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Central pain augmentation resulting from enhanced excitatory and/or decreased inhibitory neurotransmission is a proposed mechanism underlying the pathophysiology of functional pain syndromes such as fibromyalgia (FM). Multiple functional magnetic resonance imaging (fMRI) studies implicate the insula as a region of heightened neuronal activity in this condition. Since glutamate (Glu) is a major cortical excitatory neurotransmitter that functions in pain neurotransmission, we hypothesized that increased levels of insular Glu would be present in FM patients and that the concentration of this molecule would be correlated with pain report.
19 FM patients and 14 age- and sex-matched pain free controls underwent pressure pain testing and a proton magnetic resonance spectroscopy (H-MRS) session wherein the right anterior and right posterior insula were examined at rest.
FM patients had significantly higher levels of Glu (mean(SD): FM 8.09(0.72); HC 6.86(1.29); p=0.009) and combined glutamate and glutamine (e.g. Glx; mean(SD): FM 12.38(0.94); HC 10.59(1.48); p=0.001) within the right posterior insula as compared to controls. No differences were detected in any of the other major metabolites within this region (all p>0.05) and no group differences were detected for any metabolite within the right anterior insula (all p>0.10). Within the right posterior insula, higher levels of Glu and Glx were associated with lower pressure pain thresholds across both groups (Glu: r=−0.43; p=0.012; Glx: r=−0.50; p=0.003).
Enhanced glutamatergic neurotransmission resulting from higher concentrations of Glu within the posterior insula may play a role in the pathophysiology of FM and other central pain augmentation syndromes.
Although acute pain can serve a beneficial function to alert an organism of immediate or imminent tissue damage, chronic pain can often occur in the absence of tissue damage or inflammation. Functional chronic pain syndromes are a subset of pain disorders wherein patients paradoxically report frequent pain symptoms in the absence of anatomical injury or objective pathological findings (1,2). As such these disorders are particularly troublesome for patients and clinicians alike. Although new treatment options exist (3-5), significant disability and dysfunction are prevalent.
Fibromyalgia (FM) is the prototypical functional chronic pain condition that afflicts approximately 2-4% of individuals (6-8). Although the etiology of this disorder remains largely unknown, emerging data suggests that FM arises through augmentation of central pain processing pathways. This hypothesis is based largely upon findings of previous functional neuroimaging studies showing that FM patients display augmented neuronal responses to both innocuous and painful stimuli (9,10), corroborating the allodynia and hyperalgesia seen in this condition (11).
A growing body of literature suggests that glutamate (Glu), an excitatory neurotransmitter, within the central nervous system may play a role in FM pathology. A study by Peres et al. found that cerebrospinal fluid levels of Glu were elevated in FM patients possibly having consequences for glutamatergic neurotransmission (12). In a separate line of inquiry, the concentration of glutamine (a precursor in Glu biosynthesis) in the cerebrospinal fluid of FM patients was positively correlated with a number of evoked pain measures: greater glutamine levels were associated with greater pain (13). Moreover, administration of ketamine, a glutamate channel blocker, has been found to reduce experimental (14) and clinical pain (15) in FM. While these studies are informative they do not identify specific brain region(s) which are either the origin or the target for Glu in FM.
We recently demonstrated that long-term treatment of FM patients with acupuncture and/or sham acupuncture lead to changes in Glu levels within the posterior insula which were correlated with changes in experimental and clinical pain (16). Patients displaying greater reductions in Glu also had greater decreases in both experimental and clinical pain.
Here we extend these findings by investigating the relationship between insular Glu and combined glutamate + glutamine (i.e. Glx) in individuals with FM and age- and sex-matched pain free controls. We hypothesized that if insular hyperactivity is due to enhanced glutamatergic neurotransmission in FM, patients should display elevated levels of Glu as compared to controls. Furthermore if these levels are indicative of augmented pain processing, Glu and Glx levels should be negatively correlated with evoked pressure pain thresholds.
19 female FM patients (mean(SD) age in years: 45.2(15.0)) and 14 age- and sex-matched healthy controls (HC; mean(SD) age in years: 45.9(11.1); p-value = 0.89) were studied. All participants gave written informed consent and all study protocols were approved by the University of Michigan Institutional Review Board.
All FM participants: 1) met the American College of Rheumatology (1990) criteria (17) for the diagnosis of FM for at least 1 year; 2) had continued presence of pain more than 50% of days; 3) were willing to limit the introduction of any new medications or treatment modalities for control of FM symptoms during the study; 4) were over 18 and under 75 years of age; 5) were female; 6) were right handed; and 7) were capable giving written informed consent. FM participants were excluded if they: 1) had current use or a history of use of opioid or narcotic analgesics; 2) had a history of substance abuse; 3) had the presence of concurrent autoimmune or inflammatory disease such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, etc. that causes pain; 4) had concurrent participation in other therapeutic trials; 5) were pregnant and nursing mothers; 6) had severe psychiatric illnesses (current schizophrenia, major depression with suicidal ideation, substance abuse within two years); or 7) had current major depression. Concomitant medications for FM participants are listed in Supplementary Table 1. Longitudinal H-MRS data from 10 of the FM patients has been reported previously (16).
All HCs were: 1) between 18 and 75 years of age; 2) female; 3) capable of giving written informed consent; 4) right handed; and 5) willing to complete all study procedures. Exclusion criteria for HCs were: 1) having met the ACR criteria for FM; 2) having any chronic medical illness including psychiatric disorders (psychosis, schizophrenia, delusional disorder etc.); and 3) subjects who were pregnant.
All subjects underwent conventional magnetic resonance imaging of the brain on a General Electric 3.0 Tesla MR scanner (GE, Milwaukee, USA). Single voxel spectroscopy (SVS) was performed using the following parameters: PRESS, TR 3000ms/TE 30ms, 90 degree flip angle, NEX 8, FOV 16, with a volume of interest (VOI) of 2×2×3cm. During each session, two separate SVS sequences were performed, once with the VOI placed in the right anterior insula and once in the right posterior insula (Figure 1A). The approximate Montreal Neurological Institute (MNI) coordinates for the center of the anterior and posterior voxels were: 34,19,0 and 38,-17,8 respectively. These coordinates include regions shown previously to be activated during acute pain (18). Also functional magnetic resonance imaging (fMRI) trials in FM have shown augmented pain activity in these regions (9,10). Given the time constraints for our H-MRS session, we examined the right insula since it was contralateral to the pain stimuli previously used in our fMRI trials of FM (9,16). Participants were at rest during the H-MRS session. The raw data from each single voxel MR spectroscopy sequence underwent manual post-processing using H-MRS software (LCModel, Oakville, ON, Canada). LCModel uses a linear combination of individual spectra obtained from pure molecular species to fit the experimental spectra (Figure 1B). Values for Glu, glutamine (Gln), combined Glu+Gln (i.e. Glx), and other metabolites including: N-acetyl aspartate (NAA), choline compounds (Cho), creatine (Cr), and myo-inositol (myoI) were calculated as absolute concentrations using the water signal for normalization (19). Resulting metabolite absolute concentrations were reported in arbitrary institutional units (AIUs). Since our voxels incorporated cerebrospinal fluid (CSF) and the volume of CSF dilutes H-MRS derived metabolite values, we corrected our metabolite levels for CSF volume for each participant. For this we used Voxel Based Morphometry (VBM), a toolbox which operates within the image analysis program Statistical Parametric Mapping (SPM; http://www.fil.ion.ucl.ac.uk/spm/software). High resolution T1 images were segmented into gray matter, white matter, and CSF and then regions of interest within the anterior and posterior insula were used to extract gray matter, white matter, and CSF volumes from these images using the SPM2 toolbox Marsbar (http://marsbar.sourceforge.net). Metabolite values were corrected by dividing the observed concentration in AIU by the percentage of volume of the entire voxel that was not occupied by CSF (i.e. the percentage of voxel volume occupied by gray matter plus white matter). Corrected metabolite concentrations were entered into SPSS v.16 (Chicago, Il) for calculation of differences between FM and HC groups and correlational analyses with pain outcomes.
Pressure pain tenderness was assessed prior to the H-MRS session as described previously (20,21). Briefly, discrete pressure stimuli were applied to the subject's thumbnail using a stimulation device which eliminates any direct examiner/subject interaction. Pain intensity ratings were recorded on the Gracely Box Scale questionnaire (GBS) using a random presentation paradigm (21). During the testing, stimulus pressures were determined interactively: a computer program continuously adjusted stimulus pressure levels (low=GBS 0.5; medium=GBS 7.5; and high=GBS 13.5) to produce the same response distribution in each subject. Pressure pain thresholds were then correlated with H-MRS derived metabolite levels using SPSS v.16.
Metabolite levels and pain ratings were entered into SPSS v.16 (Chicago IL). We performed two-way ANOVAs to determine differences in metabolite levels with group (FM versus HC) and age strata as fixed factors. Since there was evidence of difference in variability in Glu and Glx between the HC and FM groups, we preformed an additional analysis using weighted least squares, with weights equaling the inverse of the corresponding estimated group variances. We next correlated pressure pain thresholds with Glu and Glx levels from the posterior insula as these levels were found to be elevated in the FM participants. Pearson's correlations were calculated on the combined group of FM and HC participants. Separate multiple linear regression models were constructed with Glu or Glx as dependent variables, and group (HC versus FM) medium pressure threshold, and the age strata as independent variables. Significance was set at a p-value of 0.05.
As evidenced by Table 1 and Figure 2A, individuals with FM displayed elevated levels of both Glu (p=0.009) and Glx (p=0.001) within the posterior insula. Glu and Glx remained significantly elevated in similar analyses that used weighted least squares (Glu p=0.008; Glx p=0.001). 18 of the 19 FM patients had Glu and Glx levels that were higher than the healthy control mean. FM patients also had a trend towards higher NAA in the posterior insula (p=0.06) however this did not meet statistical significance. There were no differences between FM and HC groups in the other major metabolites (e.g. Gln, myoI, Cr, and Cho) within the posterior insula (all p≥0.10). These data suggest a relatively specific elevation of Glu and Glx in right posterior insula for the FM group.
As evidenced by Table 1 and Figure 2B, there were no significant group differences in Glu and Glx or other metabolites within the anterior insula (all p>0.11). These data suggest that the elevated levels of Glu are specific for the posterior insula and do not extend into the anterior regions.
Significant negative correlations between pressure pain thresholds and posterior insula Glu and Glx levels were observed when both groups were combined (Table 2). A scatter plot of posterior insula Glx values versus medium pressure pain thresholds is illustrated in Figure 3. These data suggest that, regardless of whether an individual is a FM patient or control, individuals with higher levels of Glu and/or Glx also have enhanced sensitivity to experimentally induced pressure pain.
Since group status (FM versus HC) and pressure pain thresholds were both related to Glu and Glx levels in the posterior insula, we constructed separate linear regression models with either Glu or Glx as dependent variable, and group (FM versus HC) and medium pressure pain threshold as independent variables. Since the FM and HC were age-matched, we further used age as a stratum variable (factor) in the regression model. This is akin to the stratified analysis traditionally carried out in case-control designs. As evidenced by Table 3, both group and pressure pain threshold were significant predictors of Glx and these factors trended towards significance for Glu. For both Glu and Glx, FM patients exhibited higher values than the controls. For example, the FM patients on an average had 1.16 units higher Glx values compared to the healthy controls, for a fixed pressure pain level and age-stratum. Further, medium pressure pain threshold was negatively associated with each of the outcomes. No significant group X pressure pain interaction term was detected for either model (both p>0.25) and for this reason the interaction term was not included in the final models. Similar results were obtained in analyses using weighted least squares (Glx: group beta=1.28, p=0.009; medium pressure beta=−0.47, p=0.007; Glu: group beta=0.78, p=0.07; medium pressure beta=−0.41, p=0.007). Similar effects were also obtained when using general linear models with the high pressure threshold (Glx: group beta=1.23, p=0.01; high pressure beta=−0.37, p=0.008; Glu: group beta=0.89, p=0.05; high pressure beta=−0.18, p=0.17).
Overall these data indicate that FM patients have elevated Glu and Glx levels within the posterior insula and that these levels are associated with pressure pain thresholds. Since there was no significant group X pressure threshold interaction term, the relationship between Glu and Glx levels and pain threshold was similar across groups. Although the relationship was similar it was shifted towards higher metabolite levels for the patient group.
These findings point towards a potential role of insular Glu in the pathophysiology of fibromyalgia. The levels of glutamate in the posterior insula were higher for individuals with FM as compared to controls, and the levels of glutamate were negatively correlated with pressure pain thresholds. This suggests that the “left-ward shift” in the stimulus response function seen in both experimental pain testing and functional imaging in FM (i.e. hyperalgesia) is associated with higher levels of glutamate in certain brain regions involved in pain processing, such as the posterior insula (9,11). The posterior insula is known to play a prominent role in pain and interoceptive sensory processing (22,23), whereas the anterior insula is involved in the affective processing of pain and other subjective feelings (22,24). Since the levels of Glu in the anterior insula were no different in the FM group, this could suggest that a component of this disorder involves an amplification in sensory but not affective processing.
Our findings are entirely consistent with the broader literature and knowledge regarding FM and related syndromes, which suggests that individuals with these conditions are at the far right end of the bell shaped curve of pain and sensory processing in the population (25). Our data suggest that glutamate is playing a role in this augmented pain processing, in those individuals who have elevated glutamate. Since greater Glu was associated with lower pain thresholds, this suggests that Glu in the posterior insula is related to pain processing. The elevated levels of Glu in the FM group could raise the set point of baseline neural activity in this region which could result in augmented responses to painful stimuli. In a related line of inquiry, cold pain has been shown to increase Glu within the cingulate of pain free controls (26).
FM patients may have more glutamate within their synaptic vesicles, higher numbers or densities of glutamatergic synapses, or even less uptake of glutamate from the synaptic cleft. Any of these changes would be consistent with the hypothesis that there is augmentation of pain and sensory processing in FM. If true, this aspect of the pathophysiology of FM may be more similar to conditions such as epilepsy or neurodegenerative diseases than to the rheumatic syndromes which it has historically been associated with. For example, in epilepsy cortical and sub-cortical neurons appear to be hyper-excitable as a result of elevated concentrations of glutamate (27). These clusters of excited neurons are thought to form a locus of heightened activity which can then initiate a spreading wave of action potentials which propagate to other connected brain regions. FM may simply represent a condition wherein glutamatergic “hyperactivity” occurs within brain regions devoted to processing and modulating pain. This could arise from local increases in Glu or enhanced ascending activity to this area. This hypothesis is consistent with the fact that one of the FDA approved medications for FM is pregabalin, a drug whose action is thought to involve inhibition of presynaptic glutamate release (28). Interestingly this drug is also used in the treatment of epilepsy (29).
As with any trial, our study has limitations. The voxels used during H-MRS include multiple cell types. Our metabolite estimates of Glu and Glx reflect an ensemble average of all cell types (i.e. neurons, astrocytes, and glia) within the tissue samples. As such our findings must be interpreted with the knowledge that the cellular and sub-cellular location of the elevated glutamate is unknown. That said our methods have been empirically validated by other reported single voxel spectroscopy studies (30,31) indicating that this approach is “state of the art” for non-invasive assessment of molecular concentrations within the brain. We also recognize that our findings pertain only to the insula. Future studies that detect Glu levels in other pain processing structures such as the secondary somatosensory cortex, amygdala, cingulate etc. are needed to determine the spatial extent of elevated Glu levels. Of note a recent H-MRS study has shown decreased NAA within the hippocampus of individuals with FM (32) whereas we observed increased NAA in the posterior insula albeit at the trend level. In addition, our patient population excluded individuals with current major depression. It is possible that Glu levels within the anterior insula of depressed FM patients might be elevated, since it is known that the anterior insula is more involved in emotional processing of sensory information. Thus, our lack of group differences in anterior insula Glu may be due to the absence of depression in our sample. Finally although our results are significant, they originate from a relatively small number of participants. Validation of these findings from other studies could be made with larger study populations.
Overall we find that glutamate within the posterior insula is a potential pathologic factor in FM. The previously observed allodynia and hyperalgesia seen in these patients may be due to elevated excitatory glutamatergic neurotransmission within the posterior insula. Future studies are needed to confirm whether these findings are observed in other functional pain syndromes.
Funding for this study came from Department of Army grant DAMD-Award Number W81XWH-07-2-0050, NIH/NCRR grant M01-RR000042, and Dana Foundation Award in Brain and Immuno-imaging. REH was supported by a grant from the NIH/NCCAM K01 AT01111-01 and VN was supported by NIH/NCCAM K01-AT002166. We acknowledge Keith Newnham for his expertise in collecting H-MRS spectra. There are no conflicts of interest for any of the authors with the material presented.