Computational protocol: Influence of full length dystrophin on brain volumes in mouse models of Duchenne muscular dystrophy

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Protocol publication

[…] Images of the brain were acquired on a 7 Tesla PharmaScan® (Bruker BioSpin, Ettlingen, Germany) equipped with a 370 mT/m gradient system with ParaVision® 5.1 software. The skull was placed in a 15 mL Falcon™ tube filled with a proton-free fluid, Fomblin® (Solvay, Belgium), to prevent susceptibility artefacts induced by tissue-air interfaces. A 23-mm diameter volume coil was used to acquire high resolution 3-dimentional gradient echo (FLASH) images with echo time 5.3 ms; repetition time = 15 ms; flip angle = 30°; FOV = [18 x 13 x 13]mm; zero-filling = 1.34; image matrix size = [256 x 186 x 186]; acquisition matrix size = [256 x 140 x 140]; isotropic spatial resolution = 0.070 mm; number of averages = 12; number of dummy scans = 10; receiver bandwidth = 50,000 Hz.Four mice were excluded from analysis; two mdx and two mdx-XistΔhs mice. The brain of one mdx mouse was not completely located inside the field of view and the scans of the other three mice contained imaging artefacts. All remaining brains were successfully processed. [...] The registration scheme included registration of each subject brain to a template brain. For the template brain, volumes of interest (VOIs) for the whole brain and 22 anatomical structures were manually segmented based on the Waxholm mouse brain atlas [], the Franklin and Paxinos atlas [,] and the Allen Brain Atlas [] using AMIRA (v5, FEI Software, Oregon, USA) []. Next to this, the 22 anatomical structures were either classified as white or grey matter, according to . The whole brain volume was defined as the brain tissue limited caudally by the cerebellum and rostrally by the rhinal fissure. Using the information provided by the inverse deformation field for each subject-to-template registration, the template VOIs were propagated to the individual datasets, enabling quantitative comparison of corresponding areas. The VOIs were evaluated quantitatively for volume change.Two independent observers verified the quality of the registration by visual inspection, using a custom-made graphic user interface built with MeVisLab (v2.7, MeVis Medical Solutions AG, Bremen, Germany) []. The registration was implemented using the open source image registration toolbox Elastix [] and performed in a coarse-to-fine process. Initially, rigid registration was performed to compensate for translation and rotation. Afterwards, an affine registration was conducted to compensate for differences in brain size, followed by a non-rigid B-spline registration to compensate for local changes. A Gaussian image pyramid was employed in all registration steps, applying four resolutions for the rigid and B-spline and two for the affine registration. Mutual information was used as a similarity metric. The two independent observers were not blinded; however, no manual alterations were needed as the registration of all subject brains passed the quality control. Detailed information on the used registration parameters can be found on the Elastix website ( [...] To assess skull morphology, the ratios of the length of the major and minor axis of the skull of mdx and BL10 mice were assessed on axial and coronal MRI planes in Osirix Lite v7.5. This ratio is directly related to skull eccentricity which is known to be different in DMD patients. The coronal plane was set to mid-corpus callosum and the axial plane was rostrally set to the mid-olfactory bulb and caudally set to the second white matter branching of the arbor vitae in the cerebellum (). […]

Pipeline specifications

Software tools ParaVision, MeVisLab, elastix, OsiriX
Databases Allen Brain Atlas
Application Magnetic resonance imaging
Organisms Mus musculus, Homo sapiens
Diseases Muscular Dystrophy, Duchenne