algetische und analgetische Mediatoren moduliert. Zu den algetischen Mediatoren aus Leukozyten gehören z.B. Protonen, Zytokine, Chemokine und Bradykinin. Gleichzeitig werden aber von Leukozyten auch analgetische Mediatoren wie Opioidpeptide (Beta-Endorphin, Met-Enkephalin und Endomorphine produziert, die an Opioidrezeptoren an periphere sensorische Neuronen binden und so Analgesie auslösen. Stress oder bestimme Hormone wie Corticotrophin releasing factor (CRF) können Freisetzung von Opioidpeptiden auslösen. In der vorliegenden Arbeit wurden 4 Fragestellungen im Entzündungsschmerzmodell durch intraplantare Injektion von Freunds Adjuvans (CFA) untersucht: (i) Die Rolle von Granulozyten in der Schmerzentstehung, (ii) den Einfluss von Entzündung auf die Transkription von Opioidrezeptorgenen in Hinterwurzelganglien (DRG), (iii) periphere Opioidanalgesie durch Endomorphine und (iv) die Signalübertragungswege in der Freisetzung von Opioidpeptiden aus Granulozyten. Es wurde gezeigt, dass (i) Granulozyten per se keine Schmerzen auslösen, denn die selektive Rekrutierung durch Chemokine führt zu keiner Änderung der Schmerzschwellen. (ii) Kappa-Opioidrezeptor mRNA sowie Protein werden in der CFA Entzündung über IL-1beta hochreguliert. (iii) Die neu entdeckten Endomorphine können ebenfalls eine potente Analgesie auslösen. Stress und CRF induzierte Analgesie sind sowohl von der Ausschüttung klassischer Opioidpeptide als auch von Endomorphinen abhängig. (iv) Opioidpeptidfreisetzung aus Granulozyten nach CXCR1/2 Stimulation hängt von der intrzellulären Mobilisation von Kalzium sowie der Aktivierung von Phosphoinositol-3-Kinase (PI3K) und p38 mitogen activated kinase (MAPK) ab. In vivo können CXCR2 Liganden eine Analgesie in der entzündeten Pfote auslösen. Granulozyten spielen eine Rolle bei der peripheren Opioidanalgesie in der frühen Entzündungsphase während ihre Rolle bei der Schmerzentstehung begrenzt zu sein scheint. Anti-inflammatorische Medikamente, die auf Granulozytenfunktion oder die intrazellulären Signalkaskaden zielen, könnten unbeabsichtigter Weise daher die endogenen analgetischen Mechanismen des Körpers beeinträchtigen

CXCL10 Controls Inflammatory Pain via Opioid Peptide- Containing Macrophages in Electroacupuncture

Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund's adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao - gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3+ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3+ macrophages. In summary, we describe a novel function of the chemokine CXCL10 - as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture

Pain Control by Targeting Oxidized Phospholipids: Functions, Mechanisms, Perspectives

Within the lipidome oxidized phospholipids (OxPL) form a class of chemically highly reactive metabolites. OxPL are acutely produced in inflamed tissue and act as endogenous, proalgesic (pain-inducing) metabolites. They excite sensory, nociceptive neurons by activating transient receptor potential ion channels, specifically TRPA1 and TRPV1. Under inflammatory conditions, OxPL-mediated receptor potentials even potentiate the action potential firing rate of nociceptors. Targeting OxPL with D-4F, an apolipoprotein A-I mimetic peptide or antibodies like E06, specifically binding oxidized headgroups of phospholipids, can be used to control acute, inflammatory pain syndromes, at least in rodents. With a focus on proalgesic specificities of OxPL, this article discusses, how targeting defined substances of the epilipidome can contribute to mechanism-based therapies against primary and secondary chronic inflammatory or possibly also neuropathic pain

The Connection of Monocytes and Reactive Oxygen Species in Pain

The interplay of specific leukocyte subpopulations, resident cells and proalgesic mediators results in pain in inflammation. Proalgesic mediators like reactive oxygen species (ROS) and downstream products elicit pain by stimulation of transient receptor potential (TRP) channels. The contribution of leukocyte subpopulations however is less clear. Local injection of neutrophilic chemokines elicits neutrophil recruitment but no hyperalgesia in rats. In meta-analyses the monocytic chemoattractant, CCL2 (monocyte chemoattractant protein-1; MCP-1), was identified as an important factor in the pathophysiology of human and animal pain. In this study, intraplantar injection of CCL2 elicited thermal and mechanical pain in Wistar but not in Dark Agouti (DA) rats, which lack p47phox, a part of the NADPH oxidase complex. Inflammatory hyperalgesia after complete Freund's adjuvant (CFA) as well as capsaicin-induced hyperalgesia and capsaicin-induced current flow in dorsal root ganglion neurons in DA were comparable to Wistar rats. Macrophages from DA expressed lower levels of CCR2 and thereby migrated less towards CCL2 and formed limited amounts of ROS in vitro and 4-hydroxynonenal (4-HNE) in the tissue in response to CCL2 compared to Wistar rats. Local adoptive transfer of peritoneal macrophages from Wistar but not from DA rats reconstituted CCL2-triggered hyperalgesia in leukocyte-depleted DA and Wistar rats. A pharmacological stimulator of ROS production (phytol) restored CCL2-induced hyperalgesia in vivo in DA rats. In Wistar rats, CCL2-induced hyperalgesia was completely blocked by superoxide dismutase (SOD), catalase or tempol. Likewise, inhibition of NADPH oxidase by apocynin reduced CCL2-elicited hyperalgesia but not CFA-induced inflammatory hyperalgesia. In summary, we provide a link between CCL2, CCR2 expression on macrophages, NADPH oxidase, ROS and the development CCL2-triggered hyperalgesia, which is different from CFA-induced hyperalgesia. The study further supports the impact of CCL2 and ROS as potential targets in pain therapy

Pain, disability, and lifestyle: Patients with complex regional pain syndrome compared to chronic musculoskeletal pain-A retrospective analysis

Background Complex regional pain syndrome (CRPS) is an orphan disease occurring as a complication after trauma. Due to its acute onset and the typical clinical presentation of the inflammatory and autonomous signs, it is an eye-catching chronic pain disease affecting also young and working people. In social media and the internet, high pain severity and the unfavourable prognosis are often empathized. Methods Here, we compared epidemiological, pain and lifestyle factors of 223 CPRS patients from the “ncRNAPain” cohort with 255 patients with chronic musculoskeletal pain (MSK). MSK patients were recruited at the beginning of a multimodal pain therapy programme. We searched for factors predicting pain intensity. Results Both chronic pain diseases affected women in middle age. Patients with MSK were more obese, drank more alcohol, and were less educated (Pearson chi-square Test or Mann–Whitney/U-Test). Both groups smoked more than healthy people in the OECD (Organization for Economic Cooperation and Development). Mann–Whitney/U-Test confirmed that CRPS patients did not have more severe pain and did not suffer more from pain-related disability than patients with MSK. CRPS patients also had less psychiatric comorbidities. Multiple linear regression analysis revealed that group assignment, depressive characteristics, body mass index, average alcohol consumption and smoking predicted higher pain ratings, while disease duration, anxiety symptoms or gender had no influence on pain intensity. Conclusion In summary, our study supports a more optimistic view on pain in CRPS patients in comparison to MSK and identifies lifestyle factors that might contribute to the pathophysiology like smoking and drinking. Important next steps are the identification of CRPS patients at risk for chronification or—vice versa—with protective factors for pain resolution. Significance This study compares complex regional pain syndrome (CRPS) and chronic musculoskeletal pain and questions previously reported pain, disability and lifestyle factors associated with CRPS