DOI: 10.5061/dryad.djh9w0w4v
title: Ex vivo 100 μm isotropic diffusion MRI‐based tractography of connectivity changes in the end‐stage R6/2 mouse model of Huntington's disease

description: Background: Huntington's disease is a progressive neurodegenerative
 disorder. Brain atrophy, as measured by volumetric magnetic resonance
 imaging (MRI), is a downstream consequence of neurodegeneration, but
 microstructural changes within brain tissue are expected to precede this
 volumetric decline. The tissue microstructure can be assayed
 non-invasively using diffusion MRI, which also allows a tractographic
 analysis of brain connectivity. Methods: We here used ex vivo diffusion
 MRI (11.7T) to measure microstructural changes in different brain regions
 of end‐stage (14 weeks of age) wild type and R6/2 mice (male and female)
 modeling Huntington's disease. To probe the microstructure of
 different brain regions, reduce partial volume effects and measure
 connectivity between different regions, a 100 μm isotropic voxel
 resolution was acquired. Results: Although fractional anisotropy did not
 reveal any difference between wild‐type controls and R6/2 mice, mean,
 axial, and radial diffusivity were increased in female R6/2 mice and
 decreased in male R6/2 mice. Whole brain streamlines were only reduced in
 male R6/2 mice, but streamline density was increased. Region‐to‐region
 tractography indicated reductions in connectivity between the cortex,
 hippocampus, and thalamus with the striatum, as well as within the basal
 ganglia (striatum—globus pallidus—subthalamic nucleus—substantia
 nigra—thalamus). Conclusions: Biological sex and left/right hemisphere
 affected tractographic results, potentially reflecting different stages of
 disease progression. This proof‐of‐principle study indicates that
 diffusion MRI and tractography potentially provide novel biomarkers that
 connect volumetric changes across different brain regions. In a
 translation setting, these measurements constitute a novel tool to assess
 the therapeutic impact of interventions such as neuroprotective agents in
 transgenic models, as well as patients with Huntington's disease.
description: <strong>2.2 | MRI</strong> For MR
 imaging, mouse heads were immersed into proton‐free FluorInert (Sigma) in
 a syringe to immobilize the head for long scanning times at a high
 resolution, where minimal movement between gradient directions can affect
 image quality. Images were acquired with a Bruker AV3 HD 11.7T/89mm
 vertical bore microimaging system, with a Micro 2.5 gradient insert
 (capable of up to 150 G/cm) in a 25 mm quadrature birdcage coil (Bruker).
 Sample temperature was maintained at 21 ± 0.1°C with a Bruker SmartCooler
 BCU‐1 40/50 air chiller and probe heater with a thermocouple feedback
 loop.39 Scanning parameters were defined in ParaVision 6.0. A 3D spin‐echo
 diffusion MRI scan was acquired (repetition time, TR = 700 ms, echo time,
 TE = 24 ms, diffusion time = 12 ms, diffusion encoding duration 5
 ms, b value = 1401.57 s/mm<sup>2</sup>, with 12 non-collinear
 diffusion directions and one A<sub>0</sub> image, number of
 averages, NA = 1, matrix 196 × 128 × 128, 100 μm isotropic resolution, and
 31 h 3 min acquisition time), followed by a coregistered
 3D T<sub>2</sub>‐weighted spin‐echo scan at the same
 resolution (TR = 3500, TE = 40, NA = 1, and 15 h 55 min acquisition time).
 We used a 3D spin‐echo diffusion sequence to achieve high isotropic
 spatial resolution for investigating connectivity between small anatomical
 structures in the mouse. This approach is time‐consuming, which limits the
 diffusion paradigm, but we chose this method over an EPI approach to avoid
 potential image distortion from susceptibility artifacts at
 high field. <strong>2.3 | MR image
 processing</strong> Diffusion MR images were
 processed using Diffusion Spectrum Imaging (DSI) studio (available
 at: http://dsi-studio.labsolver.org/dsi-studio-download). The brain was masked, and the rest of the head was removed as background before processing. Reconstruction of the DTI was achieved by performing an Eigenvector analysis on the calculated tensor. Mean (MD), radial (RD), and axial diffusion (AD) maps were computed for measurement of scalar indices, as was fractional anisotropy (FA). Streamlines reflecting connectivity were mapped for the whole brain using a multidirectional deterministic fiber tracking algorithm using 10 seeds/voxel with random subvoxel positioning of seeds. Trilinear interpolation in all orientations with a step size of 0.05 mm (1/2 voxel length) was performed with an FA of 0.02 serving as a tracing endpoint of streamlines at the end of the sample, as fiber tracing was not restricted to only white matter. A streamline length of 0.2 mm (2× voxel length) was considered the minimum length of streamlines, with a maximum length of 10 mm (15 mm sample sagittal length) and an angular threshold of 60° being used to end connectivity tracing. Total number of streamlines for whole brain and individual ROIs were recorded to calculate a streamline density (streamlines/mm<sup>3</sup>) for group comparisons. Streamline length was also recorded, as there is some evidence that it is associated with regional atrophy in premanifest HD patients. <strong>2.4 | ROIs</strong> ROIs were defined manually based on anatomical structures known to be affected by HD (Figure 1), notably whole cerebral cortex (Ctx), motor cortex (MC), somatosensory cortex (SMC), corpus callosum (CC), striatum (Str), globus pallidus (GP), nucleus accumbens (NAc), thalamus (Th), substantia nigra (SN), subthalamic nucleus (STN), hippocampus (HC), and olfactory bulb (OB). Anatomical structures were defined based on those defined in the standard mouse atlas. To establish if there was a differential loss of connectivity within the basal ganglia, point‐to‐point connections between ROIs (e.g., cortico‐striatal tract) were also compared with “seeds” in one ROI (e.g., cortex) and those “ending” in another (e.g., striatum). To account for distance between ROIs, maximum streamline length was set to 10 mm for connections between cortex with the HC and striatum, 7 mm for streamlines between the thalamus and striatum, and 3 mm to map tracts from the striatum to the GP, GP to STN, and STN to SN, as well as SN to thalamus. We here analyzed both the left and right hemispheres, as there is evidence of brain asymmetry in manifest cases of HD, although this was not the case in premanifest HD.
description: DSI Studio