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Biomarkers for Better Understanding of the Pathophysiology and Treatment of Chronic Pain: Investigations of Human Biofluids
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chronic pain affects 20 % of the global population, causes suffering, is difficult to treat, and constitutes a large economic burden for society. So far, the characterization of molecular mechanisms of chronic pain-like behaviors in animal models has not translated into effective treatments.

In this thesis, consisting of five studies, pain patient biofluids were analyzed with modern proteomic methods to identify biomarker candidates that can be used to improve our understanding of the pathophysiology chronic pain and lead to more effective treatments.

Paper I is a proof of concept study, where a multiplex solid phase-proximity ligation assay (SP-PLA) was applied to cerebrospinal fluid (CSF) for the first time. CSF reference protein levels and four biomarker candidates for ALS were presented. The investigated proteins were not altered by spinal cord stimulation (SCS) treatment for neuropathic pain. In Paper II, patient CSF was explored by dimethyl and label-free mass spectrometric (MS) proteomic methods. Twelve proteins, known for their roles in neuroprotection, nociceptive signaling, immune regulation, and synaptic plasticity, were identified to be associated with SCS treatment of neuropathic pain. In Paper III, proximity extension assay (PEA) was used to analyze levels of 92 proteins in serum from patients one year after painful disc herniation. Patients with residual pain had significantly higher serum levels of 41 inflammatory proteins. In Paper IV, levels of 55 proteins were analyzed by a 100-plex antibody suspension bead array (ASBA) in CSF samples from two neuropathic pain patient cohorts, one cohort of fibromyalgia patients and two control cohorts. CSF protein profiles consisting of levels of apolipoprotein C1, ectonucleotide pyrophosphatase/phosphodiesterase family member 2, angiotensinogen, prostaglandin-H2 D-isomerase, neurexin-1, superoxide dismutases 1 and 3 were found to be associated with neuropathic pain and fibromyalgia. In Paper V, higher CSF levels of five chemokines and LAPTGF-beta-1were detected in two patient cohorts with neuropathic pain compared with healthy controls.

In conclusion, we demonstrate that combining MS proteomic and multiplex antibody-based methods for analysis of patient biofluid samples is a viable approach for discovery of biomarker candidates for the pathophysiology and treatment of chronic pain. Several biomarker candidates possibly reflecting systemic inflammation, lipid metabolism, and neuroinflammation in different pain conditions were identified for further investigation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , 89 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1342
Keyword [en]
chronic pain, neuropathic pain, lumbar radicular pain, amyotrophic lateral sclerosis, radiculopathy, fibromyalgia, pathophysiology, spinal cord stimulation, mechanism of action, disc herniation, cerebrospinal fluid, plasma, biomarker, human, protein, chemokines, cytokines, inflammation, neuroinflammation, mass spectrometry, proximity ligation assay, proximity extension assay, antibody suspension bead array, protein profiling, molecular medicine, personalized medicine
National Category
Anesthesiology and Intensive Care Clinical Laboratory Medicine Biomedical Laboratory Science/Technology Analytical Chemistry
Research subject
Anaesthesiology and Intensive Care; Biomedical Laboratory Science; Chemistry with specialization in Analytical Chemistry; Medical Science; Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-326180ISBN: 978-91-513-0006-1 (print)OAI: oai:DiVA.org:uu-326180DiVA: diva2:1119341
Public defence
2017-09-15, Enghoffsalen, Ingång 50, bv, Akademiska sjukhuset, Uppsala, 09:00 (Swedish)
Opponent
Supervisors
Projects
Uppsala Berzelii Technology Centre for Neurodiagnostics
Funder
Swedish Research CouncilVINNOVA
Available from: 2017-08-24 Created: 2017-07-03 Last updated: 2017-09-08
List of papers
1. A Multiplex Protein Panel Applied to Cerebrospinal Fluid Reveals Three New Biomarker Candidates in ALS but None in Neuropathic Pain Patients
Open this publication in new window or tab >>A Multiplex Protein Panel Applied to Cerebrospinal Fluid Reveals Three New Biomarker Candidates in ALS but None in Neuropathic Pain Patients
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 2, e0149821Article in journal (Refereed) Published
Abstract [en]

The objective of this study was to develop and apply a novel multiplex panel of solid-phase proximity ligation assays (SP-PLA) requiring only 20 μL of samples, as a tool for discovering protein biomarkers for neurological disease and treatment thereof in cerebrospinal fluid (CSF). We applied the SP-PLA to samples from two sets of patients with poorly understood nervous system pathologies amyotrophic lateral sclerosis (ALS) and neuropathic pain, where patients were treated with spinal cord stimulation (SCS). Forty-seven inflammatory and neurotrophic proteins were measured in samples from 20 ALS patients and 15 neuropathic pain patients, and compared to normal concentrations in CSF from control individuals. Nineteen of the 47 proteins were detectable in more than 95% of the 72 controls. None of the 21 proteins detectable in CSF from neuropathic pain patients were significantly altered by SCS. The levels of the three proteins, follistatin, interleukin-1 alpha, and kallikrein-5 were all significantly reduced in the ALS group compared to age-matched controls. These results demonstrate the utility of purpose designed multiplex SP-PLA panels in CSF biomarker research for understanding neuropathological and neurotherapeutic mechanisms. The protein changes found in the CSF of ALS patients may be of diagnostic interest.

National Category
Neurology Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-281233 (URN)10.1371/journal.pone.0149821 (DOI)000371175700030 ()26914813 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, European Research Council, 316929EU, European Research Council, 294409
Available from: 2016-03-21 Created: 2016-03-21 Last updated: 2017-07-03Bibliographically approved
2. Spinal Cord Stimulation Alters Protein Levels in the Cerebrospinal Fluid of Neuropathic Pain Patients: A Proteomic Mass Spectrometric Analysis
Open this publication in new window or tab >>Spinal Cord Stimulation Alters Protein Levels in the Cerebrospinal Fluid of Neuropathic Pain Patients: A Proteomic Mass Spectrometric Analysis
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2016 (English)In: Neuromodulation (Malden, Mass.), ISSN 1094-7159, E-ISSN 1525-1403, Vol. 19, no 6, 549-562 p.Article in journal (Refereed) Published
Abstract [en]

ObjectivesElectrical neuromodulation by spinal cord stimulation (SCS) is a well-established method for treatment of neuropathic pain. However, the mechanism behind the pain relieving effect in patients remains largely unknown. In this study, we target the human cerebrospinal fluid (CSF) proteome, a little investigated aspect of SCS mechanism of action. MethodsTwo different proteomic mass spectrometry protocols were used to analyze the CSF of 14 SCS responsive neuropathic pain patients. Each patient acted as his or her own control and protein content was compared when the stimulator was turned off for 48 hours, and after the stimulator had been used as normal for three weeks. ResultsEighty-six proteins were statistically significantly altered in the CSF of neuropathic pain patients using SCS, when comparing the stimulator off condition to the stimulator on condition. The top 12 of the altered proteins are involved in neuroprotection (clusterin, gelsolin, mimecan, angiotensinogen, secretogranin-1, amyloid beta A4 protein), synaptic plasticity/learning/memory (gelsolin, apolipoprotein C1, apolipoprotein E, contactin-1, neural cell adhesion molecule L1-like protein), nociceptive signaling (neurosecretory protein VGF), and immune regulation (dickkopf-related protein 3). ConclusionPreviously unknown effects of SCS on levels of proteins involved in neuroprotection, nociceptive signaling, immune regulation, and synaptic plasticity are demonstrated. These findings, in the CSF of neuropathic pain patients, expand the picture of SCS effects on the neurochemical environment of the human spinal cord. An improved understanding of SCS mechanism may lead to new tracks of investigation and improved treatment strategies for neuropathic pain.

Keyword
Cerebrospinal fluid, mechanism of action, neuropathic pain, spinal cord stimulation
National Category
Neurology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-304434 (URN)10.1111/ner.12473 (DOI)000382755300001 ()27513633 (PubMedID)
Funder
VINNOVASwedish Research Council
Available from: 2016-10-05 Created: 2016-10-05 Last updated: 2017-10-17Bibliographically approved
3. Inflammatory Serum Protein Profiling of Patients with Lumbar Radicular Pain One Year after Disc Herniation
Open this publication in new window or tab >>Inflammatory Serum Protein Profiling of Patients with Lumbar Radicular Pain One Year after Disc Herniation
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2016 (English)In: INTERNATIONAL JOURNAL OF INFLAMMATION, ISSN 2090-8040, UNSP 3874964Article in journal (Refereed) Published
Abstract [en]

Earlier studies suggest that lumbar radicular pain following disc herniation may be associated with a local or systemic inflammatory process. In the present study, we investigated the serum inflammatory protein profile of such patients. All 45 patients were recruited from Oslo University Hospital, Ulleval, Norway, during the period 2007-2009. The new multiplex proximity extension assay (PEA) technology was used to analyze the levels of 92 proteins. Interestingly, the present data showed that patients with radicular pain 12 months after disc herniation may be different from other patients with regard to many measurable serum cytokines. Given a false discovery rate (FDR) of 0.10 and 0.05, we identified 41 and 13 proteins, respectively, which were significantly upregulated in the patients with severe pain one year after disc herniation. On the top of the list ranked by estimated increase we found C-X-C motif chemokine 5 (CXCM5; 217% increase), epidermal growth factor (EGF; 142% increase), and monocyte chemotactic protein 4 (MCP-4; 70% increase). Moreover, a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated. Thus, the present results may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment. We conclude that serum proteins may be measurable molecular markers of persistent pain after disc herniation.

National Category
Family Medicine
Identifiers
urn:nbn:se:uu:diva-317717 (URN)10.1155/2016/3874964 (DOI)000394106300001 ()27293953 (PubMedID)
Funder
VINNOVASwedish Research Council, P29797-1
Available from: 2017-03-17 Created: 2017-03-17 Last updated: 2017-07-03Bibliographically approved
4. Affinity Proteomics Applied to Patient CSF Identifies Protein Profiles Associated with Neuropathic Pain and Fibromyalgia
Open this publication in new window or tab >>Affinity Proteomics Applied to Patient CSF Identifies Protein Profiles Associated with Neuropathic Pain and Fibromyalgia
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Objective: Today, there are no biological tests on which to base pain diagnoses, treatment choices or to understand the biological processes underlying and accompanying chronic pain for the individual pain patient. Relevant biological markers would greatly aid in diagnosis and treatment of patients with chronic pain. Our study aimed to find proteins in CSF associated with fibromyalgia and neuropathic pain, two common and poorly understood chronic pain conditions.

Methods: We have performed CSF protein profiling of 55 proteins using a 100-plex antibody suspension bead array. We collected, analyzed and compared CSF samples from 25 patients with neuropathic pain (two independent sets, n=14 patients for discovery and n=11 for verification), 40 patients with fibromyalgia and 135 controls without neurological disease from two different populations.

Results: We found significant differences in CSF protein levels between patients and controls (p<0.05). Among these proteins, Apolipoprotein C1 (APOC1) was found to be increased in CSF of neuropathic pain patients compared to controls and there was a non-significant trend for increased levels also in fibromyalgia patient CSF. Ectonucleotide pyrophosphatase (ENPP2, Autotaxin) was increased in the CSF of fibromyalgia patients compared to all other groups including neuropathic pain patients.  Multivariate analysis revealed partially overlapping and partially distinct CSF profiles in neuropathic pain patients compared with fibromyalgia and controls for several other proteins including angiotensinogen (AGT), prostaglandin-H2 D-isomerase (PTGDS), neurexin-1 (NRXN1), superoxide dismutase 1 (SOD1) and superoxide dismutase 3 (SOD3).

Conclusions: Our results, suggest that the CSF protein profiles of neuropathic pain and fibromyalgia patients may be different from each other and from those of controls. CSF levels of APOC1, ENPP2, AGT, PTGDS, NRXN1, SOD1 and SOD3 should be further investigated for their potential to serve as biomarkers of different kinds of pain pathophysiology.

Keyword
cerebrospinal fluid, biomarker, human, chronic pain, neuropathic pain, fibromyalgia, spinal cord stimulation, mechanism of action, radiculopathy, protein, inflammation, neuroinflammation, mass spectrometry, antibody suspension bead array, protein profiling
National Category
Neurosciences Clinical Laboratory Medicine Health Sciences Anesthesiology and Intensive Care Biomedical Laboratory Science/Technology
Research subject
Anaesthesiology and Intensive Care; Bioinformatics; Biochemistry; Biology with specialization in Molecular Biology; Biomedical Laboratory Science; Chemistry with specialization in Analytical Chemistry; Clinical Chemistry; Medical Science; Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-326158 (URN)
Projects
Berzelii Technology Centre of Neurodiagnostics
Funder
AFA Insurance, 140341VINNOVASwedish Research Council
Available from: 2017-07-03 Created: 2017-07-03 Last updated: 2017-07-03
5. High Levels of Cerebrospinal Fluid Chemokines Point to the Presence of Neuroinflammation in Peripheral Neuropathic Pain: A Cross-Sectional Study of Two Cohorts of Patients Compared to Healthy Controls
Open this publication in new window or tab >>High Levels of Cerebrospinal Fluid Chemokines Point to the Presence of Neuroinflammation in Peripheral Neuropathic Pain: A Cross-Sectional Study of Two Cohorts of Patients Compared to Healthy Controls
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Animal models suggest that chemokines are important mediators in the pathophysiology of neuropathic pain. Indeed, these substances have been called “gliotransmitters”, a term that illustrates the close interplay between glial cells and neurons in the context of neuroinflammation and pain. However, evidence in humans is scarce. The aim of the study was to determine a comprehensive cerebrospinal fluid (CSF) inflammatory profile for neuropathic pain patients. Our hypothesis was that we would thereby find indications of a postulated on-going process of central neuroinflammation.  

CSF samples were collected from two cohorts of patients with neuropathic pain (n=11 and n=16, respectively) and healthy controls (n=11). The samples were analyzed with a multiplex proximity extension assay in which 92 inflammation-related proteins were measured simultaneously (Proseek® Multiplex Inflammation I, Olink Bioscience, Uppsala, Sweden). Univariate testing with control of false discovery rate, as well as orthogonal partial least squares – discriminant analysis, were used for statistical analyses.

CSF levels of chemokines CXCL6, CXCL10, CCL8, CCL11, CCL23, as well as protein LAPTGF-beta-1, were significantly higher in both neuropathic pain cohorts compared to healthy controls, pointing to neuroinflammation in patients. These 6 proteins were also major results in a recent similar study in fibromyalgia patients. The findings need to be confirmed in larger cohorts, and the question of causality remains to be settled. Since it has been suggested that prevalent co-morbidities to chronic pain (e.g., depression, anxiety, poor sleep, and tiredness) also are associated with inflammation, it will be important to determine whether inflammation is a common mediator.

Keyword
biomarker; cerebrospinal fluid; chemokines; cytokines; human; inflammation; neuroinflammation; neuropathic pain; protein profile; proximity extension assay (PEA), chronic pain
National Category
Anesthesiology and Intensive Care Clinical Laboratory Medicine Biomedical Laboratory Science/Technology Neurosciences Medical Laboratory and Measurements Technologies
Research subject
Anaesthesiology and Intensive Care; Biomedical Laboratory Science; Chemistry with specialization in Analytical Chemistry; Medical Science; Medical Biochemistry; Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-326161 (URN)
Projects
Uppsala Berzelii Technology Centre for NeurodiagnosticsAFA Insurance (140341)Swedish Research Council (grant no. K2015-99x-21874-05-4)the Swedish Research Council (grant no. P29797-1).Swedish Governmental Agency for Innovation Systems (Vinnova)County Council of Östergötland (LIO-35923, SC-2013-00395-36)
Funder
AFA Insurance, 140341VINNOVA, P29797-1Swedish Research Council, P29797-1Swedish Research Council, K2015-99x-21874-05-4
Available from: 2017-07-03 Created: 2017-07-03 Last updated: 2017-07-03

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