3.1. Patients
Thirty patients (15 RRMS and 15 PPMS) with clinically definite MS according to McDonald’s criteria (16) were recruited. Patients who had received corticosteroids within the previous 3 months were excluded because of its potential effect on the enhancement of acute plaques. All patients underwent a full neurological examination and their expended disability status scale (EDSS) was measured. The joint ethics committee at Tehran University of Medical Sciences approved the study. Prior to image acquisition, all subjects provided written informed consent.
3.2. Magnetic Resonance Imaging
All MRI and MRS acquisitions were acquired on a 3-Tesla scanner (Magnetom Tim Trio 3T, Siemens, Erlangen, Germany) with an 8-channel head coil in our tertiary university hospital. Axial and sagittal T2-weighted images (TE/TR=3000/96 ms) were acquired. Contrast T1-weighted images (TE/TE=650/14 ms) were obtained in patients 10 minutes after intravenous administration of paramagnetic contrast at a dose of 0.1 mg/kg (Magnevist, Bayer Schering Pharma, Germany).
MRS was performed using multivoxel chemical-shift imaging (CSI) and employing the point-resolved spectroscopy (PRESS) technique in the periventricular region. MRS acquisition was done at the volume of interest (VOI) with the dimension of 8×8 voxels and the field of view (FOV) with the dimension of 16×16 array of spectra, resulting in the voxel size of 1×1×2 cm3. The acquisition parameters were spectral bandwidth of 2000 Hz, number of spectral points of 1024, TR equal to 1000 ms and TE equal to 135 ms. In order to perform absolute quantification and to reform signal phase distortion, a single voxel spectroscopy (SVS) was acquired from an external reference phantom with a known concentration using PRESS technique without water suppression. Prior to acquiring the MRS data, manual shimming was performed to optimize magnetic field homogeneity and calibration. The entire protocol took less than 45 minutes.
3.3. Voxel Selection
A single radiologist, who was blind to the MS subtypes, identified periventricular MS lesions on T2-weighted MRI. Figure 1 shows the area over which spectroscopic imaging was performed with the rectangles on the midsagittal image hyper-intensities.
Figure 1.
Periventricular lesions marked with black rectangles
To minimize partial cerebrospinal fluid volume effects, we considered voxels that were completely within a lesion, without any apparent involvement of the ventricles or sulci. Only non-enhancing lesions that occupied at least 60% of the voxel were studied in both groups (6). We assessed all non-enhancing legions fulfilling the mentioned inclusion criteria. Overall, there were 94 included lesions for RRMS patients (mean number of plaques±SD: 7.8±3.09) and 83 lesions for PPMS patients (mean number of plaques±SD: 7.5±3.2).
3.4. MRS Quantification
Quantification of the spectra was performed in the time domain using subtract-QUEST (17). In subtract-QUEST MRSI, we used an optimized metabolite basis-set to estimate the metabolites as well as the base-line. The simulated basis-set was created in NMR-SCOP of the jMRUI software. Fitted peaks for the metabolites included the range of chemical-shift as follows: NAA (1.9, 2.1), Cr (2.9, 3.1) and Cho (3.1, 3.3) (17). All metabolite concentrations were expressed relative to Cr. We performed external referencing to compute the absolute concentration of metabolites against an external reference phantom containing the known concentration of water (18).
The result of quantification was evaluated using Cramer-Rao bound (CRB) that is a method to describe unreliability of estimation. According to some criteria such as Cramer-Rao minimum variance bound (17), the accurate peak fitting reliability was confirmed.
3.5. Statistical Analysis
All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) for Windows Ver. 17.0 (SPSS Inc., Chicago, Illinois, USA). Statistical differences were evaluated with Student’s T or the Mann-Whitney U tests depending on the normality of data. In addition to comparing the metabolite concentrations and their ratios, we evaluated the differences in important variables (age, disease duration and EDSS) between two MS subtypes. P values of less than 0.05 were considered significant for all analyses.
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