Subjects and clinical information
The study populations consisted of 32 right-handed epilepsy children aged 5 to 14 years old (mean age 8.6± 2.7 years, 24 males and 8 females). Epilepsy children were further divided into 18 right-handed children patients with focal epilepsy (mean age 8.6± 2.4 years, 14 males and 4 females) and 14 right-handed children patients with generalized epilepsy(mean age 8.6± 3.0 years, 10 males and 4 females). The diagnoses of epilepsy were determined in Guangzhou Women and Children's Medical center according to the criteria by the International League Against Epilepsy in 2017[4]. All patients had had MRI scans and had long-term EEG records. The inclusion criteria were (1) diagnosed by an Epileptologist based on clinical, EEG and MRI findings; (2)age between 5 and 14 years; (3)attendance at standard schools; (4) high-resolution MR images acquired on the same type 3T research scanner. The exclusion criteria were (1) existence of organic brain disorder; (2) abnormalities in brain MRI scans; (3) ‘Combined Generalized and Focal Epilepsy’ and ‘Unknown’ were not included.
For group comparison, 18 healthy children (mean age 8.3± 1.8 years, 11 males and 7 females) who were similar in age, gender and education level were recruited as control group. Children with IQ < 70, a past history of neurologic or psychiatric disorders, a family history of epilepsy or a history of usage of medications damaging nervous system were excluded. This study was approved by the local ethics committee and informed consent was obtained for all participants from their parents.
MRI Acquisition
All of the participants underwent high-resolution brain MRI on a Siemens Trio 3T scanner. For VBM analysis, high-resolution three-dimensional magnetization prepared rapid gradient echo (MPRAGE) sequence was acquired for each subject using the following parameters: TR = 1160ms, TE = 4.19ms, 3TI = 600ms, field of view = 140 × 250mm2, matrix size =256 × 192, slice thickness = 1.2mm, and flip angle = 15∘). During the process of scanning, all subjects were asked to be relaxed and keep their eyes closed but stay awake.
Voxel-based morphometry
The quantitative analysis of MRI was performed with the VBM8 Toolbox within SPM8 (Wellcome Trust Centre for Neuroimaging, https://www.fil.ion.ucl.ac.uk/spm/) running under MATLAB R2013b (The MathWorks, Natick, MA, USA). According the standard protocol, the VBM analysis comprised the following steps.
The quality of each image was inspected and the image origin was set to the anterior commissure. A customized template appropriate to the study population was created because of the wide range of age of participants. All native images were normalized to the same stereotactic space by registering each to the customized template image for spatial normalization. The normalized images were then segmented into gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF). Finally, the normalized, segmented GM images were smoothed with an isotropic Gaussian kernel with a sigma of 8 mm for statistical analyses. Finally, differences in cerebral GM density between the patient and control groups were evaluated using the voxelwise generalised linear model applied using permutationbased non-parametric testing (5000 permutations).
Statistical analyses
Prior to statistical analyses, differences in demographic were identified using an independent two-sample t-test for continuous variables and x2 tests for dichotomous variables. The analyses were performed using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) and p values of <0.05 was considered indicative of statistical significance.
Using the general linear model, voxel-wise comparisons of GMV were performed between patients and controls, with age and gender as confounding covariates. The significance of group differences was estimated with the theory of p<0.001 uncorrected and the cluster size was set at >100 voxels.
To investigate the relationship between duration of epilepsy or age at epilepsy onset and GMV, the general linear model was applied. A statistical significance was accepted if p<0.05.