Azadeh Nazemorroaya1, Ali Aghaeifar1, Hildegard Schulz1, Thomas Shiozawa-Bayer2, Bernhard Hirt2, Klaus Scheffler3,4, and Gisela Hagberg4,5
1Max Planck Institute for Biological Cybernetics, Tuebingen, Germany, 2Clinical Anatomy, University of Tübingen, Tuebingen, Germany, 3High Field Magnetic Resonance,, Max Planck Institute for Biological Cybernetics, Tuebingen, Germany, 4Biomedical Magnetic Resonance, University of Tübingen, Tuebingen, Germany, 5High Field Magnetic Resonance, Max Planck Institute for Biological Cybernetics, Tuebingen, Germany
Synopsis
Post-mortem
brain MRI can yield
valuable information. However, tissue preservation and MR-compatibility of
fixation agents are challenging. Here we investigated the effect of four
MR-compatible formalin-based fixatives on the MR properties of pig brains at
several timepoints after start of fixation up to one month. The inclusion agents known to improve the dielectric
properties of the fixatives lead to greater R2* difference
between GM-WM than conventional fixatives. Vice versa these agents lead to a
decrease in T1 contrast.
Introduction
Magnetic
resonance imaging of post-mortem brain permits long imaging sessions without
movement artifacts. Acquired data can reveal valuable information about
different brain properties and clinical disease1-3. However, there are
challenges in terms of ex-vivo brain tissue preservation and their MR imaging, e.g.,
MR properties. These can be altered through factors
like natural tissue decomposition, chemical fixation, post-mortem interval
(PMI), fixation time, and temperature change. Significant reductions in the MR
relaxation times (T1 and T2) have been reported for
formalin-fixed-brains4-5 and may be reversed by washing out the
formalin from the fixed tissue5. Additionally,
the fixative agent should be MR-compatible and not degrade image quality. The chemicals used should not cause any
chemical shift artifacts6 and the magnetic
susceptibility and dielectric properties should be adjusted to achieve a good B0
and B1 homogeneity7. Here we developed four formalin-based
fixative agents with some additives to assure tissue fixation while maintaining
MRI-compatibility. The fixation effect of these fixatives on the MR properties
of pig brain was investigated up to one month after immersion fixation.Method
Four
formalin-based fixative agents (Fix01, Fix02, Fix03 and Fix04) were developed
considering MRI-compatibility. The NMR spectra of the
additives were checked by the online NMR spectra predictor tool provided in www.nmrdb.org. The
dielectric properties of final solutions were measured using SPEAG Dielectric Assessment
Kit, Probe DAK-12. Measurements were carried out for four independently
prepared set of each fixative. The averaged results are listed in figure 1.B.
Four
pig brain samples were bought from a slaughter house and placed in fixative
solutions with a PMI of less than 6h. An elliptical container divided into four
equal segments (figure 1.A) was filled with roughly 700 ml of each fixative, and the
sample was fixed in the middle of the segment with gauze. This setup enabled simultaneous
scanning of all samples at different times after start of fixation (12 hours,
1, 2, 3, 4, 13, 19, and 28 days). MRI measurements were performed at 9.4T in a Siemens
whole body MR scanner using a 16-channel transmit array in combination with a
31-element receive array
8. The data acquired on different days were co-registered
using SPM12 and were mapped to the same spatial grid using nearest neighbor
interpolation. The employed acquisition parameters were as follows:
-
T1 sequence: MP2RAGE [9]; Inversion times, TI1/TI2=900/3500 ms;
TE/TR=2.3/6ms; volume TR=9s; FA=4/6°; voxel size=0.8 mm isotropic, GRAPPA factor=3; 6/8 partial
Fourier factor
-
R2* sequence: Multi-echo GRE; TE= 6.03,
12, 18, 24, 30 ms; TR=34 ms; FA =15°;
voxel size=0.4 mm isotropic; FoV=204×165.8×46 mm3
R
2*
maps were generated using the numeric method described in [10] and the T
1
maps as described in
9. We used in-house developed Matlab codes to analyze
the data. The percent change in T
1 and R
2* between
12h and day 28 was calculated pixel-by-pixel as the difference in relaxation
normalized to the 12 hours values.
Results
Figure
2 shows an increase of gray matter (GM)-white matter (WM) R2* contrast
and WM R2* values with fixation time. This effect is greater
in Fix01 and Fix02 fixed-brains than in Fix03 and Fix04 fixed-brains. A
progressive reduction in T1 and GM-WM contrast occurred in all samples
but was most prominent for Fix01 and Fix02-immersed-brains (Figure 3). T1
values highly depend on molecular tumbling of water molecules in the tissue. With
increasing fixation time, fixative penetration and thus water immobilization
and protein cross-linking improve which affect molecular tumbling and
consequently T1 values. The R2*
and T1 variation rate slows down after day 13 which means the
fixation procedure takes roughly 3 weeks to complete.
Figure
4 demonstrates that the overall shape of R2* and T1
histograms of four fixed brains after 12 hours of fixation are similar except that
they are scaled with the sample size. By fixation time, R2*
and T1 value of GM, and WM change. This evolution causes relevant changes
in the histogram shape, in terms of distinct peaks and shoulder emerging in the
R2* and T1 histograms of day 28. The leftward
shift of the R2* and T1 histogram during
fixation time are depicted in Figure 5.A. It shows that Fix01 and
Fix02-immersed-brains had a smaller R2* reduction and a
larger T1 reduction than the Fix03 and Fix04-immersed-brains.
Pixel-wise
T1 and R2* differences between 12 hours and 28
days are shown in Figure 5.B. demonstrating prominent R2* increase and T1 reduction in the WM, especially for Fix01 and
Fix02-immersed brains. Several of the GM pixels appeared with decreased T1
and R2* values.Discussion and conclusion
We
investigated the effect of four MR-compatible formalin-based fixatives on the
MR properties of post mortem pig brain. The results demonstrated a progressive
T1 and R2* decrease with fixation time. The
reduction rate was different for different fixatives and was stabilized after 3
weeks of immersion. An increased in WM R2* values Future
work could be the segmentation of pig brain tissue classes (GM, WM and apparent
CSF in the fixative-filled ventricles) using probabilistic atlases to plot related
T1 and R2* histograms individually and track
the changes by fixation time. Another suggestion would be washing out the
fixatives from the fixed brains using Phosphate-buffered saline (PBS) to
investigate any possible changes in their MR properties.Acknowledgements
No acknowledgement found.References
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