MRI analysis
All MR Imaging experiments were performed on a Bruker 70/30 7T microMRI scanner equipped with
a 60 mm I.D. 1000 mT/mm gradient and shim coil. MR signal transmission and reception was
performed with a quadrature 35 mm I.D. volume Radio Frequency (RF) coil (magnet, gradient/shim
coil, and RF coil are all from Bruker, Billerica, MA). Diseased mice or non-diseased age matched
controls (C57/B6) 23 week old were group housed. The mice were fed Prolab RMH 3000 5P00 chow
ad libitum. Chemical composition of the chow included: 22.5% protein, 5.4% fat (ether extract), 6.4%
Fat (acid hydrolysis), and 4% fiber (crude).
Anesthesia was induced by placing the mouse in an induction chamber initially filled with room air,
then flowing a mixture of isoflurane (3%) and O2 (3 L/min) into the induction chamber. When the
animal was anesthetized, it was moved from the induction chamber into the magnet room, where it
was placed in a mouse bed fitted with a nose cone that continued to supply isoflurne (1.5%) and O2
(1 L/min) to maintain anesthesia during scanning. The mouse was placed in a supine position so that
its paws were accessible for the application of ECG leads. Neonatal ECG leads (3M, St. Paul MN)
were attached to the mouse’s front right paw and left rear paw. The leads were connected to an
ECG/T module that was interfaced with a PC running small animal monitoring software (SAIIInstruments, Stony Brook, NY). A respiration pillow was placed over the abdomen to monitor the
respiration rate. The animal’s vital signs were monitored and isoflurane levels were adjusted to keep
the respiration rate between 35 and 45 breaths per minute. The ECG wave form and respiration wave
form also facilitated gated acquisition of MR data to reduce motion artifacts that are caused by
cardiac and respiratory motion. The animal’s body temperature was monitored and maintained by
blowing thermostatically controlled warm air into the bore of the magnet during the scan.
When the animal was anesthetized and connected to vital sign monitoring equipment, the mouse bed
with the mouse secured in it was placed in the RF coil so that the aortic arch was at the iso center of
the RF coil. The RF coil was tuned and matched to 300.2 MHz. Then a three plane localizer scout
scan was acquired to verify that the mouse’s aortic arch was centered in the RF coil and in the
magnet. An ungated Rapid Acquisition with Relaxation Enhancement (RARE) pulse sequence with an
echo train length of eight was used for the three plane scout. Parameters for the RARE scans were:
Repetition Time (TR) = 1500 ms, Echo Time (TE) = 25 ms, Field of View (FOV) =3.0 cm, Matrix =
128x128, slice thickness (thk) = 2.0 mm Number of Averages (NEX) = 1. If necessary, the mouse
was repositioned in the RF coil or the RF coil was repositioned in the magnet or both. A follow up
three plane scout scan was acquired to confirm that the mouse’s aortic arch was centered. When the
mouse was in position, a series of ECG and respiratory gated low resolution Fast Low Angle Shot
(FLASH) scout scans were acquired to facilitate the slice plane positioning for the high resolution.
Parameters of the FLASH scout scans were: TR/TE/Flip Angle(FA)/FOV/Matrix/thk/NEX =
44ms/5ms/300/3.0cm/128x128/1.5mm/1. All gated scans were triggered when an R-wave peak was
detected that fell in between breaths. The maximum width of the breath trigger window was monitored
throughout scanning and was adjusted so that it started 100 ms after each breath and ended sooner
than 400 ms before the next breath. Low resolution scout scans and the high resolution aorta images
were acquired using a saturation slice and a fat suppression prepulse. The saturation slice was
positioned to cover the ventricles to saturate the blood that would be flowing through the image plane
during acquisition to suppress signal from flowing blood. The fat suppression pre-pulse was used to
reduce chemical shift artifact that can be caused by fat surrounding the heart. The FLASH scout
scans were acquired to generate an oblique coronal view of the aortic arch that clearly showed the
three branches that exit the top of the aortic arch. This scout scan was used to place the high
resolution ECG and respiratory gated FLASH sequence slice planes so that they covered the 3 mm
proximal to the brachiocephallic artery branch point. The parameters for the high resolution FLASH
sequence were: TR/TE/FA/FOV/Matrix/thk/NEX= 120ms/4.9ms/300/3.0cm/256x256/0.38mm/4 (giving
a pixel size of 117 µm).
Experiments were performed using two personnel. The first injected either control or ATCA view tail
vein. The second, blinded to the injections, performed the MRI scans and analysis. After the baseline
high resolution scan was acquired, mice were injected with 100uL of ATCA (100ug) via tail vein. This
concentration was determined to be optimal in initial experiments (not shown). The animal was placed
back into the RF coil, the RF coil was placed back in the magnet and the protocol to locate the aortic
arch and brachiocephalic artery was performed again to allow the acquisition of images at 30
minutes, 60 minutes, and 120 minutes post injection. The use of the brachiocephalic artery branch
point as an anatomical reference allowed reproducible slice placement for the pre injection scan and
the first post injection scan. All the post injection scans were acquired with the mouse and slice
planes in the same position so they were the same for the post injection time points.
Animals were scanned at 30 minutes, 60 minutes, and 120 minutes post injection. The post injection
scans were performed without moving the mouse so the animal position and slice positions for all the
post injection scans were the same for each animal. The animals were euthanized using CO 2 and
cervical dislocation following scanning. The aortas were removed for analysis.
For quantification of the ATCA signal in the aorta two separate methods were used. First, the ROI of
the brightest spots in the aorta walls were quantified (Figure 4B) using Image J Software. As a
comparison, each reviewer (three) blinded to the compound being measured, quantified whole aorta
walls from 4 separate 0.3 mm2 slices (slices 2-5 as slices 1 and 6 are too narrow) manually drawing
the whole aorta wall and normalized to the surrounding muscle (same area measured). One reviewer
drew the areas to be measured, the second reviewer read out the quantifiable numbers (See Table
below), and the third reviewer recorded the numbers. When measuring the entire ROI of each slice
(four ) at each time point (four) the mean intensity normalized to thorasic muscle is very similar to
what was measured originally (Figure 4C)-the brightest ROI’s that was presented in Figure 4B. Both
approaches equally quantify the significant enhanced contrast using ATCA. Statistical analysis is
shown in the text and each experiment represents five separate animals.
Supplementary Table; Quantification of ATCA aorta MRI sices
Aorta
Mouse 1 (pre)
Mouse 1 (30 min)
Mouse 1 (60 min)
Mouse 1 (120 min)
Thorasic area
Slice #
2
3
4
5
area
0.44
0.49
0.47
0.45
mean
3966
3547
5620
6167
max
10247
12248
11092
9425
min
1308
1579
606
1316
SD
1409
1722
2033
1844
area (mm2)
0.46
0.43
0.42
0.51
mean
7935
7725
7338
7343
max
9298
9003
9752
8708
min
6188
6315
5558
5478
SD
698
730
746
778
Slice #
2
3
4
5
area
0.55
0.61
0.47
0.48
mean
7384
8153
8367
6722
max
12326
12581
17607
14692
min
1677
689
1733
633
SD
1377
1820
2584
2503
area (mm2)
0.46
0.43
0.42
0.4
mean
7488
7688
7343
7319
max
10232
9690
9595
9164
min
4967
6034
5532
4876
SD
920
929
801
856
Slice #
2
3
4
5
area
0.68
0.67
0.54
0.54
mean
11580
9669
9280
12318
max
12776
13468
17196
14472
min
1433
1060
2828
1267
SD
1725
1896
2517
2766
area (mm2)
0.46
0.5
0.45
0.4
mean
8359
9067
9110
9215
max
10129
11430
11764
11621
min
6341
7296
7073
7479
SD
799
800
858
714
Slice #
2
3
4
5
area
0.71
0.65
0.52
0.49
mean
8643
9625
10175
9213
max
11626
14096
18304
17349
min
1041
906
1176
1328
SD
1587
1748
2669
2473
area (mm2)
0.42
0.46
0.56
0.46
mean
8465
8432
8323
8315
max
10568
10608
10456
11293
min
6230
5896
6015
5769
SD
874
1081
883
1130