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2) The study found that SPIO nanoparticles were taken up by macrophages within inflamed plaques, darkening the plaques on MRI.
3) Histological analysis confirmed iron particle presence in plaques, correlated with macrophage density and plaque inflammation. This non-invasive MRI method after SPIO injection shows promise for detecting vulnerable human plaques.
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Advanced benchtop microscopes can deliver resolution on the order of nanometers. However, they are highly invasive and require hours to days of tedious sample preparation, increasing the possibility of artifacts. Imagine a microscope that can capture images in real time directly from a living animal!
Befitting this, laser confocal endomicroscopy (LCE), with its probe-based imaging approach, delivers cellular resolution, while being minimally invasive. Many scientists and imaging core facility managers refer to it as “virtual histology”; it captures real time microscale images like classical histology, in a non-destructive manner. It is a cutting-edge imaging modality with endless possibilities in preclinical imaging.
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Topics to be discussed in this webinar included:
Hardware functionality and software operation of the FIVE2 (ViewnVivo) – fluorescence in vivo endomicroscope
How to use the FIVE2 (ViewnVivo) for imaging tissue architecture with cellular resolution and for optical sectioning
Examples of normal and abnormal tissues in the abdominal cavity using a variety of fluorophores
Overview of the approach to image analysis
An understanding of applications of the FIVE2 (ViewnVivo) in preclinical research
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052 non invasive high resolution detection
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
5. 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…
6. Current Methods for DetectionCurrent Methods for Detection
of Vulnerable Plaquesof Vulnerable Plaques
lOptical coherence
tomography
lRadioscintigraph
lThermography
lNear infrared
spectroscopy
lMagnetic resonance
imaging
8. 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- …
11. Current applications of SPIO in MR imaging:
-Detection of Hepatic Lesions
(primary and metastatic cancers)
-Experimental Nephritic syndrome in Laboratory animals
-Monitoring rejection of transplanted heart or kidney in
the animal model of allograft transplantation.
-Experimental detection of CNS lesions in laboratory
animals.
12. USPIOs Enter the AtheroscleroticUSPIOs Enter the Atherosclerotic
Plaque ThroughPlaque Through
lMacrophages that engulfed
them
lFissured or thin cap
l Extensive angiogenesis
l vasa vasorum leakage
l Intra plaque hemorrhage
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.
All SPIO were labeled by a fluorescent dye
(DCFA).
15. Double DAPI Staining with Fluorescence-labeled SPIO Macrophages
after 24hr Incubation
16. HypothesisHypothesis
Active macrophages residing inside
the inflamed vulnerable plaques can
be visualized following injection of
SPIO or USPIOs into the systemic
circulation by virtue of a decrease in
the magnetic resonance signal
intensity of the plaque,and correlate
with histopathologic characteristics of
vulnerability to rupture.
17. 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
18. SPIO and T2 Effect
In-vitro study to show the effect of
macrophage SPIO uptake on their
T2 relaxation time
21. 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.
•The SPIO that we used was Feridex (Berlex) injectable
solution.
•Animals were sacrificed on day 3 and 5 after injection.
22. 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.
23. •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.
24. Histopathologic study of the Mouse injected
With SPIO (Thoracic Aorta)
ApoE KO mouse, Movat staining,
proximal aorta
Coronary
Cross section
Atherosclerosis
plaque
25. Histopathologic study of ApoE KO Mouse injected
With SPIO (Thoracic Aorta)
CD68 staining
(aortic plaque)
Iron Staining (aortic plaque) Iron Staining (coronary section)
Iron particles Iron particles
26. Histopathologic study of ApoE KO Mouse
injected With SPIO (Abdominal Aorta)
H&E staining
Iron Staining CD 68 staining
Iron particles
27. Histopathologic study of wild type Mouse
injected With SPIO (Thoracic Aorta)
H&E staining
CD68 stainingIron staining
28. 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
29. MR Image of Abdominal Aorta After SPIO
Injection in ApoE and Control Mice
ApoE
deficien
t 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
30. 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).
31. 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
32. Histopathologic studies of Thoracic aorta in Watanabe
Hereditary Hypercholesterolemic rabbit after SPIO injection
H&E staining
Iron staining
Iron staining
33. Histopathologic studies of Thoracic aorta in Watanabe
Hereditary Hypercholesterolemic rabbit after SPIO injection
H&E staining
Iron staining Iron staining
Iron particles
34. Plaque Cell Density vs SPIO
0
10
20
30
40
50
60
0 10 20 30 40 50 60 70
Cell Denity in H&E staining
SPIOpositivecell-Iron
staining
Series1
R=0.956
Correlation between Iron positive cells in Iron
staining and cell density in H&E staining in rabbit
atherosclerotic aorta.
35. MR Angiography 3D with Gadolinium-DTPA in
Watanabe Rabbit
Before SPIO injection After SPIO injection
36. Ex-vivo MR study of the thoracic aorta in Watanabe and
Wild type rabbit after SPIO injection compared to control.
3D MR Angiography with Gadolinium-DTPA
Watanabe rabbit
Post-SPIO
Watanabe rabbit
control
NZW rabbit
control
NZW rabbit
Post-SPIO
37. Ex-vivo MR study of the thoracic aorta in Watanabe and
Wild type rabbit after SPIO injection compared to control.
(Gradient echo)
Watanabe rabbit
Post-SPIO
Watanabe rabbit
control
NZW rabbit
Post-SPIO
NZW rabbit
control