We recruited participants at the headache clinic of Longhua Hospital, Shanghai University of Traditional Chinese Medicine. Migraine patients were diagnosed based on the criteria from The International Classification of Headache Disorders, 3rd edition (ICHD-Ⅲ)(2). The study was approved by the Ethics Committee of Longhua Hospital that is affiliated with the Shanghai University of Traditional Chinese Medicine (2015LCSY012), which was conducted in accordance with the Helsinki Declaration of 1964 with later revisions. Participants provided written informed consent after receiving detailed oral and written information regarding the study.
All participants were aged between 19 and 64 years. Participants in the migraine patient group were included if they were diagnosed with migraine by their attending neurologist/physician because they fulfilled the ICHD-Ⅲ criteria for migraine with or without aura and had at least two migraine attacks per month for the last 3 months, and if they had an established migraine diagnosis for at least 3 years. The exclusion criteria included the following: medication use (migraine prophylactic drugs, antidepressants, narcotics, antiepileptic drugs, mood stabilizers, and anxiolytics); pregnant or breast-feeding women; significant acute or chronic neurologic, psychiatric, endocrine, or medical problems; a history of conditions that could compromise the spectroscopic data (e.g., implants, dental braces, tattoos); another primary chronic headache disorder, any secondary headache disorder, or headache medication overuse; a history of having used medications known to alter GABA and GLX levels; and a history of neck injury or claustrophobia. Participants in the control group were aged-matched healthy individuals with no history of migraine. Participants were included in the healthy control (HC) group if they did not experience regular headaches and had no headache in the last 3 months. The exclusion criteria were the same as those applied in the migraine patient group with the added criterion of positive family history of migraine among first-degree relatives.
A total of 32 participants were enrolled in the study, and MRI data were collected. Four datasets were discarded due to poor spectral quality. Twenty-eight participants, including 19 in the patient group and 9 in the HC group, were considered in the final analysis. Patients were allowed to take analgesic medications, such as non-steroidal anti-inflammatory drugs. They were all assessed interictally at least 24 h after their last attack.
MRI data were obtained using a 3 Tesla Siemens Verio MR scanner with a 32-channel head coil (Siemens AG, Erlangen, Germany). The head position was fixed with foam padding to minimize movement artifacts.
Anatomical T1-weighted images were acquired using a three-dimensional magnetic preparation fast gradient echo (3D-MPRAGE) sequence with the following parameters: echo time (TE) = 3.65 ms, repetition time (TR) = 2530 ms, field of view (FOV) = 240 × 240 mm2, 256 × 256 matrix, slice thickness = 1.0 mm, and 224 continuous sagittal slices. The T1-weighted images were used to localize the voxels-of-interest (VOIs) in the following MRS acquisition.
The MRS data were acquired using a Mescher-Garwood point resolved spectroscopy (MEGA-PRESS) sequence with the following parameters: TR = 1500 ms, TE = 68 ms, 328 averages with water suppression, and eight averages without water suppression. The VOIs in the SG ACC (25 × 25 × 25 mm3), left thalamus (25 × 25 × 18 mm3), and right thalamus (25 × 25 × 18 mm3) were localized in the mid-sagittal and transverse slices, respectively (Figure 1). Six orthogonal fat saturation bands were placed surrounding the VOIs to avoid signal interference. Automated shimming followed by manual shimming were conducted to reduce the water signal full width at half maximum (FWHM) below 25 Hz.
MRS raw data were processed using the LCModel software, version 6.3-1P Stephen (Provencher, 2016)(23, 24). Water-scaled GABA and GLX concentrations were quantified based on the LCModel package and LCMgui. The edited spectra were fitted using LCM-basis functions that were generated from phantom measurements using the MEGA-PRESS sequence with the appropriate acquisition parameters(25). The GABA peak arose at 3.01 ppm, and the GLX peak, at 3.74 ppm(26) (Figure 1). The levels of GABA and GLX were expressed as ratios with respect to the N-acetylaspartate (NAA) peak at 2.01 ppm. The criteria for reliable metabolite concentrations were based on the % standard deviation (SD) of the fit for each metabolite, reflecting the Cramer-Rao lower bounds (CRLB) in the LCModel analysis(24, 27, 28). Only results with a %SD below 30% were included in the following analysis(23, 26).
To calculate the proportion of grey matter (GM), white matter (WM), and cerebrospinal (CSF) contained in the VOIs, the 3D MPRAGE T1 images were segmented using the SPM8 software (The Wellcome Centre for Human Neuroimaging, 2008) (29). The voxel CSF content in each subject was evaluated by extracting the location of the voxel from the spectra file headers and using an in-house software developed in Visual Studio 2013 (Microsoft, 2013, USA) to calculate the proportions of GM, WM, and CSF contents based on the segmented T1-weighted images. To correct the spectroscopy results for the partial volume effect of CSF contamination, each metabolite value was corrected for the CSF content in the VOI using the following formula: corrected metabolite level = uncorrected metabolite level/(1 − C), where C is the fractional CSF content in the VOI(30).
All the statistical analyses were conducted using SPSS v.22.0 (IBM Inc, 2014, Armonk, NY, USA). The normality of the distribution of all the independent variables, including age, weight, height, and metabolite concentrations, was examined using Kolmogorov-Smirnov tests. Demographic variables and GM, WM, and CSF volumes within the VOIs were compared between the two groups using a t test. Chi-squared tests were performed to assess the group effect on sex and handedness. An independent sample t test was performed to compare group differences in the GABA and GLX levels in each VOI. All tests were two tailed and the level of statistical significance was set at P < 0.05.