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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 andTexas Heart Institute, Houston, Texas
Rupture Prone Inflamed Plaque?
Atherosclerotic plaques which are characterized by:
1) Active inflammation (i.e. macrophage infiltration)2) Extensive angiogenesis,3) Thin permeable cap4) Large lipid core
that are prone to rupture and cause sudden luminal clot formation and lead to heart attack and stroke.
Rupture Prone Inflamed Plaque
Monocyte / Macrophage Recruitment into Atherosclerotic plaques
Review of Prior Studies
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.
Figure:Macrophages labeled with fluorescent microspheres adhere to atherosclerotic plaques.
-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.
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.
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).
For the atherosclerotic plaque 2 different settings wereSelected, Fatty streaks and more advanced lesions.
They concluded that 623 per million cells in the earlyFatty 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.
SPIOSPIOSuper Paramagnetic Super 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 timelEngulfed by and accumulated inside cells with phagocytic activity
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?
FL-labeled SPIO Incubated Macrophages 24hr
Atherosclerotic mice not injected with cytokinesBut received SPIO showing iron particles in the Monocytes in a clot
Iron staining H & E Staining
H&E Staining
Apo E-deficient mouse injected with SPIO No cytokines
Iron Staining
Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO Cytokines added
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
Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection
Perls’ Staining H&E Staining X10 X10
Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection
Perls’ Staining H&E Staining X40 X40
Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection
Perls’ Staining H&E Staining X10 X10
Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection
Perls’ Staining H&E Staining X40 X40
Histopathologic Studies of Thoracic Aorta in WatanabeHereditary Hypercholesterolemic Rabbit after SPIO Injection
H&E staining
Iron staining Macrophage staining
Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO injection
H&E staining
Macrophage staining Iron staining
Electron Microscopy evidence of IntracellularSPIO in the Rabbit Aorta
Endothelial cell, x7500 Foamy cell, x4000
0
10
20
30
40
50
60
0 10 20 30 40 50 60 70
macrophage (foam cell) density
SP
IO p
ositi
ve c
ell -
Iron
st
aini
ng
Series1
Correlation between Iron positive cells in Iron staining and foam cell density in H&E staining in rabbit
atherosclerotic aorta.
R=0.956
MR Angiography 3D with Gadolinium-DTPA in Watanabe Rabbit
3D-TOFTR=59msTE=7.0msFlip=30
3D-TOF
TR=59ms
TE=7.0ms
Flip=30
After SPIO injectionBefore SPIO injection
Baseline Day 5
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.
Ex-vivo MR Study of Thoracic Aorta in SPIO-injected Atherosclerotic and Normal Rabbits after Compared to
Non-injected Controls.
Watanabe rabbitpost-SPIO
Watanabe rabbitwithout SPIO
NZW rabbit
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.
Center for Vulnerable Plaque Research Houston, Texas