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

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 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