SHAPE Society

1. Non-Invasive High Resolution Detection of
Active Atherosclerotic Plaque in ApoE
Deficient Mice by Magnetic Resonance
Imaging after Injection of Super
Paramagnetic Iron Oxide (SPIO)
Center for Vulnerable Plaque
Research
University of Texas at Houston
Texas Heart Institute

2. Vulnerable Plaque?
Atherosclerotic plaques which are
characterized by active inflammation
(macrophage infiltration), extensive
angiogenesis, thin permeable cap and
large lipid core that are prone to
rupture and cause sudden luminal clot
formation and lead to heart attack and
stroke.

3. Plaque Morphology
vs.
Plaque Activity
Why do we need both?
The short answer is: because not all plaques with
similar morphology would result in similar
outcome.

4. Morphology vs. Activity Imaging
Inactive and
non-inflamed
plaque
Active and
inflamed
plaque
Appear Similar in
IVUS OCT MRI
w/o CM
Morphology
Show Different
Activity
Thermography, Spectroscopy,
immunoscintigraphy, MRI with
targeted contrast media…

5. Human Carotid Plaque
Courtesy of
Dr. Chun Yuan
University of Washington

6. ?

7. We need MRI with vulnerable plaque
targeted contrast media that identifies:
1- Inflammation (macrophage infiltration),
2- Fissured/Permeable Cap,
3- Leaking Angiogenesis and
4- Intra-Plaque Hemorrhage
5- …?

8. Super ParamagneticSuper Paramagnetic
andand
Ultra SmallUltra Small
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 in cells
with phagocytic activity

9. Particle Core Size Particle Size Blood
(nm) (nm) Half-life
Combidex 5-6 20-30 8h
Feridex 4-6 35-50 2.4±0.2h
DDM 43/34/102 6.4 20-30 6h
Clariscan
MION 4-6 17 varies
Feruglose
--- --- --- ---
Examples of commercially available SPIOs

10. Emerging Applications of SPIO in MR Imaging
of Inflammation
-Monitoring rejection of transplanted heart in the
animal model of allograft transplantation.
-Kidney allograft transplant rejection model in animal
Models.
-Experimental detection of encephalitis in laboratory
animals.

11. HypothesisHypothesis
1- SPIO nanoparticles are
accumulated in atherosclerotic plaque
and their accumulation correlates with
histologic evidence of macrophage
infiltration.
2- Accumulated SPIO in plaque can
be detected by MRI comparing the
signal intensity of plaque before and
after SPIO injection.

12. vasa vasorum
Over magnification is a major advantage of SPIO
Darkening property of SPIO in the white background of fat
and water of plaque is another advantage

13. In-vitro Study of Macrophage
SPIO Uptake
 In a series of in-vitro studies we have tested
the rate of SPIO uptake by human activated
monocytes in different conditions regarding
incubation time and concentration of SPIO.
SPIO particles were labeled by a fluorescent
dye (DCFA).

14. FL-labeled SPIO Incubated Macrophages 24hr

15. Double DAPI Staining with Fluorescence-labeled SPIO Macrophages
after 24hr Incubation

16. SPIO and T2 Effect
In-vitro study to show the effect of
macrophage SPIO uptake on their
T2 relaxation time

18. 0
10
20
30
40
50
60
70
80
90
50 250 control
20 min
60 min
6 hours
24 hours
Macrophage Uptake of Feridex with Time
and Concentration Shown by T2 Reduction
Concentration µmol/ml

19. •Intra-macrophage SPIO
reduces T2 relaxation
time similar to extra-
cellular free SPIO.

20. In-vivo distribution of SPIO in ApoE
deficient and wild type mice:
•For the initial study, we use the mouse model of
atherosclerosis.
•
•ApoE deficient mouse has similar atherosclerotic
lesions to human and the lesions are more common in
the aortic arch and thoracic aorta.
• We used ApoE deficient mice and normal variant
(C57BL mice) as control.
•Animals were sacrificed on day 3 and 5 after injection.

21. Pre and Post-SPIO Enhanced Magnetic
Resonance Imaging of ApoE K/O and Wild
Type Mice:
We used 4.7 tesla MRI unit (University of Texas,
Galveston MRI Unit, Galveston, TX) in our study.
After baseline MR imaging with respiratory gating, we
injected 1mMolFe/kg super paramagnetic iron oxide to
six ApoE deficient and two C57bl mice through the tail
vein.
Post-contrast MR imaging were performed in day 5 with
the same parameters (TR=2.5 sec, TE=0.012 sec,
FOV=6.6 cm, slice thickness=2.0mm, flip angle
(orient)=trans, and matrices=256x256).
We selected the aorta at the level of kidney for
comparison of the baseline and post-contrast images.

22. •Tissues from different organs including liver,
kidneys, lung, heart, spleen and aorta
(including valve region ascending, descending
and abdominal) was obtained and stained.
• We used Hematoxyline and Eosin (H&E), Iron,
CD68 and Movat staining.
• After Iron staining, aortic wall of the ApoE
deficient mice and normal variant were
compared based on the number of Iron
particle.
• Also different doses of Feridex and The
timing between injection and tissue obtaining
was considered.

23. Histopathologic Study of the Mice Injected
with SPIO (Aortic Root)
ApoE KO mouse, Movat staining,
proximal aorta
Coronary
Cross section
Atherosclerosis
plaque

24. Histopathologic study of ApoE KO Mice injected
With SPIO (Aortic Root)
CD68 staining
(coronary plaque)
Iron Staining (aortic plaque) Iron Staining (coronary section)
Iron particles Iron particles

25. Histopathologic Study of ApoE KO Mice
Injected With SPIO (Abdominal Aorta)
H&E staining
Iron Staining CD 68 staining
Iron particles

26. Histopathologic Study of Wild Type Mice
Injected with SPIO (Thoracic Aorta)
H&E staining
CD68 stainingIron staining

27. Comparison of the Number of the Iron Particles (per
HPF) in ApoE KO Mice Plaque vs. Normal Wall
0
5
10
15
Atherosclerotic
Aorta
Average
number of iron
particles per
sample
P <0.001

28. MR Image of Abdominal Aorta After SPIO
Injection in ApoE and Control Mice
ApoE
deficient
mouse
C57B1
(control)
mouse
Before Injection After Injection (5 Days )
Dark (negatively enhanced) aortic wall, full of iron particles
Bright aortic lumen and wall without negative
enhancement and no significant number of iron particles

29. Typical in vivo MR images of a live mouse at the heart (left)
and renal level (right). Various vessels and aortic arch can
easily e seen in these images. The slice thickness is 0.5 mm
and the in-plane resolution is 50 µm (7.1 T MR system).

30. 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
MRI system at the University of Texas, MD Anderson,Houston,Texas).
Then we compared the images in hypercholesterolemic rabbits with
the normal,wild type NZW rabbits.
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.
SPIO-Enhanced MRI study in Rabbits

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

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

33. 0
10
20
30
40
50
60
0 10 20 30 40 50 60 70
macrophage (foam cell) density
SPIOpositivecell-Iron
staining
Series1
R=0.956
Correlation between Iron positive cells in Iron
staining and foam cell density in H&E staining in rabbit
atherosclerotic aorta.

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

35. Ex-vivo MR Study of Thoracic Aorta in SPIO-injected
Atherosclerotic and Normal Rabbits after Compared to
Non-injected Controls from both.
(3D MR Angiography with Gadolinium-DTPA)
Watanabe rabbit
post-SPIO
Watanabe rabbit
without SPIO
NZW rabbit
without SPIO
NZW rabbit
post-SPIO

36. Ex-vivo MR Study of Thoracic Aorta in SPIO-Injected
Atherosclerotic and Normal Rabbits compared to Non-
Injected Controls from both.
(Gradient Echo MRI)
Watanabe rabbit
Post-SPIO
Watanabe rabbit
Without SPIO
NZW rabbit
Post-SPIO
NZW rabbit
Without SPIO

37. MR Angiography 3D with Gadolinium-DTPA in
Watanabe Rabbit
Before SPIO injection After SPIO injection

38. Conclusion:
1) SPIO nanoparticles are actively accumulating
n some (not all) areas of atherosclerotic lesions
in rabbits and mice.
2) There is a strong correlation between the areas of foamy
cells infiltration and SPIO uptake in the mice and rabbit
plaques.
3) MR imaging of these atherosclerotic plaques show
Significant changes after SPIO injection compared to the
baseline, suggesting a possible role for iron particles
o detect the areas of active atherosclerotic plaque with
nflammation; although other possibilities could not be
ruled out at this time and warrants further studies.

39. Human Clinical Trial
Detection of Vulnerable Carotid Plaque using SPIO
Enhanced MRI
Carotid Coil
MRI of first carotid patient
Patients undergoing
Carotid surgery
Baseline MR imaging
SPIO Injection
Post-contrast MR imaging
Histologic study of
the plaque after
surgery
Currently underway…
MRI Pathway Inc.