Alan C Seifert1,2,3, Joseph A Borrello1,2,3, Jessie Laffey4, Etty Cortes4, Tamjeed Sikder4, Enna Selmanovic5, Bradley N Delman2, John Crary4, Kristen Dams-O'Connor5, and Junqian Xu1,2,3,6
1Biomedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 2Department of Radiology, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 3Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 4Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 5Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 6Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, United States
Synopsis
Whole-brain ex vivo
MRI has proven extremely valuable in neuroscience research, but detailed
descriptions of the specific preparation and packaging steps to minimize
motion, susceptibility artifacts, and background signal are often missing from
reports of experimental findings. We
present the design of a reproducible container and a detailed protocol that
yield whole-brain images uncorrupted by susceptibility artifacts. The most important steps to eliminate
susceptibility artifacts due to residual air bubbles are removal of the
leptomeninges, application of vacuum to dislodge bubbles, immersion in Fluorinert,
and rolling of the sealed container, although removal of leptomeninges may be
undesirable in some cases.
Introduction
Whole-brain or
whole-hemisphere ex vivo MRI has yielded incredibly detailed information on neuroanatomy
and neurological disorders1–5. However,
detailed descriptions of the specific preparation and packaging steps to
minimize motion, susceptibility artifacts, and background signal are, with few
exceptions6–10, often omitted in reports focused on
experimental findings. We therefore present
the design of an easily reproducible container and a detailed protocol that
yield whole-brain images almost completely free of susceptibility and motion
artifacts.Methods
Container: Parts for an acrylic imaging container
were cut from cylinder, sheet, and rod stock and assembled using acrylic
welding solvent. The outer vessel has
8in outer diameter, 7.5in inner diameter, 8.625in outside length, and 8.25in
inside length. The brain is stabilized
by movable acrylic rods 7.875in long and 0.25in in diameter, anchored by two
perforated end plates 0.4375in thick with 0.25in diameter holes at 0.5in
on-center spacing. Three acrylic rods
are permanently attached to one endplate, so that the endplate at the closed
end of the vessel can be remotely manipulated without placing pressure on the
brain tissue. The lid is constructed of
three 0.156in thick acrylic plates, and is attached to a 0.5x0.5in flange on the
vessel using twelve glass-filled nylon nuts and 1in long machine screws with
10-32 threads and 0.156in diameter. A 0.5in
wide, 0.094in thick neoprene gasket provides a liquid-tight seal.
Tissue preparation: Human brains were extracted and immersed
in formalin for >1 month. Seven
brains were scanned for the purpose of iteratively developing this protocol:
five brains with intact leptomeninges; one hemi-brain with leptomeninges
removed from the frontal lobe, slit over sulci in the temporal lobe, and intact
in the parietal and occipital lobes; and one brain with leptomeninges removed. Each brain was agitated under a vacuum10 of -1 bar for 15min while
immersed in formalin. A platform of rods
was constructed between the two endplates, the brain was placed onto this
platform, the endplates were slid against the brain, and additional rods were
inserted to form a cage around the brain (Figure 1b,c). The caged brain was slid into the vessel,
which was then filled with Fluorinert11,12 (FC-770, TMC
Industries). Residual formalin, which is
less dense than and immiscible with Fluorinert, was removed using a syringe, then
the container was sealed and rolled through a full revolution >10 times to
dislodge any remaining air and formalin.
Imaging: Imaging was performed on a 3T human scanner (Skyra, Siemens) using a
20-channel head/neck coil. The protocol
included FLAIR (0.63mm isotropic, TR/TI/TE=4800/1650/291ms, 2.3hr),
susceptibility-weighted imaging (0.63mm isotropic,
TR/TE1/TE2=29.00/20.00/24.88ms, 55min), and five multi-echo GRE (ME-GRE)
acquisitions (0.39mm isotropic,
TR/TE1/TE2/TE3/TE4=28.0/2.95/8.80/14.60/20.40ms, FA=30°/25°/20°/15°/10°, 2hr
each). ME-GRE images were combined by
root-sum-of-squares, and T1, T2*, and apparent proton density (S0) maps were fitted.Results
Relaxometry and SNR
measurements are tabulated (Figure 2) in the best-prepared brain (Figure 3). When leptomeninges are removed,
susceptibility artifacts arising from residual air bubbles in sulci are nearly
completely eliminated; only a single bubble was visible (Figure 3). If the entire protocol is followed, except for
removal of the leptomeninges, susceptibility artifacts overwhelm the images (Figure
4). In a hemi-brain in which portions of
the leptomeninges were removed, slit, or left intact, more air bubbles remain
in the areas covered by intact or slit leptomeninges than areas where the
leptomeninges were removed (Figure 5).Discussion
The T1 and T2* values for formalin-fixed brain tissues are
consistent with reported values13–15, providing additional
confirmation that Fluorinert does not alter brain tissue’s relaxation
properties. Fluorinert was chosen as the
susceptibility-matching solution rather than Fomblin due to its lower
viscosity, which allows it to fill the sulci and ventricles, but its low
boiling point precludes its exposure to vacuum.
Fluorinert also conducts heat more effectively than Fomblin, which mitigates
tissue warming during long scans with high RF duty cycles.
The echo-combined ME-GRE image provides the best resolution
and yields parametric maps from the same data, but the acquisition time for
this sequence is long. FLAIR provides
excellent contrast and high resolution in a 2.3hr acquisition. The 20-channel head/neck coil and the longer
RF wavelength at 3T provide excellent image uniformity.
Successful elimination of air is challenging and requires
removal of the leptomeninges, which is a tedious process and is inevitably
incomplete for deep fissures and the cerebellum. This step is also undesirable when preservation
of the leptomeningeal-parenchymal interface is of histological interest. Slitting the leptomeninges over sulci was found
to be no more effective in allowing air to escape than leaving the leptomeninges
intact.
Opportunities remain to improve this container
and protocol. There is little margin of
error when tightening the screws to seal the container. If too loose, the container leaks; if
overtightened, the threads strip. Larger
fasteners or hardware made from a stronger material should be used in future
designs. Also, the container does not
fit into a standard 32ch 7T head coil. A
smaller container with a hemispherical end would enable imaging at 7T, but
these changes would significantly increase the complexity of manufacturing and
assembling the container.Conclusion
We have presented a simple
and reproducible container design and preparation protocol that enables ex vivo
human brain imaging at 3T with minimal susceptibility artifacts.Acknowledgements
This study was
supported by NIH/NINDS U01-NS086625 (KDO).References
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