Jennifer Lefeuvre1,2, Joseph R Guy1, Nick Luciano1, Emily Leibovitch1, Mathieu D Santin2, Afonso C Silva1, Steve Jacobson1, Stéphane Lehericy2, Daniel S Reich1, and Pascal Sati1
1NINDS, NIH, Bethesda, MD, United States, 2INSERM U1127/CNRS UMR7225, CENIR, Brain and Spine Institute, Paris, France
Synopsis
Multiple
sclerosis (MS) is a demyelinating disease that affects the entire central
nervous system, with more than 90% of patients showing focal or diffuse
abnormalities in the spinal cord (SC). Experimental autoimmune
encephalomyelitis (EAE) in marmosets is an attractive animal model of MS due to
its radiological presentation with brain lesions that mimic MS1. However,
spinal cord lesions in marmoset EAE have not yet been well characterized. The
proposed methodology allows high-quality, high-resolution imaging of SC lesions
in autopsied marmosets with EAE. The artifact-free images allowed accurate
detection of focal and confluent rim lesions along the edges of the SC.Purpose
To
enable an accurate assessment of disease-related abnormalities in the SC of marmosets
with EAE.
Methods
Five
SCs were collected following perfusion fixation for healthy (n=1) and EAE
animals (n=4) immunized with 200 mg fresh-frozen human white matter homogenate.
Both upper and lower spine tissues were stored in 4% formaldehyde (Figure 1A). Each
SC sample was inserted inside a 50 ml Falcon tube filled with Fomblin and imaged
on a 7T animal scanner (30 cm USR/AVIII Bruker Biospin) using a 35 mm inner
diameter (ID) volume coil (Bruker Biospin). The imaging protocol included a 3D
T2*-weighted gradient-echo (TR/TE: 80/15 ms, FA: 20°) using 100 μm isotropic
voxel size. To obtain high-quality images of the entire spine, the total
acquisition time was ~30 h. After this first imaging session, the SC tissues were
extracted from the spine samples by removing the surrounding tissue, including vertebrae
and muscles (Figure 1B). A 3D-printed SC holder was created
2 (netfabb
Professional 5.0, Projet 6000 HD printer with Visijet Sl Tough material)
(Figure 1C) and was used to insert all the extracted SC tissues in a 15 ml Falcon tube filled with
Fomblin (Figure 1D). These extracted SC tissues were imaged on the same scanner
with a 25 mm ID volume coil (Bruker Biospin). Images were acquired using a 3D
T2* weighted gradient-echo (TR/TE: 50/10 ms, FA: 12°) with 70 μm isotropic in-plane
resolution and 200 μm slice thickness. The total scan time for the entire spine
was 12 h in one session. All images were processed offline (MIPAV and ImageJ,
NIH) to generate volumes of the entire SC.
Results/Discussion
At
equivalent signal-to-noise level (SNR
non-extracted
= 39.7 versus SNR
extracted = 35.5) and similar voxel size (1 nL
for non-extracted versus 0.98 nL for extracted), it was possible to image the extracted
SC tissues in a substantially shorter acquisition time (30 hours for
non-extracted versus 12 hours for extracted). Due to the presence of various tissues
(nerve roots, vertebrae, muscles) around the spinal cord, images of the
non-extracted SC displayed non-negligible signal dropout on the edges of the SC
white matter (Figure 2, top). These image artifacts were absent from the
extracted SC images (Figure 2, bottom), thus enabling better visualization of
the SC white matter. Another benefit of the extraction was to correct for the
curvature of the SC (Figure 3, sagittal views). SC images from the healthy
control displayed excellent gray-to-white matter contrast and homogeneous signal
intensity across the white matter (Figure 3, left). SC images from EAE animals also
showed excellent lesion-to-white matter contrast,
which
allowed for accurate detection of focal area of abnormal signal and confluent rim-like
abnormal signal rims along the edges of the SC (Figure 3, right).
Conclusion
Using
our proposed methodology, we were able to obtain high-quality artifact-free
images of the entire ex vivo marmoset spinal cord with an overnight scan. This methodology
opens the door for accurate evaluation of the spinal cord lesion distribution
in this animal model of MS.
Acknowledgements
No acknowledgement found.References
1. Gaitan
MI et al. Perivenular brain lesions in a primate multiple sclerosis model at
7-tesla magnetic resonance imaging.
Multiple Sclerosis Journal 2014, Vol 20(1) 64–71
2. Guy
JR et al. Custom fit 3D-printed brain holders for comparison of histology with
MRI in marmosets. J Neurosci Methods. 2015 Sep 10;257:55-63