The role of myelination in measures of white matter integrity: Combination of diffusion tensor imaging and two-photon microscopy of CLARITY intact brains

Abstract

Diffusion tensor imaging (DTI) is used extensively in neuroscience to noninvasively estimate white matter (WM) microarchitecture. However, the diffusion signal is inherently ambiguous because it infers WM structure from the orientation of water diffusion and cannot identify the biological sources of diffusion changes. To compare inferred WM estimates to directly labeled axonal elements, we performed a novel within-subjects combination of high-resolution ex vivo DTI with two-photon laser microscopy of intact mouse brains rendered optically transparent by Clear Lipid-exchanged, Anatomically Rigid, Imaging/immunostaining compatible, Tissue hYdrogel (CLARITY). We found that myelin basic protein (MBP) immunofluorescence significantly correlated with fractional anisotropy (FA), especially in WM regions with coherent fiber orientations and low fiber dispersion. Our results provide evidence that FA is particularly sensitive to myelination in WM regions with these characteristics. Furthermore, we found that radial diffusivity (RD) was only sensitive to myelination in a subset of WM tracts, suggesting that the association of RD with myelin should be used cautiously. This combined DTI-CLARITY approach illustrates, for the first time, a framework for using brain-wide immunolabeling of WM targets to elucidate the relationship between the diffusion signal and its biological underpinnings. This study also demonstrates the feasibility of a within-subject combination of noninvasive neuroimaging and tissue clearing techniques that has broader implications for neuroscience research.

Keywords: Fractional anisotropy; Multimodal imaging; Myelin basic protein; Neuroimaging; Tissue clearing; Whole brain immunolabeling.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Fig. 1

Brain-wide analyses combining ex vivo DTI and CLARITY. (A) Schematic of experimental design for DTI-CLARITY multimodal approach. (B) DTI images were registered to a single reference brain within DTI space. CLARITY MBP images were registered to the same reference brain within CLARITY space. WM ROIs were manually delineated in DTI FA space (TrackVis) and CLARITY MBP space (Imaris). Our procedure yielded corresponding WM ROIs that correlated in a near linear relationship between volume comparisons (Fig. 3B). Displayed ROI volumes are: hippocampal commissure (green), stria medullaris (blue), and fasciculus retroflexus (yellow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Whole mouse brain CLARITY MBP immunolabeling. (A) Images of CLARITY MBP whole brain in 3D showing a top-down view (left) and rotated horizontal view (right). Scale bars, 2 mm. (B) 1500 µm sagittal optical section near the midline of a CLARITY MBP brain shows many of the major myelinated WM structures within the mouse brain. Labeled WM tracts are: corpus callosum (cc), fimbria (fi), fornix (fx), anterior commissure (ac), stria medullaris (sm), posterior commissure (pc), fasciculus retroflexus (fr), mammillothalamic tract (mmt), and cerebellar WM (cb). Scale bar, 1.0 mm. (C) Representative sagittal image slice from FA map showing some of the same WM structures.

Fig. 3

FA is correlated with MBP immunofluorescence. (A) Images show ROI-based comparisons of WM ROIs in DTI space (left) and MBP-positive WM structures in CLARITY space (right). Displayed ROIs are corpus collosum (genu, body, splenium) in orange and anterior commissure (posterior aspect) in green. Scale bar, 1000 µm. (B) DTI volumes significantly correlated with CLARITY volumes across all quantified WM structures (P<0.00001). (C) Mean MBP immunofluorescence correlated significantly with mean FA (P< 0.01). However, MBP did not correlate with measures of directional diffusivity, mean AD (D) or mean RD (E) across all WM ROIs measured. Linear regressions are shown with 95% confidence intervals. Each data point is a mean MBP immunofluorescence and mean FA measure from a single WM ROI, in one mouse brain. MBP immunofluorescence values were normalized to a global average. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

RD does not correlate with myelination in a subset of WM tracts. (A) Within a subset of mainly commissural WM tracts, FA and RD were significantly correlated with MBP immunofluorescence. (B) RD did not correlate with MBP in the remaining WM tracts, indicating that other factors such as fiber architecture, axon density, or axon caliber may be more significant contributors to diffusion anisotropy and directional diffusivity in these WM regions. Linear regressions are shown with 95% confidence intervals. Each data point is a mean MBP immunofluorescence and mean FA measure from a single WM ROI, in one mouse brain. MBP immunofluorescence values were normalized to a global average.

Fig. 5

Spatial resolution differences and within-tract analyses. (A) Comparison of coronal FA and direction-encoded color (DEC) FA maps to CLARITY MBP microscopy. The low spatial resolution of the DTI images, in comparison to microscopy, becomes evident when moving from macroscopic to microscopic scales. Large, coherent WM structures (e.g. corpus callosum) are well represented by DTI, but more complex fiber