A total of 42 female subjects (28 FM patients and 14 healthy controls) were analyzed in the present study. Patients were recruited as part of a pharmacological multicenter study (EudraCT # 2004-004249-16). All three sites – one in England, Sweden and Germany, respectively - also performed the experiment in healthy controls, consecutively recruited throughout the study. A total of 92 female FM patients aged 25 to 55 years, mean age 44 (SD = 8.2) were enrolled. Of the 92 patients 9 were excluded from fMRI analyses due to image artifacts or en passant findings of intracranial anomalies, leaving 83 patients for matching with healthy controls. A total of 14 healthy female controls were available, aged 24 to 48 years, mean age 34 (SD = 8.6). Due to the relatively large number of patients, each healthy control could be age-matched with two FM patients (n = 28, range 24–48 years, mean 38, SD = 6.8). In cases where there were more than two patients with the same age, the two patients with the most similar duration of FM symptoms were chosen. The study was performed in accordance with the Helsinki Declaration and was approved by the local Ethical Committee at each of the three participating sites. All patients and healthy controls gave written informed consent. Parts of the present dataset were used in 2 previous publications; see Jensen et al. 2010  where all 92 available FM patients were analyzed, and Jensen et al. 2009 , where 11 FM patients and 14 controls from the present study were included.
All subjects were first screened and then scheduled for one behavioral session and one fMRI session on two adjacent days. On day one, subjects were familiarized with the equipment that was used to evoke experimental pain, calibrated for subjective pain ratings and asked to fill out self-report questionnaires. FMRI scanning was performed on day two.
All subjects were right-handed. The study protocol required that all medications that could influence pain perception, including psychopharmacological medications, were washed out. Patients had to withdraw from all central nervous system acting therapies, including antidepressants, anticonvulsants, mood stabilizers, opioids, and narcotic patches, and to discontinue treatment with transcutaneous electrical nerve stimulation, biofeedback, tender and trigger point injections, acupuncture, and anesthetics. Required periods off medication were dependent on the actual pharmacological characteristics to ensure complete washout. All analgesics were prohibited during the study, except for paracetamol, dipyrone and nonsteroidal anti-inflammatory agents (NSAIDs), which were used as rescue medications. The rescue medications had to be prescribed at the lowest available dose and for the shortest period of time necessary to manage the patient’s acute pain. Use of any analgesic or narcotic drug had to be discontinued 48 hours prior to the assessments of pain sensitivity. Zolpidem was allowed for treatment of insomnia. Exclusion criteria included the following: severe psychiatric illness (including severe melancholic depressive episode); serious suicide risk; history or behavior that would prohibit study compliance; history of substance, drug, or alcohol abuse; heavy cigarette smoking (> 25 cigarettes/day); presentation of an intracranial anomaly; significant cardiovascular, pulmonary, gastrointestinal, hepatic, or renal disease; history of autoimmune disease; current systemic infection; active cancer (except basal cell carcinoma) or current cancer therapy; unstable endocrine disease; severe sleep apnea; or pregnancy or breastfeeding. FM patients were mainly recruited from primary care and were diagnosed according to the 1990 American College of Rheumatology criteria . All patients had a self-reported average pain intensity of at least 40 mm on a 100 mm visual analogue scale (VAS) over the previous week. For healthy controls, the exclusion criteria included presence and/or history of any clinical pain problem.
Calibration of pain and stimulations during fMRI were performed using a laptop-controlled tool that delivered pressure to the right thumbnail. This specially designed tool, which consists of a plastic piston that applies pressure via a 1 cm2 hard rubber probe, has been proven effective in several previous publications [9, 31, 32]. Calibration was performed one day before fMRI scanning. Subjects were instructed to rate the intensity of the pain evoked by each stimulus by putting a mark on a 0–100 mm horizontal VAS ranging from “no pain” to “worst imaginable pain”. Each subject’s pain threshold and tolerance were determined in an ascending series of pressures, using 50 kPa increments (tolerance was determined as the first pressure where the patient rated > 60 mm VAS). These values were then used to create a randomized series of five different pressure intensities within the range of each subject’s threshold and tolerance. For example, if the pain threshold was represented by 200 kPa, and the maximum pain rating by 600 kPa, the randomized series would consist of pressures of 200 kPa, 300 kPa, 400 kPa, 500 kPa and 600 kPa. In total, 15 stimuli were delivered in a randomized order at 30 seconds intervals. The duration of each pressure stimulus was 2.5 seconds. Pain ratings from the randomized series were used to calculate each subject’s calibrated pressure at 50 mm VAS. This calibrated pressure was used during fMRI scanning on the following day.
Imaging with fMRI
Images were collected using three different 1.5 Tesla scanners: in London a General Electric HDx scanner was used, in Stockholm a General Electric Twinspeed Signa Horizon, and in Cologne a PHILIPS scanner was used. Multiple T2*-weighted single-shot gradient echo EPI sequences were used to acquire blood oxygen level dependent (BOLD) contrast images. The following parameters were used: repetition time: 3000 ms (35 slices acquired), echo time: 40 ms, flip angle: 90 degrees, field of view: 24 × 24 cm, 64 × 64 matrix, 4 mm slice thickness with 0.4 mm gap and sequential image acquisition order. In the scanner, cushions and headphones were used to reduce head movement and dampen scanner noise. Placing a blank screen in front of the patient’s field of view from inside the scanner minimized visual distraction during scans.
Two types of stimulations were used during the functional scans: individually calibrated painful pressure, representing each subject’s 50 mm VAS, and a non painful pressure perceived as light touch, representing 0 mm VAS. All stimulations were randomly jittered over the scanning time, preventing subjects from anticipating the onset time and event type. The time interval between consecutive events was randomized with a mean stimulus onset asynchronicity (SOA) of 15 seconds (range 10–20 seconds). The total duration of the scans was approximately 35 minutes. Before scanning, subjects were instructed to focus on the pressures on the thumb and not to use any distraction or coping techniques.
In addition to the functional scans, high-resolution T1-weighted structural images were acquired in coronal orientation for anatomical reference purposes and screening for cerebral anomalies. Parameters were: Spoiled Gradient Recalled 3D sequence, repetition time: 24 ms, echo time: 6 ms, flip angle 35 degrees with a voxel size of 0.9 × 1.5 × 0.9 mm3.
The scanning procedure was standardized between sites by the use of written manuscripts for the oral instructions as well as practical training for all investigators involved in the study. In order to ensure calibrated experimental procedures and scanner settings, several visits were done at the three sites by a central coordinator.
The day before fMRI-scanning, all patients used a 0–100 mm VAS to rate their average clinical pain during the previous week and their current pain. Additionally, patients completed the Fibromyalgia Impact Questionnaire (FIQ); a 20-question questionnaire that assesses the overall symptom severity in patients with FM . A high score on the FIQ corresponds to high self-reported FM severity.
Analysis of behavioral data
The differences between FM patients and healthy controls regarding age and pressure pain sensitivity were assessed using two independent samples t-tests (two-tailed). A polynomial regression was used to determine each individual’s representation of VAS 50 mm, built on the 15 ratings from the randomized series of thumb pressures. The SPSS statistics software, version 18, was used for statistical analyses of behavioral data.
Functional connectivity analysis
The connectivity fMRI analyses were performed in line with the methods previously described by our group [26, 27, 34] and other groups [35, 36]. In brief, functional data was preprocessed to decrease image artifacts, between-slice timing differences, and differences in odd/even slice intensity. The data was then spatially smoothed, using a Gaussian kernel of 6 mm full-width at half-maximum and temporally filtered (0.009 Hz < f < 0.08 Hz). Several spurious or nonspecific sources of variance were removed by including the following variables in the overall statistical model: 1) six movement parameters computed by rigid body translation and rotation during preprocessing, 2) mean whole brain signal, 3) mean brain signal within the lateral ventricles, and 4) the mean signal within a deep white matter ROI. Inclusion of the first temporal derivatives of these regressors within the linear model accounted for the time-shifted versions of spurious variance. The functional connectivity analysis produced coefficients for each seed-to-voxel correlation, and Fisher’s r-to-z transformation was used to convert these correlation maps into z maps. Group effects were tested with random-effects analyses using Matlab 7 (Mathworks) and the Statistical Parametric Mapping 5 software (SPM5) (http://www.fil.ion.ucl.ac.uk/spm/). Two separate one-sample t-tests were performed for the rACC and thalamus, assessing the connectivity between the seeds and the rest of the brain in FM patients (n = 28) and healthy controls (n = 14), respectively. Differences in rACC and thalamus connectivity between FM patients and healthy controls were assessed by two separate two-sample t-tests. The two seeds used in this study were 1) the rACC, centered at x = 8, y = 46, z = 4 (Montreal Neurological Institute [MNI] coordinates) with a 2 mm radius, and 2) the thalamus, centered at x = −14, y = −34, z = 12 MNI with a 2 mm radius. The coordinates were based on the results from a previous publication where we compared the pain-evoked fMRI signal in FM patients and healthy controls .
According to our hypothesis and previous studies of the DPMS , the PAG/brain stem , amygdala , nucleus accumbens , hippocampus , and prefrontal cortex  were defined as regions of interest. For predefined anatomical regions of interest (ROI’s), the statistical threshold was set at voxel-wise p < .005, uncorrected, with a minimum cluster size of 20 contiguous voxels. For all other brain regions, a threshold of voxel-wise p < .05, corrected for multiple comparisons, was used.