Ex vivo 100 μm isotropic diffusion MRI-based tractography of connectivity changes in the end-stage R6/2 mouse model of Huntington's disease

Abstract

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 (11.7 T) 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.

Keywords: Huntington’s disease; MRI; connectome; diffusion tensor imaging; mouse; tractography.

FIGURE 1

Anatomical segmentation of ROIs in Huntington’s disease. To investigate the differential effects of Huntington’s disease on CC, cortical areas, and HC, as well as structures of the basal ganglia, ROIs were manually defined in both control and R6/2 mice. A manual segmentation ensures that a differential atrophy of structures did not affect the anatomical definition of these ROIs. CC, corpus callosum; HC, hippocampus; ROIs, regions-of-interest.

FIGURE 2

Diffusion directionality and tractography. (A) DEC images overlaid on T₂-weighted structural images reveal the directionality of individual voxel water diffusion in the R6/2 mouse brain. (B) Exploiting this diffusion directionality, streamlines tracking fibers connecting different parts of the brain can inform on brain connectivity. (C) Parcelation of connectivity based on the origin of streamlines probes regional connectivity with fibers originating in the Ctx (red), striatum (green), HC (orange), or CC (blue). (D) Separation of the regions with streamlines colored for fiber direction provides a deeper understanding of how these regions and their connectivity are impacted by Huntington’s disease. CC, corpus callosum; DEC, diffusion-encoded color; HC, hippocampus.

FIGURE 3

Whole brain volume and tractography. (A) Representative whole brain tractograms of WT and R6/2 male and female mice. (B) Whole brain volume was significantly reduced in male R6/2 mice, but not in female R6/2 mice. This difference was also evident in the total number of streamlines for whole brain. By considering brain volume and calculating streamline density, male R6/2 mice exhibited a higher streamline density, whereas female R6/2 mice had an equivalent streamline density compared to WT controls. *p ≤ 0.05, **p ≤ 0.01. WT, wild-type.

FIGURE 4

Striatal tractography. (A) Transverse connectivity of the R6/2 male striatum reveals extensive streamlines crossing the CC, projecting through the RMS into the OB, as well as the cerebellum. (B) A sagittal cut further details the subcortical connectivity of the striatum with the thalamus, GP, NAc, STN, and SN. (C). A coronal view in contrast provides a multislice view of how streamlines from the striatum connect with the SMC S1 and S2. (D) Transcallosal connectivity of the striatum highlights the complex interaction between both hemispheres. AC, anterior commissure; CC, corpus callosum; EC, external capsule; GP, globus pallidus; IC, internal capsule; NAc, nucleus accumbens; OB, olfactory bulb; RMS, rostral migratory stream; SMC, somatosensory cortex; SN, substantia nigra; STN, subthalamic nucleus.

FIGURE 5

Tractography of the Ctx and thalamus. (A) Transverse view of streamlines seeded in the Ctx reveals widespread connectivity. (B) Streamlines project from the surface of the neocortex to the CC. (C) Parcelation of the streamlines seeded in the motor (blue) and somatosensory (red) cortex indicates mostly topographically separated territories, but with some notable areas of local overlap in both the motor and SMC. (D) Tractography of the thalamus. (E) Thalamic connectivity is highest with the striatum, but also projects to the HC. (F) Thalamic connectivity color-coded based on regions where connections are terminating. (G) Visualization of thalamo-striatal connectivity with seeds placed in the thalamus and selection of streamlines ending in the striatum. CC, corpus callosum; HC, hippocampus; SMC, somatosensensory cortex.

FIGURE 6

Regional tractography. Tracing of specific connections between two anatomical structures was achieved by seeding traces in one ROI and selecting those streamlines that ended in a target region. This allowed us to quantitatively assess the impact of Huntington’s disease on the connectivity of the basal ganglia. ROI, region-of-interest.

FIGURE 7

Tractographic dissection of the basal ganglia circuitry. (A) Schematic of the basal ganglia overlaid onto a sagittal T₂-weighted female WT mouse. (B) DEC map reveals specific diffusion directionality in subcortical structures affording added contrast to distinguish small anatomical structures, such as the STN that are not easily segmented on T₂-weighted images. (C) Tractogram of the basal ganglia. (D) Input connections to the striatum from the MC, and SMC are reduced in both male and female R6/2 mice. Only female R6/2 exhibited a significantly reduced connectivity of the thalamus with the striatum in the left hemisphere. The connections between the HC and the mPFC were significantly reduced in the left hemisphere in male R6/2 and the right hemisphere for female R6/2. (E) Output projections from the striatum to GP were also affected in female R6/2 mice, whereas in males this decrease was not significant. The magnitude of decrease in the right hemisphere of female R6/2 was also more pronounced than in the left hemisphere. The connection between the STN and SN was decreased in both male and female R6/2 mice in the right hemisphere, but only female R6/2 mice exhibited a significant decrease also in the left hemisphere. In contrast, in the left hemisphere the SN to thalamus connection was decreased in female and male R6/2 mice, but only female R6/2 mice had a significant decrease in the right hemisphere. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. DEC, diffusion-encoded color; GP, globus pallidus; HC, hippocampus; MC, motor cortex; mPFC, medial prefrontal cortex; SMC, somatosensory cortex; SN, substantia nigra; STN, subthalamic nucleus; WT, wild-type.