This study investigated the use of superparamagnetic iron oxide (SPIO) nanoparticles to detect vulnerable atherosclerotic plaques via MRI. Histological analysis found higher SPIO uptake in inflamed plaques of WHHL rabbits compared to normal arterial walls. MRI of WHHL rabbits showed decreased aortic wall signal intensity after SPIO injection. Ex vivo MRI further revealed luminal irregularities and negative enhancement in WHHL aortas. The results suggest SPIO can detect plaque inflammation and vulnerability by MRI, with potential for targeted SPIO to precisely locate high-risk plaques.

1. Background:
It is now widely questioned that
atherosclerotic plaques with
hemodynamically significant stenosis
cause the majority of acute
myocardial infarction and stroke.
Rather, they are caused by the so-
called "vulnerable plaques". These
plaques, retrospectively characterized
by large lipid cores, thin fibrous caps,
dense superficial macrophage
infiltration, or endothelial denudation
with thick cap, are prone to induce
thrombosis and sudden luminal
occlusion. It is still impossible for
existing diagnostic techniques to
accurately predict which plaque is
going to cause luminal thrombosis.

2. We believe the answer may not be
found solely in the structural evaluation
of the plaques. In other words,
atherosclerotic lesions with similar
structural features may not behave
similarly. The need for assessment of
functional properties or activity of
plaques, in particular their monocyte
recruitment rate and macrophage
activity, new angiogenesis, matrix
proteolysis has led us to pursue
“functional assessment” of plaques.
We believe Magnetic Resonance
Imaging (MRI) has the potential to
fulfill all the above in addition to
illustrating the anatomy of plaque
and lumen.
We have sought a method to detect
inflammation (macrophage density),
leaking angiogenesis and
fissured/permeable cap of
atherosclerotic plaques based on their
uptake of nano-particles of Super
Paramagnetic Iron Oxide (SPIO).

3. SPIO:
SPIO and ultra-SPIO (USPIO) have a
central core of iron oxide generally
coated by a polysaccharide layer. These
nano-particles are taken up 10-100
times more by macrophages than other
cells, and also leak out through loose
endothelial junctions of new vessels. On
MRI, they shorten relaxation time by 10
folds or more and produce a sharp dark
contrast by virtue of signal reduction.
Here, we present our preliminary
findings on detection of atherosclerotic
plaques in atherosclerotic rabbits using
SPIO nano-particles.

4. Hypothesis:
We hypothesized that certain features
associated with plaques vulnerability (i.e.
Inflammation, angiogenesis, intra-plaque
hemorrhage, and fissured/permeable cap)
may cause higher uptake of SPIO by
atherosclerotic plaques compared with
normal arterial wall.
Fig 1. Introducing a novel method for MR imaging of atherosclerotic
plaque to identify plaque inflammation, angiogenesis, vasa
vasorum, fissured and permeable cap.

5. “SPIO Effect”
SPIO-induced decreased signal intensity
is not proportional to the size of SPIO. In
other words, SPIO particles produce a
big dark halo around them, much larger
than their actual size (over-
magnification), specially in T2 images.
The above schematic figures (Fig.1)
represent a vulnerable plaque taking up
more SPIO compared to a stable plaque.
Non-stenotic, yet vulnerable plaques do
not show luminal narrowing in ordinary
MRI or MRA. However, after injecting
SPIO, a plaque loaded with SPIO can be
detected as a big dark spot along the
arterial line, as if there is a stenotic
plaque obstructing blood flow. This
phenomenon may be called “SPIO
Effect”.

6. METHODS:
In order to study the distribution of iron
in different tissues, 3 WHHL rabbits and
2 NZW rabbits (controls) were injected
with SPIO (2 mmol Fe/kg) IV through an
ear vein. One WHHL and one NZW
rabbit served as untreated controls (i.e.,
they received no SPIO). Animals were
sacrificed on postinjection day 5 and 10.
Tissues from the aorta as well as liver,
spleen, kidneys, and heart were fixed
and stained for H&E, iron, and RAM 11
(rabbit anti-macrophage antibody).

7. In Vivo:
Using a 1.5T MRI system (Signa,
General Electric) equipped with a
conventional extremity coil, baseline MRI
of the aorta was done in 4 WHHL and 2
NZW rabbits (T2 gradient echo: TR =
1200 msec, TE = 6 msec, FOV = 16 x 12
cm, matrix size = 256 x 192 pixels; 3-
dimensional magnetic resonance [3D
MR] angiography with gadolinium-DTPA:
TE = 1.3 msec, TR = 5.6 msec). The
rabbits were injected with SPIO (2 mmol
Fe/kg) IV via an ear vein. Post-contrast
MRI was performed on day 5 using the
same MRI sequences. MRI was done
with respiratory and cardiac gating. The
rabbits were anesthetized with isoflurane
for the duration of their studies.

8. Ex Vivo:
All rabbits that underwent in vivo MRI
were sacrificed. The aortas were
excised, isolated, and placed in a gel
medium. Both ends of the aorta were
clamped and all side branches were
occluded. Gadolinium-DTPA was
injected inside the lumen. Then, MRI was
performed, using the 1.5T scanner used
in the in vivo experiments (Signa,
General Electric). Data on T2 gradient
echo and 3D MR angiography sequences
were recorded for each specimen.

9. RESULTS:
Histopathologic studies in rabbits revealed
accumulation of iron in the atherosclerotic
arterial wall (Fig. 2).
The correlation between iron accumulation
and macrophage accumulation in the aortic
wall was significant (r = 0.95). Actively
inflamed atherosclerotic areas of the aortic
wall showed higher uptake of SPIO than did
the normal aortic wall (RAM-11 positive) and
noninflamed atherosclerotic areas. SPIO
particles were not evenly distributed in all
plaques. Areas with thick fibrous caps
accumulate less SPIO while areas with
minimal fibrosis and an abundance of
subendothelial foamy cells accumulate more.
Electron microscopy studies showed that
almost all SPIO particles were intracellular
(Fig. 3). They also revealed sporadic
localization in endothelial cells, though this
may simply indicate diffusion into permeable
areas of the endothelium.

10. Fig. 2. Histopathology of the aortic wall in
hypercholesterolemic (WHHL) and normal (NZW)
rabbits. Shown are the aortic wall in a WHHL rabbit (A-
C) and a NZW rabbit (J-L) 5 days after SPIO injection.
Also shown are the aortic wall in a WHHL rabbit (D-F)
and a NZW rabbit (G-I) serving as untreated controls.
Staining was done for hematoxylin and eosin (panels
A, D, G, and J), iron (panels B, E, H, and K), and rabbit
anti-macrophage antibody (RAM11) (panels C, F, L,
and I). Magnification 10x for panels A, D, G, and J;
magnification 40x for all other panels.

11. Fig. 3. Electron microscopy of the aortic intima in a
WHHL rabbit, revealing iron particles inside foamy
macrophage cells (left) and inside an endothelial
cell (right). Magnification 8000x for left panel;
magnification 2400x for right panel.

12. WHHL vs. NZW Rabbits
In vivo MRI studies revealed decreased signal intensity on
3D MR angiography in the aortic wall (Fig. 4).
Fig. 4. In vivo images of the aorta in atherosclerotic
(WHHL) and normal (NZW) rabbits, obtained by 3-
dimensional (3D) TOF magnetic resonance
angiography with gadolinium-DTPA before and 5 days
after SPIO injection.
(A) WHHL rabbit before injection; (B) WHHL rabbit
after injection; (C) NZW rabbit before injection; (D)
NZW rabbit after injection.

13. Because SPIO uptake in the liver,
spleen, bone marrow, and other tissues
imposed a tissue artifact, changes in
T1- and T2-weighted images of the
aorta or other arteries could not be
appreciated.
Ex vivo MRI studies were done to
negate the effect of the tissue artifact
mentioned above. As revealed by 3D
MR angiography, there were significant
luminal irregularities in the aortic walls
of SPIO-injected WHHL rabbit. Also, as
shown by T2*- weighted images of the
SPIO-injected WHHL rabbit, SPIO had
a negative enhancement effect in the
atherosclerotic aortic wall (Fig. 5).

14. Fig. 5. (Top) Ex vivo images of the intraaortic lumen
in atherosclerotic (WHHL) and normal (NZW) rabbits,
obtained by 3-dimensional (3D) TOF magnetic
resonance angiography with gadolinium-DTPA. (A)
WHHL rabbit injected with SPIO; (B) WHHL rabbit not
injected with SPIO; (C) NZW rabbit injected with
SPIO; (D) NZW rabbit not injected with SPIO.
(Bottom) T2 gradient echo magnetic resonance
imaging sequences for the same subjects, in the axial
view (E-H, respectively).

15. Conclusion:
Histologic examination of SPIO
injected in WHHL rabbits showed a
significantly higher uptake of SPIO
particles by aortic atherosclerotic
lesions than normal arterial wall (both
within the same animals and also
compared to NZW rabbits). These
particles can be found in the plaque
as early as 3 hours (data not shown),
and as late as 10 days post injection.
MR imaging of the rabbit aorta both
in-vivo and ex-vivo revealed the
reduction in signal intensity in the
aorta after injection of SPIO. This
effect could be seen mainly in T2*
and 3D angiogram sequences.

16. Implications :
Our preliminary findings may have clinical
application in detection of vulnerable plaques
using MRI. The goal should be to achieve
plaque-targeted SPIO (i.e. ox-LDL and ICAM-1
antibody-conjugated SPIO, under development
in our laboratory). This goal holds promise for
more precisely locating vulnerable plaques by
MRI.
Since monocyte/macrophage system is the
major source for SPIO accumulation in the
plaques, histologic studies of atherosclerotic
plaques after the injection of SPIO could help
us study on the dynamics of macrophage
movement in and out of the plaque. Further
information on inhibitors and stimulators of
macrophage homing could be obtained using
this novel macrophage tracer in the near
future.