Genetics and genomics in postoperative pain and analgesia

Purpose of reviewThe review describes recent advances in genetics and genomics of postoperative pain, the association between genetic variants and the efficacy of analgesics, and the role of pharmacogenomics in the selection of appropriate analgesic treatments for postoperative pain.Recent findingsRecent genetic studies have reported associations of genetic variants in catechol-O-methyltransferase (COMT), brain-derived neurotrophic factor (BDNF), voltage-gated channel alpha subunit 11 (SCN11A) and -opioid receptor (OPRM1) genes with postoperative pain. The recent pharmacogenetics studies revealed an association of the organic cation transporter 1 (OCT1) and ATP-binding cassette C3 (ABCC3) polymorphisms with morphine-related adverse effects, an effect of polymorphisms in cytochrome P450 gene CYP2D6 on the analgesic efficacy of tramadol and no effect of CYP2C8 and CYP2C9 variants on efficacy of piroxicam.SummaryGenetic variants associate with inter-individual variability in drug responses and they can affect pain sensitivity and intensity of postoperative pain. Despite the recent progress in genetics and genomics of postoperative pain, it is still not possible to precisely predict the patients who are genetically predisposed to have severe postoperative pain or who develop chronic postoperative pain.Peer reviewe

The circadian clock at the intercept of sleep and pain

Peer reviewe

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP–5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP–5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain

Molekulare Mechanismen von Muskelschmerzen bei myotoner Myopathie Typ II

Table of contents Preface ...................................................................................................... iii Acknowledgements…………………………........................................... iv Table of contents ................................................................................... v Summary ................................................................................................ viii Zusammenfassung................................................................................. x List of figures.......................................................................................... xii List of tables........................................................................................... xii List of abbreviations ...........................................................................xiv 1\. Introduction ......................................................................................... 1 1.1 Pain ........................................................................................................ 1 1.1.1 Pain theories ........................................................................................ 1 1.1.2 Types of pain ....................................................................................... 3 1.1.3 Nociceptors .......................................................................................... 4 1.2 Peripheral and central mechanisms of nociception ......................... 6 1.2.1 Peripheral mechanisms of nociception ................................................ 7 1.2.1.1 Ion channels ............................................................................................. 8 1.2.1.2 G-protein coupled receptors ........................................................... 10 1.2.1.3 Receptors for neurotrophins, neuropeptides and cytokines ........... 10 1.2.2 Central mechanisms of nociception …………………………………… 12 1.2.2.1 Glutamate/NMDA receptor-mediated sensitization …………...…… 12 1.2.2.2 Loss of GABAergic and glycinergic inhibition ……………….……... 13 1.2.2.3 Neuro-glia interactions ………………...……………………………… 14 1.3 Skeletal muscle and associated muscle diseases …………………. 15 1.3.1 Structure and physiology of skeletal muscle ………………………….. 15 1.3.2 Muscle diseases …………………………………………………………. 16 1.4 Myotonic Dystrophy Type 2 (DM2) ……………………………………. 18 1.4.1 Clinical aspects of DM2 ……………………………………………….… 19 1.4.2 Molecular pathogenesis of DM2 ……………………………………….. 22 1.5 Muscle pain in DM2 ………….…………………………………...……… 26 1.6 Systems biology approaches to study chronic pain ………………. 27 1.6.1 Transcriptomics ………………………………………………………….. 28 1.6.2 Proteomics ………………………………………...……………………... 30 1.6.3 Lipidomics ………………………………………………………………… 32 2\. Aims of doctoral project …………………………………………….. 35 3\. Materials and methods ……………….……………………………… 36 3.1 Materials ………………………..………………………………………….. 36 3.1.1 List of chemicals and consumables …………………...………………. 36 3.1.2 List of instruments ……………………………………………………….. 36 3.2 Study design and participants ……………...………...……………….. 37 3.3 Clinical assessment of DM2 patients …………………...…………..... 37 3.4 Sensory testing protocol ……………………………………………...… 38 3.4.1 Pressure pain threshold ………………………………………………… 39 3.4.2 Thermal detection and pain thresholds ……………………………….. 39 3.4.3 Mechanical detection threshold ………………………………………... 39 3.4.4 Mechanical pain threshold ……………………………………………… 40 3.4.5 Stimulus/Response function: mechanical pain sensitivity and dynamic mechanical allodynia ……... 40 3.4.6 Wind-up ratio ……………………………………………………….…….. 40 3.5 Transcriptomic analysis of muscle tissue …………………....……... 41 3.6 Quantitative RT-PCR ……………..………………………….…...……… 41 3.7 Microdialysis …………….....……………………………………………... 42 3.8 Preparation of microdialysis samples for lipidome analysis …..… 43 3.9 LC-MS/MS analysis of eicosanoid species (lipidomics) …………... 43 3.10 Preparation of microdialysis samples for proteome analysis ..... 44 3.11 Selected reaction monitoring (targeted proteomics) …………….. 44 3.12 Label free quantification (shotgun proteomics) ………………....... 45 3.13 Statistical analysis ………………………………………………….…... 46 4\. Results ……………………….………………………..……...…………… 49 4.1 Patient characteristics ……………………...…………………………… 49 4.2 Sensory changes in patients with DM2 ………………...…………….. 54 4.3 Myalgic and non-myalgic DM2 muscle has distinct transcriptome profiles ………….. 58 4.4 DM2 patients with and without muscle pain have different muscle secretome profiles …………… 61 4.5 No association of inflammatory cytokines IL-1, IL-6 and NGF with muscle pain in DM2 patients .... 68 4.6 Anti-inflammatory eicosanoid 11,12-DHET present only in DM2 without pain ………...………….. 69 4.7 Correlation between RNA sequencing and proteome data ……..... 71 5\. Discussion ………………………….…….……………………………… 73 5.1 Somatosensory profiling of muscle pain in DM2 ……….………….. 74 5.2 Distinct DM2 muscle transcriptome profiles based on onset of muscle pain ……………………. 75 5.3 Differences in DM2 muscle secretome associated with muscle pain ......……………………….. 76 5.4 The role of lipids in the onset of muscle pain in DM2 ……...……... 78 5.5 Correlation between DM2 transcriptome and proteome profiling of muscle pain ………………... 79 5.6 Relevance of the study ……………………….…………………………. 79 6\. Future perspectives ………..…………………………………………. 81 7\. Publications …………………….……………………………………….. 82 8\. Bibliography ………………….………………………………….………. 83Muscle pain is a frequent clinical symptom that impairs the quality of life but the molecular mechanisms behind the onset of pain remain unknown. Patients with myotonic dystrophy type 2 (DM2) often develop muscle pain of moderate severity which is resistant to conventional analgesics. DM2 is a promising model for studying muscle pain since it is a monogenetic disorder caused by CCTG repeats expansion in intron 1 of transcription factor encoding CNBP which alters gene expression and splicing of downstream genes. We assumed that the expansion of repeats in CNBP could alter the gene expression and splicing of pain regulating genes in DM2. Additionally, certain myokines secreted from DM2 affected muscles during contraction could contribute to the onset of muscle pain. Considering that hyperlipidemia is frequent in DM2 and lipids are known mediators of peripheral nociception, altered levels of lipids might also modulate the nociception in DM2. In our study, we examined a cohort of 42 DM2 patients and 20 healthy controls. Patients were characterized for somatosensory profiles with a standardized QST protocol. We obtained biopsy specimens from 12 DM2 patients (6 with and 6 without muscle pain) in order to analyze their transcriptome profiles and performed microdialysis with 14 patients (7 with and 7 without muscle pain) for proteomic and lipidomic profiling of muscle pain in DM2. QST revealed significantly decreased pressure pain thresholds over proximal and distal muscles only in DM2 patients with pain indicating that the cause of the muscle pain is within the muscle. DM2 patients had distinct transcriptome profiles based on the presence of muscle pain and RNA sequencing identified 14 differentially expressed genes between patients with and without pain. Shotgun proteomic analysis of DM2 interstitial fluid revealed 22 differentially secreted proteins based on the presence of muscle pain. There was no association between muscle pain in DM2 and the known pro-inflammatory cytokines IL-1, IL-6 and NGF. LC-MS/MS analysis of DM2 lipidome profiles identified the presence of anti-inflammatory eicosanoids 11,12-DHET only in patients without pain. These results are the first evidence that the muscle pain in DM2 is caused by peripheral nociception. Furthermore, this study shows that by using OMICS techniques in homogenous groups of patients with chronic pain, it is possible to identify novel genes and molecules that might contribute to the onset of pain. Our study might also explain the pathogenic mechanisms of more common disorders with chronic muscle pain such as fibromyalgia.Muskelschmerzen sind ein häufiges klinisches Symptom, das die Lebensqualität beeinträchtigt. Trotzdem bleiben die Molekularmechanismen, die dahinterstehen, unbekannt. Bei den Patienten mit der Myotonen Dystrophie Typ 2 (DM2) treten oft Muskelschmerzen mittleren Schweregrades auf, die gegen konventionelles Analgetikum resistent sind. DM2 ist ein viel versprechendes Modell für die Studie über Muskelschmerzen, weil es eine monogenetische Krankheit ist, die durch die Zunahme der CCTG Wiederholungen, die im Intron 1 von CNBP, das die Genexpression und das Spleißen der Downstream-Gene ändert, stattfindet. Wir haben angenommen, dass die Zunahme der Wiederholungen in CNBP bei DM2 die Genexpression sowie das Spleißen der Gene, die die Schmerzen regulieren, ändern könnte. Darüber hinaus könnten gewisse Myokine, die aus den von DM2 betroffenen Muskeln während Kontraktion abgesondert wurden, zu dem Auftreten der Schmerzen beitragen. Da bei DM2 die Hyperlipidämie oft vorkommt und die Lipide bekannte Mediatoren der peripheren Nozizeption sind, könnten die geänderten Lipidspiegel auch die Nozizeption bei DM2 regeln. In unserer Studie haben wir eine Kohorte von 42 DM2 Patienten und 20 gesunden Kontrollprobanden untersucht. Die Patienten wurden für somatosensorische Profile mit standarisiertem QST Protokoll charakterisiert. Wir haben von 12 DM2 Patienten (6 mit und 6 ohne Muskelschmerzen) Biopsieproben entnommen, um ihre Transkriptomprofile zu analysieren und haben bei 14 Patienten (7 mit und 7 ohne Muskelschmerzen) die Mikrodialyse für die proteomische und lipämische Profilierung von Muskelschmerzen bei DM2 durchgeführt. QST zeigte deutlich reduzierte Grenzwerte von Druckschmerzen in proximalen und distalen Muskeln nur bei den DM2 Patienten mit Schmerzen, was darauf hinweist, dass die Ursache für die Muskelschmerzen innen im Muskel liegt. Die DM2 Patienten hatten verschiedene Transkriptomprofile, die auf der Präsenz der Muskelschmerzen basierten und die RNA-Sequenzierung identifizierte 14 differentiell bei den Patienten mit und ohne Muskelschmerzen exprimierte Gene. Die proteomische Shotgun-Analyse von der DM2 interstitiellen Flüssigkeit offenbarte 22 differentiell abgesonderte, auf der Präsenz der Muskelschmerzen basierende Proteine. Es ergab sich keine Verbindung zwischen Muskelschmerzen in DM2 und bekannten pro-inflammatorischen Zytokinen IL-1, IL-6 und NGF. LC-MS-Analyse von DM2 lipämische Profile identifizierten die Präsenz von entzündungshemmenden Eicosanoiden 11,12DHET nur bei Patienten ohne Muskelschmerzen. Dieses Ergebnis ist der erste Beweis dafür, dass die Muskelschmerzen bei DM2 ein Resultat der peripheren Nozizeption sind. Außerdem zeigt die Studie, dass durch die Nutzung von OMICS-Techniken bei homogenen Gruppen von Patienten mit chronischen Schmerzen es einfacher ist, neuartige Gene und Moleküle zu identifizieren, die zu dem Auftreten von Schmerzen beitragen können. Unsere Studie könnte auch die pathogenen Mechanismen von gewöhnlicheren Krankheiten mit chronischen Muskelschmerzen wie Fibromyalgie erklären

Expression of mitochondrial TSPO and FAM173B is associated with inflammation and symptoms in patients with painful knee osteoarthritis

Objectives To characterize the expression profiles of two nuclear-encoded mitochondrial genes previously associated with chronic pain, the translocator protein (TSPO) and family with sequence similarity 173B (FAM173B), in different knee compartments from patients with painful knee OA. Also, to examine their association with the joint expression of inflammatory cytokines/chemokines and clinical symptoms. Methods The study was performed on 40 knee OA patients and 19 postmortem (PM) controls from which we collected the knee tissues: articular cartilage (AC), synovial membrane (SM) and subchondral bone (SB). Quantitative real-time polymerase chain reaction was used to determine the relative mRNA levels of TSPO, FAM173B, and inflammatory mediators IL6, IL8, IL10, IL12, MCP1, CCL11 and CCL17. OA patients rated their pain intensity (visual analogue scale), severity of knee-related outcomes (KOOS) and pain sensitivity assessed by pressure algometry. Results The gene expression of TSPO in SM was elevated in OA patients compared with control subjects while there were no group differences in AC and SB. Expression of FAM173B was reduced in SM but elevated in SB in OA patients compared with controls. The expression of TSPO and FAM173B in SM and SB was associated with the expression of inflammatory substances, but not in AC. Synovial expression of TSPO correlated with lower pain intensity and FAM173B with increased pressure pain sensitivity in OA. Conclusion Our results suggest that altered expression of TSPO and FAM173B is associated with joint expression of inflammatory mediators and with clinical symptoms indicating the relevance for the pathophysiology of knee OA

circRNA landscape in dorsal root ganglia from mice with collagen antibody-induced arthritis

Circular RNAs are a novel class of RNA molecules that are covalently closed into a ring structure. They are an epigenetic regulatory mechanism, and their best-studied function is regulation of microRNA activity. As such circular RNAs may be involved in the switch from acute to chronic pain. They have previously been studied in the context of neuropathic pain models, but their importance in inflammation-induced chronic pain models is unexplored. Microarray analysis of dorsal root ganglia collected in the late phase of collagen antibody-induced arthritis (day 59) were used to elucidate the relevance of circular RNAs in the mechanical hypersensitivity caused by this model. 120 circular RNA genes were found to be significantly differentially regulated in female BALB/c mice with collagen antibody-induced arthritis. Six genes were chosen for RT-qPCR analysis in the late (day 54–60) as well as the inflammatory (day 11–12) phase of this model. This validated an increase in circNufip1 expression in the late phase of collagen antibody-induced arthritis. Additionally, it was found that circVps13 and circMicall1 are upregulated in the inflammatory phase. Interestingly, no changes were found in dorsal root ganglia from mice injected with Freund's Complete Adjuvant (day 3) nor mice with spared nerve injury (day 20), despite their similarities to inflammatory and late phase collagen antibody-induced arthritis, respectively. This study provides evidence that mild circular RNA changes occur in dorsal root ganglia of mice with collagen antibody-induced arthritis that are, bioinformatically, predicated to be involved in processes relevant to sensitization

Expression of mitochondrial TSPO and FAM173B is associated with inflammation and symptoms in patients with painful knee osteoarthritis

Objectives: To characterize the expression profiles of two nuclear-encoded mitochondrial genes previously associated with chronic pain, the translocator protein (TSPO) and family with sequence similarity 173B (FAM173B), in different knee compartments from patients with painful knee OA. Also, to examine their association with the joint expression of inflammatory cytokines/chemokines and clinical symptoms. Methods: The study was performed on 40 knee OA patients and 19 postmortem (PM) controls from which we collected the knee tissues: articular cartilage (AC), synovial membrane (SM) and subchondral bone (SB). Quantitative real-time polymerase chain reaction was used to determine the relative mRNA levels of TSPO, FAM173B, and inflammatory mediators IL6, IL8, IL10, IL12, MCP1, CCL11 and CCL17. OA patients rated their pain intensity (visual analogue scale), severity of knee-related outcomes (KOOS) and pain sensitivity assessed by pressure algometry. Results: The gene expression of TSPO in SM was elevated in OA patients compared with control subjects while there were no group differences in AC and SB. Expression of FAM173B was reduced in SM but elevated in SB in OA patients compared with controls. The expression of TSPO and FAM173B in SM and SB was associated with the expression of inflammatory substances, but not in AC. Synovial expression of TSPO correlated with lower pain intensity and FAM173B with increased pressure pain sensitivity in OA. Conclusion: Our results suggest that altered expression of TSPO and FAM173B is associated with joint expression of inflammatory mediators and with clinical symptoms indicating the relevance for the pathophysiology of knee OA

Elevated inflammatory proteins in cerebrospinal fluid from patients with painful knee osteoarthritis are associated with reduced symptom severity

Neuroinflammation and periphery-to-CNS neuroimmune cross-talk in patients with painful knee osteoarthritis (OA) are poorly understood. We utilized proximity extension assay to measure the level of 91 inflammatory proteins in CSF and serum from OA patients and controls. The patients had elevated levels of 48 proteins in CSF indicating neuroinflammation. Ten proteins were correlated between CSF and serum and potentially involved in periphery-to-CNS neuroimmune cross-talk. Seven CSF proteins, all with previously reported neuroprotective effects, were associated with lower pain intensity and milder knee-related symptoms. Our findings indicate that neuroinflammation in OA could be protective and associated with less severe symptoms