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Comparing DTI White Matter Integrity to Labeled Fiber Tracts in CLARITY Whole-Brains

Eric Chang*, Miklos Argyelan, Toni-Shay Chandon, Jordan Dienstag, Yifan Li, Manisha
Aggarwal, Susumu Mori, Anil Malhotra
Zucker Hillside Hospital, Manhasset, New York, United States
Background: Abnormalities in white matter integrity have been reported in several psychiatric
disorders, including schizophrenia. These deficits are typically measured using diffusion tensor
imaging (DTI), a technique that infers white matter microstructure based on directional water
diffusion through brain tissue. While DTI has proven to be a powerful non-invasive tool in both
clinical and research settings, it lacks the spatial resolution necessary to interrogate neural
circuits on the microscopic level and performs poorly in regions with complex fiber trajectories.
Also, because DTI is an indirect measure of white matter integrity, it cannot determine the cause
of anisotropy or diffusivity changes. Recently, novel brain clearing techniques such as Clear
Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining Tissue-hYdrogel
(CLARITY) allow for molecular phenotyping of neural circuits on multiple biological scales,
including brain-wide, mesoscopic, and microscopic. By combining DTI with CLARITY, we set
out to compare inferred white matter microstructure to axon-associated proteins labeled with
myelin basic protein (MBP) and neurofilament (NF).
Methods: For ex vivo DTI, we scanned six PFA-hydrogel C57BL/6J mouse brains on an 11.7 T
MR scanner for 24 hrs and 15 directions. DTI data was analyzed using FSL and TrackVis
software. Following MR scanning, samples were processed with the CLARITY protocol until
optically transparent. Whole cleared brains were then immunostained with MBP or NF primary
antibody followed by a fluorescent secondary antibody (Alexa Fluor 633). Cleared and labeled
brain samples were imaged on an Olympus Fluoview FV1000 multiphoton microscope and
analyzed using Imaris 8.0 software.
Results: We found that the liquid PFA-hydrogel solution did not adversely affect high-resolution
DTI scanning. CLARITY whole brains labeled with MBP or NF antibodies produced intact 3dimensional white matter tracts of the major commissures and fiber tracts in mice. We compared
CLARITY brains to DTI scalar metrics of fractional anisotropy (FA), mean diffusivity (MD),
axial diffusivity (AD), and radial diffusivity (RD) in specific regions of interest (ROIs) that have
been associated with decreased FA in schizophrenia patients. In the corpus collosum ROI, we
found that mean FA strongly correlated with mean MBP fluorescence intensity (Spearman’s
R=0.92, P<0.01), while RD (R=-0.64) and MD (R=-0.70) were negatively correlated. In
examining the anterior commissure with seeded tractography, we also found positive correlations
between mean FA and MBP intensity(R=0.66), and NF intensity (R=0.40).
Conclusions: This unique strategy of combining DTI and CLARITY provides insight into the
nature of white matter integrity differences that are observed in models of psychiatric disease and
in human patients. By using molecularly phenotyped CLARITY whole brains, we can now map
axon-related proteins and quantify fiber tracts on several biological scales. By understanding
how the structural connectivity of the brain is changed by psychiatric illness, we can begin to
develop improved therapeutics and drugs to remedy these deficits.
Keywords: Diffusion tensor imaging, CLARITY, mouse brain, schizophrenia, White Matter