Supplemental Digital Content 1
In Vivo MRI
Experiments were performed using a 4.7 T horizontal bore NMR spectrometer
(Varian, Palo Alto, CA). After sedation with alfaxalone anesthesia (10 mg/kg; alfaxan;
Vetoquinol, Den Bosch, The Netherlands) the animals were prepared for mechanical
ventilation by an endotracheal intubation. A tail vein was cannulated for
administration of a bolus of Gadovist (gadobutrol, Gd-BT-DO3A, Bayer Schering
Pharma AG, Leverkusen, Germany).
Animals were immobilized in a specially designed stereotactic holder and placed
in an animal cradle, which was inserted into the NMR spectrometer. During MRexperiments the animals were ventilated with isoflurane (1-2 %) in N2O/O2 (70/30).
Expiratory CO2 was monitored, and the body temperature was maintained at 37°C
with a heated water pad. An infrared sensor (Nonin Medical Inc, Plymouth, MN) was
attached to the animal hind paw for monitoring of heart rate and blood oxygen
saturation. A homebuilt Helmholtz volume coil ( 85 mm) and a self-made
inductively coupled surface coil ( 35 mm) were used for radio frequency (RF)
transmission and signal reception, respectively.
On a sagittal scout image, 35 contiguous coronal slices of 1 mm were defined
covering the complete brain with the following characteristics: field of view = 40x40
mm; matrix = 128 x 128; zero-filled = 256 x 256; voxel volume= 97x10-3mm3, two
transitions. From these slices the following MRI data sets were collected (total
duration of the experiments ~1.5 hrs):
- T2 maps [ms]. Maps were obtained by a mono-exponential fit of five multi-echo
images. TR= 2850 ms; TE = 15 + 4x15 ms. The second image in this series, i.e.
TE=30ms, was used for Regions of Interest (ROI) determination.
- Magnetization transfer ratio (MTR) maps [% decrease in signal intensity]. Maps
were calculated from two T1Weighted (T1W) gradient echo images with and
without a MT-saturation pulse, with MTR=100*((Munsaturated-Msaturated)/Munsaturated).
TR=680ms; TE=3.17ms; MT-pulse: 12 ms Gaussian shaped pulse, nominal flip
angle 500°, offset – 4.7 kHz. Pixel-intensities display the percentage decrease in
the MR signal intensity due to saturation pulse.
- Gadovist leakage images [% increase of signal intensity] were calculated from two
T1W gradient echo images (TR=200 ms, TE=2.75ms, alpha=45°) before and after
a bolus of 0.5 mmol/kg Gadovist (i.v., 10 min in circulation) with Gadovist leakage
= 100*(( T1W post
Gadovist-
T1W pre
Gadovist)/
T1W pre
Gadovist).
Pixel-intensities display the
percentage increase in the MR signal intensity due to Gadovist leakage.
T2 images are highly sensitive to the increased water content in lesions caused by
vasogenic edema. The MTR value of a tissue is calculated from the ratio of free and
lattice-bound protons. Hence, reduction of MTR value of a tissue occurs when the
tissue water content increases (inflammation) and when the density of tissue
macromolecules decreases, such as by demyelination. T1W images were recorded to
visualize active lesion with a more permeable blood-brain barrier. Increase of the
T1W signal intensity by intravenously administered Gadolinium containing contrast
agent (Gadovist), occurs when the paramagnetic probe leaks through the blood-brain
barrier, which is more permeable by local production of inflammatory mediators 1.
Postmortem MRI
Post mortem MRI was performed on a 9.4 T horizontal bore NMR spectrometer
(Varian), equipped with a quadrature coil (RAPID, Biomedical, Rimpar, Germany).
Formalin-fixed brains were submerged in a non-magnetic oil (Fomblin; perfluorinated
polyether, Solvay Solexis, Weesp, The Netherlands) to prevent unwanted
susceptibility artifacts.
On a sagittal scout image, 41 contiguous coronal slices of 0.75 mm were defined
covering the complete brain, with the following characteristics: field of view =
25x25mm; matrix = 256x256; zero-filled = 512x512; voxel volume= 7.15x10-3mm3,
two transitions. The following MRI data sets were collected:
-
T2 maps. These maps were calculated by a mono-exponential fitting of six spin
echo images with increasing TE. TR= 2650ms; TE= 10+5x10ms. The second
image of this series, i.e. TE=20 ms, was used for ROI determination.
-
MTR maps. Maps were calculated from two T1W spin echo images with and
without a MT-saturation pulse. TR=1675ms; TE=23ms; MT-pulse: 8.19ms
Gaussian shaped pulse, nominal flip angle 1000, offset –9.4kHz.
-
Inversion recovery experiment in which the signal of the grey matter was
suppressed. Fast spin echo sequence, TR=4050ms, echo train length = 9, echo
spacing = 8.93 ms, effective TE= 17.86ms, Inversion Time =425ms for nulling the
signal arising from the grey matter.
Data evaluation MRI
Calculations of T2, MTR, and Gadovist leakage were done with a homemade
software package developed in MATLAB (v. 7.2 The Mathworks Inc., Natick, MA).
ROI were defined using the free available Medical Image Processing, Analysis and
Visualization (MIPAV version 4.3.0., National Institutes of Health, Bethesda, MD)
package.
The following ROI were defined on T2W images for both in vivo (TE=30ms) and post
mortem (TE=20ms) images (Supplementary Figure 1):
-
White Matter (WM) lesions: defined as areas with abnormal high signal intensities
in the WM. WM lesions were outlined in the total brain.
-
WM: the WM area was outlined in a predefined slice, i.e. the first caudal to rostral
slice which showed a complete corpus callosum connection.
-
Grey matter (GM): GM areas were outlined in the predefined slice as described
for WM.
-
Normal Appearing WM (NAWM): the NAWM area was calculated for the
predefined slice with NAWM = WM – WM lesions.
For the post mortem images the following areas were also included:
-
GM lesions were defined as areas with abnormal signal intensities on a combined
T2W image and the inversion recovery experiment with suppression of the MR
signal arising from grey matter. The latter experiment facilitates the detection of
grey matter lesions. The total hemisphere was examined.
-
Normal Appearing GM (NAGM). The NAGM area was calculated on the same
predefined slice describe above with NAGM = GM – GM lesions.
Volumes, and MRI characteristics were calculated from all ROI.
References
1.
't Hart BA, Smith P, Amor S et al. MRI-guided immunotherapy development
for multiple sclerosis in a primate. Drug Discov Today. 2006;11:58-66