138 Chapter 5 Tractography analyses (III) Deterministic tractography was carried out using ExploreDTI’s threedimensional automated atlas-based tractography pipeline with a 0.2 FA threshold and maximum 30° angle change. Included fibres had to pass through the hypothalamus and a second pre-defined ROI (thalamus, amygdala or midbrain) in the corresponding hemisphere and not cross the midsagittal plane, except for the midbrain analyses. Tracts had to be within 10–200 mm length and only the segments between the ROIs were extracted to guarantee intersubject tract correspondence. More diffuse tracts connecting the hypothalamus with the orbitofrontal, anterior cingulate and parahippocampal WM were not found for every subject and were therefore not included in the eventual analyses. Total tract bundle volume and mean FA and MD results were automatically calculated in a voxel-by-voxel manner and served as input for statistical analysis. As the visual inspection of the corrected b0 and FA images showed misalignment of several voxels in the hypothalamus between the scans with opposite phase-encoding directions, only the anterior-to-posterior-encoded DTI series was incorporated for tractography analyses as less deformity was seen. Statistical analysis Statistical processing of demographic, clinical and diffusion measures was done in IBM SPSS Statistics version 24, with p-values < .05 being considered as statistically significant. Student’s t-tests were used for age, IQ and ESS comparisons between groups and a chi-square test to identify possible sex differences. Separate TBSS FA analyses were carried out in patients using time since EDS onset and ESS score as covariates of interest. Averaged ROI and tractography results were processed using one-way analyses of covariance (ANCOVAs). All DTI analyses were corrected for within group age and sex variability as covariates of no interest. Additional correction for whole-brain volume was performed in tractography analyses on absolute tract volume. Bonferroni correction for multiple comparisons was used per outcome measure for all ROI (p < .0029 for FA and MD and p < .0125 for AD and RD) and tractography (p < .0083) analyses.
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