Single voxel spectroscopy was performed using the following parameters: PRESS, TR 3000ms/TE 30ms, 90 degree flip angle, NEX 8, FOV 16, with a volume of interest of 2?2?3cm. During each session, bcl-2 two separate SVS sequences were performed, once with the VOI placed in the right anterior insula and once in the right posterior insula. The approximate Montreal Neurological Institute 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. Also functional magnetic resonance imaging trials in FM have shown augmented pain activity in these regions. 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.
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 uses a linear combination of individual spectra obtained from pure molecular species to fit the experimental spectra. Values for Glu, glutamine, combined GluGln, and other metabolites including: N acetyl aspartate, INCB018424 choline compounds, creatine, and myoinositol were calculated as absolute concentrations using the water signal for normalization. Resulting metabolite absolute concentrations were reported in arbitrary institutional units. Since our voxels incorporated cerebrospinal fluid 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, a toolbox which operates within the image analysis program Statistical Parametric Mapping. 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. 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. Corrected metabolite concentrations were entered into SPSS v. 16 for calculation of differences between FM and HC groups and correlational analyses with pain outcomes. Harris et al.
Page 3 Arthritis Rheum. Author manuscript, available in PMC 2010 October 1. Experimental Pain Pressure pain tenderness was assessed prior to the H MRS session as described previously. 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 using a random presentation paradigm. During the testing, stimulus pressures were determined interactively: a computer program continuously adjusted stimulus pressure levels 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. Statistical Analyses Metabolite levels and pain ratings were entered into SPSS v. 16. We performed two way ANOVAs to determine differences in metabolite levels wit