SHAPE Society

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. Hypothesis
• We proposed the use of
superparamagnetic iron oxide
(SPIO)-enhanced MRI to detect
plaque inflammation, a key
component of vulnerable plaques.
SPIO nanoparticles are taken up
by macrophages and alter MR
relaxation properties.

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

5. Rupture-Prone Inflamed Plaque

6. Monocyte / Macrophage
Recruitment
into Atherosclerotic Plaques
•Macrophage infiltration plays a central role in
plaque vulnerability and risk of thrombotic
complications.
Review of Prior Studies

7. 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.

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

9. -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.

10. Steinberg et al, in 2000 published their study
regarding a new method of detecting monocytes in
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.

11. 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).

12. 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.

13. 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

14. It is shown that SPIO particles after
injection into the body, follow the tract
of inflammation through monocyte /
Macrophage system.
Could it be used to detect the dynamic of
macrophage recruitment in the
inflammatory atherosclerotic plaque?

15. FL-labeled SPIO Incubated Macrophages 24hr

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

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

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

19. 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

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

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

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

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

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

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

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

27. 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

28. 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

29. 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.

30. 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

31. Conclusion:
1) SPIO nanoparticles profoundly accumulate
in 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.

32. Center for Vulnerable Plaque Research
Houston, Texas