This document describes a study that introduces a non-invasive method for imaging macrophage infiltration in inflamed atherosclerotic plaques using superparamagnetic iron oxide (SPIO) nanoparticles and MRI. The researchers injected SPIO into hypercholesterolemic and normal rabbits and found that SPIO profoundly accumulated in areas of macrophage infiltration in the atherosclerotic plaques, as confirmed by histology. SPIO-enhanced MRI was able to identify these inflamed plaques non-invasively. The results suggest SPIO-enhanced MRI can be a novel method for detecting rupture-prone inflamed plaques associated with heart attacks and strokes.

1. Plaque Inflammation in
Atherosclerotic Rabbits can be
Identified By SPIO; Introducing a
non-invasive method for Imaging
Macrophage Infiltration in active
and inflamed Vulnerable Plaque
Center for Vulnerable Plaque Research
University of Texas-Houston and
Texas Heart Institute, Houston, Texas

2. Rupture Prone Inflamed Plaque?
Atherosclerotic plaques which are characterized by:
1) Active inflammation (i.e. macrophage infiltration)
2) Extensive angiogenesis,
3) Thin permeable cap
4) Large lipid core
that are prone to rupture and cause sudden luminal
clot formation and lead to heart attack and stroke.

3. Rupture Prone Inflamed Plaque

4. Monocyte / Macrophage Recruitment
into Atherosclerotic plaques
Review of Prior Studies

5. In the study by S. Patel, James T. Willerson and
Edward Yeh, published in 1997, peritoneal
macrophages of mouse were labeled with fluorescent
latex microspheres and injected into the blood.
Antibodies to ICAM-1, integrin and E-selectin were
Injected 6-8 hours before macrophage injection.

6. Figure:
Macrophages labeled with
fluorescent microspheres adhere
to atherosclerotic plaques.

7. -The mean number of macrophages in
the proximal 1mm of aortic root was
estimated to be 143+17 per aortic root
-Antibodies against ICAM-1 and
integrin significantly reduced the
number of macrophage homing.

8. Steinberg et al, in 2000 published their study
regarding a new method of detecting
monocytes in the plaque.
The basic idea is the introduction into a
recipient animal of leukocytes differing from
those of the recipient by virtue of one easily
identified and quantified genetic marker.
PCR was the tool to detect the mutated leukocyte.
Due to its extreme sensitivity, this test is able to
detect a band in a dilution of 5 cells in 1 million
unmarked cells.

9. A: Time course of the disappearance of
donor monocytes purified from the blood
of a wild-type donor (NAT-R) and
injected intravenously into a mutant
(NAT-S) recipient.
B: Time course of the disappearance
of donor monocytes from the blood
of a mutant recipient (NAT-S) after
intravenous injection of 45 ml of
Whole blood from a wild type donor
(NAT-R).

10. For the atherosclerotic plaque 2 different settings were
Selected, Fatty streaks and more advanced lesions.
They concluded that 623 per million cells in the early
Fatty streaks were donor leukocytes.
In more advanced stage, the aortic arch showed a
maximum number of 3860 donor leukocytes per 1
million cells.(>1% of all the cells in aortic arch).
The rate of leukocyte infiltration and lesion expansion
will vary with time.

11. SPIOSPIO
Super ParamagneticSuper Paramagnetic
Iron OxideIron Oxide
lBlood pool magnetic resonance (MR)
imaging contrast media with a central
core of iron oxide generally coated by a
polysaccharide layer
lShortening MR relaxation time
lEngulfed by and accumulated inside
cells with phagocytic activity

12. SPIO is engulfed by monocyte/macrophage
system upon entry into the body.
Could it be used to detect the dynamic of
macrophage involvement in the
inflammatory Atherosclerotic plaque?

13. FL-labeled SPIO Incubated Macrophages 24hr

14. Atherosclerotic mice not injected with cytokines
But received SPIO showing iron particles in the
Monocytes in a clot
Iron staining H & E Staining

15. H&E Staining
Apo E-deficient mouse injected with SPIO
No cytokines
Iron Staining

16. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
Cytokines added

17. We chose Watanabe Hereditary Hypercholesterolemic
rabbits (WHHR) and New Zealand White rabbits (NZW)
for this study.
We injected them with SPIO (Feridex) 1 mMol Fe/kg and
obtained baseline as well as 5-day post-SPIO injection
MR images of the aorta (1.5 Tesla, Signa, GE systems).
Then we compared the images in hypercholesterolemic
rabbits with the normal,wild type NZW rabbits.
SPIO-Enhanced MRI study in Rabbits

18. Hypercholesterolemic Rabbit, Aorta, 4 days after
SPIO injection
Perls’ Staining H&E Staining
X10 X10

19. Hypercholesterolemic Rabbit, Aorta, 4 days after
SPIO injection
Perls’ Staining H&E Staining
X40 X40

20. Hypercholesterolemic Rabbit, Aorta, 10 days
after SPIO injection
Perls’ Staining H&E Staining
X10 X10

21. Hypercholesterolemic Rabbit, Aorta, 10 days
after SPIO injection
Perls’ Staining H&E Staining
X40 X40

22. Histopathologic Studies of Thoracic Aorta in Watanabe
Hereditary Hypercholesterolemic Rabbit after SPIO Injection
H&E staining
Iron staining Macrophage staining

23. Histopathologic studies of Thoracic aorta in Watanabe
Hereditary Hypercholesterolemic rabbit after SPIO injection
H&E staining
Macrophage staining Iron staining

24. Electron Microscopy evidence of Intracellular
SPIO in the Rabbit Aorta
Endothelial cell, x7500 Foamy cell, x4000

25. 0
10
20
30
40
50
60
0 10 20 30 40 50 60 70
macrophage (foam cell) density
SPIOpositivecell-Iron
staining
Series1
Correlation between Iron positive cells in Iron
staining and foam cell density in H&E staining in rabbit
atherosclerotic aorta.
R=0.956

26. MR Angiography 3D with Gadolinium-DTPA in
Watanabe Rabbit
3D-TOF
TR=59ms
TE=7.0ms
Flip=30
3D-TOF
TR=59ms
TE=7.0ms
Flip=30
After SPIO injectionBefore SPIO injection
Baseline Day 5

27. Rabbit ex-vivo MRI studies:
After the in-vivo MR images, we sacrificed the animals and
excised the aorta.
Then we put the isolated aorta in a gel medium, clamped
both ends and any side branches and injected gadolinium
inside the lumen.
We did the same procedure for all rabbits.
We also used 2 more rabbits, one WHHR and one NZW
that were not injected with SPIO, as control, in the ex-vivo
MR study.

28. Ex-vivo MR Study of Thoracic Aorta in SPIO-injected
Atherosclerotic and Normal Rabbits after Compared to
Non-injected Controls.
Watanabe rabbit
post-SPIO
Watanabe rabbit
without SPIO
NZW rabbit

29. Conclusion:
1) SPIO nanoparticles profoundly accumulate
n some (not all) areas of atherosclerotic lesions
in rabbits and mice.
2) There is a strong correlation between the areas of
SPIO accumulation and macrophage density
in mice and rabbit atherosclerotic plaques.
3) Non-invasive SPIO-enhanced MR imaging can
identify inflamed atherosclerotic plaques.

30. Center for Vulnerable Plaque Research
Houston, Texas