Nazdar Ghafouri

Inhibition of monoacylglycerol lipase and fatty acid amide hydrolase by analogues of 2-arachidonoylglycerol.

The pharmacology of monoacylglycerol lipase (MAGL) is not well understood. In consequence, the abilities of a series of analogues of 2‐arachidonoylglycerol (2‐AG) to inhibit cytosolic 2‐oleoylglycerol and membrane‐bound anandamide hydolysis by MAGL and fatty acid amide hydrolase (FAAH), respectively, have been investigated. 2‐AG and its 1‐regioisomer (1‐AG) interacted with MAGL with similar affinities (IC50 values 13 and 17 μM, respectively). Shorter homologues of 2‐AG (2‐linoleoylglycerol and 2‐oleoylglycerol) had affinities for MAGL similar to 2‐AG. This pattern was also seen when the arachidonoyl side chain of arachidonoyl trifluoromethylketone was replaced by an oleoyl side chain. Arachidonoyl serinol (IC50 value 73 μM) was a weaker inhibitor of MAGL than 2‐AG. The IC50 values of noladin ether towards MAGL and FAAH were 36 and 3 μM, respectively. Arachidonoyl glycine interacted with FAAH (IC50 value 4.9 μM) but only weakly interacted with MAGL (IC50 value >100 μM). α‐Methyl‐1‐AG had similar potencies towards MAGL and FAAH (IC50 values of 11 and 33 μM, respectively). O‐2203 (1‐(20‐cyano‐16,16‐dimethyl‐eicosa‐5,8,11,14‐tetraenoyl) glycerol) and O‐2204 (2‐(20‐hydroxy‐16,16‐dimethyl‐eicosa‐5,8,11,14‐tetraenoyl) glycerol) were slightly less potent, but again affected both enzymes equally. α‐Methyl‐1‐AG, O‐2203 and O‐2204 interacted only weakly with cannabinoid CB1 receptors expressed in CHO cells (Ki values 1.8, 3.7 and 3.2 μM, respectively, compared with 0.24 μM for 1‐AG) and showed no evidence of central cannabinoid receptor activation in vivo at doses up to 30 mg kg−1 i.v. It is concluded that compounds like α‐Methyl‐1‐AG, O‐2203 and O‐2204 may be useful as leads for the discovery of selective MAGL inhibitors that lack direct effects upon cannabinoid receptors.

Palmitoylethanolamide and stearoylethanolamide levels in the interstitium of the trapezius muscle of women with chronic widespread pain and chronic neck-shoulder pain correlate with pain intensity and sensitivity.

Summary The levels of palmitoylethanolamide and stearoylethanolamide differ in women with chronic neck/shoulder pain and widespread pain in response to a low‐force exercise which induces pain. ABSTRACT Chronic widespread pain (CWP) is a complex condition characterized by central hyperexcitability and altered descending control of nociception. However, nociceptive input from deep tissues is suggested to be an important drive. N‐Acylethanolamines (NAEs) are endogenous lipid mediators involved in regulation of inflammation and pain. Previously we have reported elevated levels of the 2 NAEs, the peroxisome proliferator‐activated receptor type‐&agr; ligand N‐palmitoylethanolamine (PEA) and N‐stearoylethanolamine (SEA) in chronic neck/shoulder pain (CNSP). In the present study, the levels of PEA and SEA in women with CWP (n = 18), CNSP (n = 34) and healthy controls (CON, n = 24) were investigated. All subjects went through clinical examination, pressure pain threshold measurements and induction of experimental pain in the tibialis anterior muscle. Microdialysis dialysate of the trapezius was collected before and after subjects performed a repetitive low‐force exercise and analyzed by mass spectrometry. The levels of PEA and SEA in CNSP were significantly higher post exercise compared with CWP, and both pre and post exercise compared with CON. Levels of both NAEs decreased significantly pre to post exercise in CWP. Intercorrelations existed between aspects of pain intensity and sensitivity and the level of the 2 NAEs in CWP and CNSP. This is the first study demonstrating that CNSP and CWP differ in levels of NAEs in response to a low‐force exercise which induces pain. Increases in pain intensity as a consequence of low‐force exercise were associated with low levels of PEA and SEA in CNSP and CWP. These results indicate that PEA and SEA have antinociceptive roles in humans.

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.

Chronic widespread pain: increased glutamate and lactate concentrations in the trapezius muscle and plasma.

Background:Chronic widespread pain (CWP), including fibromyalgia syndrome (FM), is associated with prominent negative consequences. CWP has been associated with alterations in the central processing of nociception. Whereas some researchers consider CWP/FM as a central hyperexcitability pain condition, others suggest that the central alterations are maintained by peripheral nociceptive input. Microdialysis can be used in vivo to study muscle alterations in chronic myalgia. Aim:The aim of the study was to investigate the plasma and interstitial concentrations of metabolites and algesics in the trapezius muscle of women with CWP and in pain-free women (CON). Materials and Methods:Seventeen women with CWP and 24 CON went through a clinical examination and completed a questionnaire; the pressure pain thresholds in the upper and lower extremities were registered. Microdialysis was conducted in the trapezius muscle, and a blood sample was drawn. Muscle blood flow, interstitial muscle concentrations, and plasma concentrations of lactate, pyruvate, glutamate, glucose, and glycerol (not in the plasma) were determined. Results:CWP patients had significantly increased interstitial muscle (P=0.02 to 0.001) and plasma (P=0.026 to 0.017) concentrations of lactate and glutamate. No significant differences existed in blood flow between CWP and CON. The interstitial concentrations—but not the plasma levels—of glutamate and lactate correlated significantly with aspects of pain such as pressure pain thresholds of the trapezius (R2=0.22) and tibialis anterior (R2=0.18) and the mean pain intensity (R2=0.10) in CWP but not in CON. Conclusions:The present study supports the suggestion that aspects of pain and central alterations in CWP/FM are influenced by peripheral tissue alterations.

High Levels of N-Palmitoylethanolamide and N-Stearoylethanolamide in Microdialysate Samples from Myalgic Trapezius Muscle in Women

Background N-acylethanolamines (NAEs) are endogenous compounds that regulate inflammation and pain. These include the cannabinoid ligand anandamide (AEA) and the peroxisome proliferator-activated receptor-α ligand palmitoylethanolamide (PEA). Little is known as to the levels of NAEs in pain states in human, particularly in the skeletal muscle. The aim of this study was to investigate the levels of these lipid mediators in muscle dialysate from women with chronic neck-/shoulder pain compared to healthy controls. Methods Eleven women with chronic neck-/shoulder pain and eleven healthy women participated in this study. All participants went through microdialysis procedures in the trapezius muscle. Muscle dialysate samples were collected during four hours and analysed by nano liquid chromatography tandem mass spectrometry (nLC-MS/MS). Results We were able to detect AEA, PEA, N-stearoylethanolamine (SEA) and 2-arachidonoylglycerol (2-AG) in a single chromatographic run. Of the NAEs studied, PEA and SEA were clearly detectable in the muscle microdialysate samples. The muscle dialysate levels of PEA and SEA were significantly higher in myalgic subjects compared to healthy controls. Conclusion This study demonstrates that microdialysis in combination with mass spectrometry can be used for analysing NAEs in human muscle tissue regularly over time. Furthermore the significant group differences in the concentration of PEA and SEA in this study might fill an important gap in our knowledge of mechanisms in chronic myalgia in humans. In the long run this expanded understanding of nociceptive and anitinociceptive processes in the muscle may provide a base for ameliorating treatment and rehabilitation of pain.

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.

Signs of ongoing inflammation in female patients with chronic widespread pain: A multivariate, explorative, cross-sectional study of blood samples

Abstract This cross-sectional study investigates the plasma inflammatory profile of chronic widespread pain (CWP) patients compared to healthy controls (CON). Rather than analyzing a relatively few substances at a time, we used a new multiplex proximity extension assay (PEA) panel that enabled the simultaneous analysis of 92 inflammation-related proteins, mainly cytokines and chemokines. Seventeen women with CWP and 21 female CON participated and a venous blood sample was drawn from all subjects. Pain intensity and pain thresholds for pressure, heat, and cold were registered. A PEA panel (92 proteins) was used to analyze the blood samples. Multivariate data analysis by projection was used in the statistical analyses. Eleven proteins significantly differentiated the CON and CWP subjects (R2 = 0.58, Q2 = 0.37, analysis of variance of cross-validated predictive residuals P = 0.006). It was not possible to significantly regress pain thresholds within each group (CON or CWP). Positive significant correlations existed between several proteins and pain intensities in CWP, but the model reliability of the regression was poor. CWP was associated with systemic low-grade inflammation. Larger studies are needed to confirm the results and to investigate which alterations are condition-specific and which are common across chronic pain conditions. The presence of inflammation could promote the spreading of pain, a hallmark sign of CWP. As it has been suggested that prevalent comorbidities to pain (e.g., depression and anxiety, poor sleep, and tiredness) also are associated with inflammation, it will be important to determine whether inflammation may be a common mediator.

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.