Patrik Olausson

Identification of Proteins from Interstitium of Trapezius Muscle in Women with Chronic Myalgia Using Microdialysis in Combination with Proteomics

Background Microdialysis (MD) of the trapezius muscle has been an attractive technique to investigating small molecules and metabolites in chronic musculoskeletal pain in human. Large biomolecules such as proteins also cross the dialysis membrane of the catheters. In this study we have applied in vivo MD in combination with two dimensional gel electrophoresis (2-DE) and mass spectrometry to identify proteins in the extracellular fluid of the trapezius muscle. Materials and Methods Dialysate from women with chronic trapezius myalgia (TM; n = 37), women with chronic wide spread pain (CWP; n = 18) and healthy controls (CON; n = 22) was collected from the trapezius muscle using a catheter with a cut-off point of 100 kDa. Proteins were separated by two-dimensional gel electrophoresis and visualized by silver staining. Detected proteins were identified by nano liquid chromatography in combination with tandem mass spectrometry. Results Ninety-seven protein spots were identified from the interstitial fluid of the trapezius muscle; 48 proteins in TM and 30 proteins in CWP had concentrations at least two-fold higher or lower than in CON. The identified proteins pertain to several functional classes, e.g., proteins involved in inflammatory responses. Several of the identified proteins are known to be involved in processes of pain such as: creatine kinase, nerve growth factor, carbonic anhydrase, myoglobin, fatty acid binding protein and actin aortic smooth muscle. Conclusions In this study, by using in vivo microdialysis in combination with proteomics a large number of proteins in muscle interstitium have been identified. Several of the identified proteins were at least two-fold higher or lower in chronic pain patients. The applied techniques open up for the possibility of investigating protein changes associated with nociceptive processes of chronic myalgia.

Multivariate proteomic analysis of the cerebrospinal fluid of patients with peripheral neuropathic pain and healthy controls : a hypothesis-generating pilot study

Pain medicine lacks objective biomarkers to guide diagnosis and treatment. Combining two-dimensional gel proteomics with multivariate data analysis by projection, we exploratively analyzed the cerebrospinal fluid of eleven patients with severe peripheral neuropathic pain due to trauma and/or surgery refractory to conventional treatment and eleven healthy controls. Using orthogonal partial least squares discriminant analysis, we identified a panel of 36 proteins highly discriminating between the two groups. Due to a possible confounding effect of age, a new model with age as outcome variable was computed for patients (n=11), and four out of 36 protein spots were excluded due to a probable influence of age. Of the 32 remaining proteins, the following seven had the highest discriminatory power between the two groups: an isoform of angiotensinogen (upregulated in patients), two isoforms of alpha-1-antitrypsin (downregulated in patients), three isoforms of haptoglobin (upregulated in patients), and one isoform of pigment epithelium-derived factor (downregulated in patients). It has recently been hypothesized that the renin–angiotensin system may play a role in the pathophysiology of neuropathic pain, and a clinical trial of an angiotensin II receptor antagonist was recently published. It is noteworthy that when searching for neuropathic pain biomarkers with a purely explorative methodology, it was indeed a renin–angiotensin system protein that had the highest discriminatory power between patients and controls in the present study. The results from this hypothesis-generating pilot study have to be confirmed in larger, hypothesis-driven studies with age-matched controls, but the present study illustrates the fruitfulness of combining proteomics with multivariate data analysis in hypothesis-generating pain biomarker studies in humans.

Protein alterations in women with chronic widespread pain – An explorative proteomic study of the trapezius muscle

Chronic widespread pain (CWP) has a high prevalence in the population and is associated with prominent negative individual and societal consequences. There is no clear consensus concerning the etiology behind CWP although alterations in the central processing of nociception maintained by peripheral nociceptive input has been suggested. Here, we use proteomics to study protein changes in trapezius muscle from 18 female patients diagnosed with CWP compared to 19 healthy female subjects. The 2-dimensional gel electrophoresis (2-DE) in combination with multivariate statistical analyses revealed 17 proteins to be differently expressed between the two groups. Proteins were identified by mass spectrometry. Many of the proteins are important enzymes in metabolic pathways like the glycolysis and gluconeogenesis. Other proteins are associated with muscle damage, muscle recovery, stress and inflammation. The altered expressed levels of these proteins suggest abnormalities and metabolic changes in the myalgic trapezius muscle in CWP. Taken together, this study gives further support that peripheral factors may be of importance in maintaining CWP.

Intramuscular pain modulatory substances before and after exercise in women with chronic neck pain

In peripheral tissue, several substances influence pain and pain modulation. Exercise has been found to decrease pain and improve function for chronic pain conditions, but how and why exercise produces beneficial effects remains unclear. This study investigates whether aspects of pain and concentrations of substances with algesic, analgesic and metabolic functions differ between women with chronic neck shoulder pain (CNSP) and healthy women (CON) and whether changes are found after an exercise intervention for CNSP.

Relative recovery over time - an in vivo microdialysis study of human skeletal muscle.

Abstract Background. The microdialysis technique is a method for sampling endogenous molecules from the interstitial compartments of varying tissues and relies on diffusion of molecules between the tissue and a perfusate via a membrane. Such samples do not allow determination of the true interstitial concentration but only a certain percentage. This gives rise to one of the most crucial parameter that needs to be considered for a dependable microdialysis; the relative recovery. Relative recovery states the efficiency of which an analyte is extracted from its external medium. Aim. To investigate the relative recovery of small molecules (< 20 kDa) such as lactate, fluid recovery and the reproducibility of the relative recovery at group and individual level of the microdialysis technique applied in muscle. Materials and methods. Using in vivo microdialysis of the trapezius muscle of 65 women from two separate occasions 4–6 months apart. Relative recovery of small molecules was measured from samples collected every 20 min during a period of 220 min. Results. Good reproducibility at group level of catheters with cut-offs 100 and 20kDa were found. Furthermore, there was a high and steady relative recovery with an overall good fluid recovery. Poor reproducibility was found at the individual level for both catheters. Conclusions. This study demonstrates that when using microdialysis in skeletal muscle relative recovery is stable over time and is not affected by low-force exercise. Although there is a good reproducibility at group level this is not the case at the individual level. Thus in vivo, the relative recovery should be determined for each test subject and at each test occasion.

The proteomic profile of whole and glandular saliva in healthy pain-free subjects

Determination of the variability in the salivary proteome is a prerequisite for the development of saliva as a diagnostic and prognostic tool in particular physiological states. In this context, it is important that technical variability induced by sample collection and processing is kept at minimum to be able to reproducibly assess variability in states of health and disease. In the current study, the proteome profile in unstimulated and stimulated whole, parotid and sublingual saliva was investigated using two-dimensional gel electrophoresis. Saliva samples were structurally collected from ten examined and characterized healthy individuals during the exactly same conditions. The results demonstrated that different collection methods provide clear differences in the snapshot of the salivary proteome and also in the relative amount of specific proteins. The variable nature of the salivary proteome suggests that different approaches may have to be adopted when studying its composition or its possible role as an indicator for particular physiological states. The results emphasize the importance of consistency when collecting saliva samples for proteomic analysis.

Specific proteins of the trapezius muscle correlate with pain intensity and sensitivity – an explorative multivariate proteomic study of the trapezius muscle in women with chronic widespread pain

Chronic widespread pain (CWP) including fibromyalgia syndrome (FMS) has a high prevalence and is associated with prominent negative consequences. CWP/FMS exhibits morphological and functional alterations in the central nervous system. The importance of peripheral factors for maintaining the central alterations are under debate. In this study, the proteins from biopsies of the trapezius muscle from 18 female CWP/FMS patients and 19 healthy female controls were analyzed. Pain intensity and pressure pain thresholds (PPT) over the trapezius muscles were registered. Twelve proteins representing five different groups of proteins were important regressors of pain intensity in CWP/FMS (R2=0.99; P<0.001). In the regression of PPT in CWP/FMS, it was found that 16 proteins representing six groups of proteins were significant regressors (R2=0.95, P<0.05). Many of the important proteins were stress and inflammation proteins, enzymes involved in metabolic pathways, and proteins associated with muscle damage, myopathies, and muscle recovery. The altered expression of these proteins may reflect both direct and indirect nociceptive/inflammatory processes as well as secondary changes. The relative importance of the identified proteins and central alterations in CWP need to be investigated in future research. Data from this and the previous study concerning the same cohorts give support to the suggestion that peripheral factors are of importance for maintaining pain aspects in CWP/FMS.

Systemic alterations in plasma proteins from women with chronic widespread pain compared to healthy controls: a proteomic study

Chronic widespread pain (CWP) is a complex pain condition that is difficult to treat. The prevalence of CWP approximates ~10% of the general population, with higher prevalence in women. Lack of understanding of molecular mechanisms has been a challenge for diagnosis and treatment of chronic pain. The aim of this study was to explore the systemic protein changes in CWP compared to those in healthy controls (CON). By applying 2-dimensional gel electrophoresis, we analyzed the protein pattern of plasma samples from women with CWP (n=16) and healthy women (n=23). The proteomic data were analyzed using multivariate statistical models, and altered proteins were identified using mass spectrometry. The proteome analysis was further validated by gel-free Western blot. Multivariate statistical data analysis of quantified proteins revealed 22 altered proteins in women with CWP, compared to CON group. Many of the identified proteins are previously known to be involved in different parts of the complement system and metabolic and inflammatory processes, e.g., complement factor B, vitamin D-binding protein, ceruloplasmin, transthyretin and alpha-2-HS-glycoprotein. These results indicate that important systemic protein differences exist between women with CWP and healthy women. Further, this study illustrates the potential use of proteomics to detect biomarkers that may provide new insights into the molecular mechanism(s) of chronic pain. However, further larger investigations are required in order to confirm these findings before it will be possible to identify proteins as potential pain biomarkers for clinical use.

Clear differences in cerebrospinal fluid proteome between women with chronic widespread pain and healthy women – a multivariate explorative cross-sectional study

Introduction Frequent chronic local pain can develop into chronic widespread pain (CWP). The spread of pain is correlated with pain intensity, anxiety, and depression, conditions that ultimately lead to a poor quality of life. Knowledge is incomplete about CWP’s etiology, although it has been suggested that both central hyperexcitability and/or a combination with peripheral factors may be involved. Cerebrospinal fluid (CSF) could act as a mirror for the central nervous system as proteins are signal substances that activate the formation of algesics and control nociceptive processes. To this end, this study investigates the CSF protein expression in women with CWP and in female healthy controls. Materials and methods This study included 12 female patients with CWP diagnosed according to the American College of Rheumatology criteria with 13 healthy age- and sex-matched pain-free subjects. All subjects went through a clinical examination and answered a health questionnaire that registered sociodemographic and anthropometric data, pain characteristics, psychological status, and quality of life rating. CSF was collected by lumbar puncture from each subject. Two-dimensional gel electrophoresis in combination with mass spectrometry was used to analyze the CSF proteome. This study identifies proteins that significantly discriminate between the two groups using multivariate data analysis (MVDA) (i.e., orthogonal partial least squares discriminant analysis [OPLS-DA]). Results There were no clinically significant levels of psychological distress and catastrophization presented in subjects with CWP. MVDA revealed a highly significant OPLS-DA model where 48 proteins from CSF explained 91% (R2) of the variation and with a prediction of 90% (Q2). The highest discriminating proteins were metabolic, transport, stress, and inflammatory. Conclusion The highest discriminating proteins (11 proteins), according to the literature, are involved in apoptotic regulations, anti-inflammatory and anti-oxidative processes, the immune system, and endogenous repair. The results of this explorative study may indicate the presence of neuro-inflammation in the central nervous system of CWP patients. Future studies should be larger and control for confounders and determine which alterations are unspecific/general and which are specific changes.

Protein profiles of nasal lavage fluid from individuals with work‐related upper airway symptoms associated with moldy and damp buildings

Upper airway irritation is common among individuals working in moldy and damp buildings. The aim of this study was to investigate effects on the protein composition of the nasal lining fluid. The prevalence of symptoms in relation to work environment was examined in 37 individuals working in two damp buildings. Microbial growth was confirmed in one of the buildings. Nasal lavage fluid was collected from 29 of the exposed subjects and 13 controls, not working in a damp building. Protein profiles were investigated with a proteomic approach and evaluated by multivariate statistical models. Subjects from both workplaces reported upper airway and ocular symptoms. Based on protein profiles, symptomatic subjects in the two workplaces were discriminated from each other and separated from healthy controls. The groups differed in proteins involved in inflammation and host defense. Measurements of innate immunity proteins showed a significant increase in protein S100-A8 and decrease in SPLUNC1 in subjects from one workplace, while alpha-1-antitrypsin was elevated in subjects from the other workplace, compared with healthy controls. The results show that protein profiles in nasal lavage fluid can be used to monitor airway mucosal effects in personnel working in damp buildings and indicate that the profile may be separated when the dampness is associated with the presence of molds.