Positron Emission Tomography Imaging of
Poststroke Angiogenesis
Weibo Cai, PhD; Raphael Guzman, MD; Andrew R. Hsu, BS; Hui Wang, PhD; Kai Chen, PhD;
Guohua Sun, MD, PhD; Atul Gera, BS; Raymond Choi, BS; Tonya Bliss, PhD; Lina He, MD;
Zi-Bo Li, PhD; Anne-Lise D. Maag, BS; Nobutaka Hori, MD, PhD; Heng Zhao, PhD;
Michael Moseley, PhD; Gary K. Steinberg, MD, PhD; Xiaoyuan Chen, PhD
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Background and Purpose—Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important
roles during neurovascular repair after stroke. In this study, we imaged VEGFR expression with positron emission
tomography (PET) to noninvasively analyze poststroke angiogenesis.
Methods—Female Sprague-Dawley rats after distal middle cerebral artery occlusion surgery were subjected to weekly
MRI, 18F-FDG PET, and 64Cu-DOTA-VEGF121 PET scans. Several control experiments were performed to confirm the
VEGFR specificity of 64Cu-DOTA-VEGF121 uptake in the stroke border zone. VEGFR, BrdU, lectin staining, and
125
I-VEGF165 autoradiography on stroke brain tissue slices were performed to validate the in vivo findings.
Results—T2-weighed MRI correlated with the “cold spot” on 18F-FDG PET for rats undergoing distal middle cerebral
artery occlusion surgery. The 64Cu-DOTA-VEGF121 uptake in the stroke border zone peaked at ⬇10 days after surgery,
indicating neovascularization as confirmed by histology (VEGFR-2, BrdU, and lectin staining). VEGFR specificity of
64
Cu-DOTA-VEGF121 uptake was confirmed by significantly lower uptake of 64Cu-DOTA-VEGFmutant in vivo and intense
125
I-VEGF165 uptake ex vivo in the stroke border zone. No appreciable uptake of 64Cu-DOTA-VEGF121 was observed in
the brain of sham-operated rats.
Conclusions—For the first time to our knowledge, we successfully evaluated the VEGFR expression kinetics noninvasively in a rat stroke model. In vivo imaging of VEGFR expression could become a significant clinical tool to plan and
monitor therapies aimed at improving poststroke angiogenesis. (Stroke. 2009;40:270-277.)
Key Words: positron emission tomography 䡲 stroke 䡲 vascular endothelial growth factor receptor
S
creased expression of vascular endothelial growth factor
(VEGF) and VEGF receptors (VEGFRs) plays important
roles during neurovascular repair after stroke.11 Human neural stem cells overexpressing VEGF have been shown to
provide neuroprotection and enhance angiogenesis.12 Because ischemia and cell transplantation can enhance VEGFR
expression,13,14 in vivo imaging of VEGFR could be a
valuable indicator for the repair mechanisms occurring after
stroke.
Positron emission tomography (PET) has been widely used
in the clinic for diagnosis and treatment monitoring.15 We
recently developed a tracer, 64Cu-DOTA-VEGF121 (DOTA
denotes 1,4,7,10-Tetraazacyclododecane-N,N⬘,N⬙,N⵮tetraacetic acid), which can enable PET imaging of VEGFR
expression in various disease models.16,17 Herein, we demonstrate for the first time to our knowledge the in vivo imaging
of VEGFR expression after acute experimental stroke.
troke remains the number one cause of disability among
Americans each year. Besides thrombolytic treatments,
which have a very narrow time window and limited availability,1 there are currently no other therapeutic approaches.
Promising novel therapeutic strategies, such as cell transplantation,2 are moving toward clinical trials, but there is a
significant lack of clinically applicable methods to guide and
monitor such therapies. Molecular imaging, a recently
emerged field that seeks to quantitatively measure biological
processes at the cellular and molecular level in vivo,3 can be
a valuable tool for these applications. Major areas of molecular imaging in stroke research include metabolic imaging
without contrast agents,4 labeling methods for tracking stem
cells and macrophages migrating into ischemic brain regions,5 probes that can reflect the metabolic status,6,7 and
probes that binding specifically to stroke markers.8
One potentially important repair mechanism after stroke is
angiogenesis,9,10 the formation of new blood vessels. In-
Received February 14, 2008; final revision received April 24, 2008; accepted May 29, 2008.
From The Molecular Imaging Program at Stanford (W.C., A.R.H, H.W., L.H., K.C., Z-B.L., M.M., X.C.), Department of Radiology, Stanford
University, Calif; Departments of Radiology and Medical Physics (W.C.), University of Wisconsin–Madison, Wis; Department of Neurosurgery (R.G.,
G.S., A.G., R.C., T.B., A-L.D.M., N.H., H.Z., G.K.S.), Stanford University, Calif.
W. Cai and R. Guzman contributed equally to this work.
Correspondence to Xiaoyuan Chen, PhD, The Molecular Imaging Program at Stanford, Department of Radiology, Stanford University, Stanford, CA.
E-mail shawchen@stanford.edu
© 2008 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.108.517474
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Figure 1. Characterization of the stroke model. A, Axial and coronal T2-weighted MR images of the rat brain at various days after surgery. B, The stroke size based on MRI. C, Hematoxylin and eosin–stained rat brain tissue after dMCAo surgery. D, Weekly 18F-FDG
PET scans of the dMCAo rats. E, 18F-FDG uptake in the stroke area and the contralateral brain. Arrowheads indicate the stroke area in
all cases. n⫽4; *P⬍0.05; **P⬍0.01; ***P⬍0.001.
Materials and Methods
Distal Middle Cerebral Artery Occlusion Surgery
All experimental protocols performed on animals were approved by
the Stanford University Administrative Panel on Laboratory Animal
Care. Focal cerebral ischemia (distal middle cerebral artery occlusion [dMCAo] model) was generated as described previously.18
Sham-operated animals (controls) underwent the same surgery without occlusion of the blood vessels. Daily intraperitoneal injections
with 5-bromo-2⬘-deoxyuridine (BrdU) were administered in 3 mice
for 7 days after dMCAo.
Receptor Binding Assay
The detailed procedure of a competitive cell binding assay has been
reported previously.16,19 VEGF121, VEGFmutant (the amino acid residues 63, 64, 67, 82, 84, and 86 in the VEGFR binding sites were
mutated to alanine), and their DOTA conjugates were prepared as
previously reported.16,20 VEGFR-1 and VEGFR-2 binding affinities
of VEGF121, VEGFmutant, and the DOTA conjugates were evaluated
by porcine aorta endothelial cells transfected with human VEGFR-1
and porcine aorta endothelial cells transfected with human VEGFR-2
cell-binding assays using 125I-VEGF165 (GE Healthcare) as the
radio-ligand. The best-fit IC50 values were calculated by fitting the
data by nonlinear regression using GraphPad Prism Software. Experiments were performed with triplicate samples.
MRI Protocol
The MR frame consisted of a nonmagnetic stereotactic wrist coil
with a cylindrical surface coil (5-cm internal diameter) positioned
directly over the rat head. MR imaging was performed in a 3-T
whole-body MR scanner (GE Healthcare). Rats were anesthetized
during imaging using 1% to 2% inhaled isoflurane. The brain of each
rat was imaged weekly with T2-weighted fast-spin echo sequence
(receiver bandwidth⫽16 kHz; repetition time⫽5000 ms; echo
time⫽86 ms; echo train length⫽8; field of view⫽4⫻4 cm; ma-
trix⫽256⫻256; 16 slices; slice thickness⫽1 mm; number of signals
averaged⫽3; total imaging time⫽8 min). The MR images were
analyzed using Image J software (http://rsb.info.nih.gov/ij/). The
stroke area in each MR image was traced and measured to calculate
the stroke volume.
MicroPET Studies
Weekly PET scans of the rats were performed on a microPET R4
rodent model scanner (Siemens Medical Solutions).16,21 64CuCl2 (10
mCi) was diluted in 300 ␮L of 0.1 mol/L sodium acetate buffer
(pH⫽6.5) and added to 100 ␮g DOTA-VEGF121 or DOTAVEGFmutant.16,22 The reaction mixture was incubated for 1 hour at
40°C with constant shaking. 64Cu-DOTA-VEGF121 and 64Cu-DOTAVEGFDEE were then purified by size exclusion chromatography using
phosphate-buffered saline as the mobile phase. The radiochemical
purity was ⬎98%.
Each rat was intravenously injected with ⬇1 mCi tracer (18F-FDG,
64
Cu-DOTA-VEGF121, or 64Cu-DOTA-VEGFmutant). For 18F-FDG, the
rats were scanned at 1 hour after injection. For 64Cu-DOTA-VEGF121
or 64Cu-DOTA-VEGFmutant, the rats were scanned at multiple time
points in pilot studies (1, 2, and 4 hours after injection); 2 hours after
injection was chosen because of the best contrast observed. For each
PET scan, 3-dimensional regions of interest were drawn over the
stroke area on decay-corrected images. The average radioactivity
concentration was obtained from the mean pixel values within the
region of interest volume, which were converted to counts per
milliliter per minute by use of a predetermined conversion factor.
Given a tissue density of 1 g/mL, the counts per milliliter per minute
were converted to counts per gram per minute, and the values were
divided by the injected dose to obtain the imaging region of interest
derived percentage injected dose per gram (%ID/g).
Autoradiography
Detailed procedure of autoradiography was reported earlier.23 The
perfused and acetone fixed rat brain slices (with or without the stroke
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Figure 2. 64Cu-DOTA-VEGF121 PET and autoradiography of the dMCAo rats. A, Axial and coronal PET images of the dMCAo rat
brain at 2 hours postinjection of ⬇1 mCi of 64Cu-DOTA-VEGF121. B, 64Cu-DOTA-VEGF121 uptake in the stroke area and the contralateral brain. C, The ratio of 64Cu-DOTA-VEGF121 uptake in the stroke area vs the contralateral brain. n⫽4; *P⬍0.05; **P⬍0.01;
***P⬍0.001. D, 125I-VEGF165 autoradiography of the dMCAo rat brain.
area; 20-␮m thick) were incubated with 125I-VEGF165 (0.1 ␮Ci per
slice) for 1 hour at 20°C. After washing with phosphate-buffered
saline 3 times, the slices were placed onto a chilled autoradiography
cassette containing a Super Resolution screen (Packard) and stored
for 3 days at ⫺20°C. Screens were then laser-scanned with the
Packard Cyclone system and displayed at pseudocolor scale.
Histology
Stroke rats at different days after surgery were transcardially
perfused with saline, followed by 3% paraformaldehyde. The brains
were removed and postfixed for 24 hours in 3% paraformaldehyde,
followed by cryo-preservation in 3% paraformaldehyde/20% sucrose
for 48 hours. Subsequently, the brains were embedded in optimal
cutting temperature compound (OCT) and cut into 20-␮m sections.
All primary antibodies were diluted in phosphate-buffered saline/
0.3% Triton containing 1% bovine serum albumin (BSA). The
following antibodies were used: rabbit anti-VEGFR-1 (Laboratory
Vision Corp; 1:50), rabbit anti-VEGFR-2 (Laboratory Vision Corp;
1:50), mouse anti-lectin (Vector Labs; 1:250), mouse anti-BrdU
(Vector Labs; 1:250), mouse anti-GFAP (Advanced ImmunoChemicals; 1:800), mouse anti-NeuN (R&D Systems; 1:500), FITC donkey
anti-rabbit (Jackson ImmunoResearch; 1:500), and Cy3 goat antimouse (Jackson ImmunoResearch; 1:500). For double staining of
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Figure 3. Control experiments with 64Cu-DOTA-VEGFmutant. A, Cell-binding assay reveals that VEGF121 has high affinity binding to
VEGFR-2 (IC50: 2.8 nmol/L), whereas VEGFmutant does not. B, Both VEGF121 and VEGFmutant bind to VEGFR-1, with the IC50 values being
5.4 and 11.3 nM, respectively. C, T2-weighted MRI and 18F-FDG PET confirmed stroke presence. D, Axial and coronal PET images of
the dMCAo rat brain at 2 hours postinjection of ⬇1 mCi of 64Cu-DOTA-VEGFmutant. Note that there is virtually no uptake in the stroke
area. n⫽3.
lectin and BrdU, the lectin stain was performed and the tissue
postfixed in 3% paraformaldehyde for 10 minutes at 4°C. After
thorough washes, the sections were treated with 3 mol/L HCl for 30
minutes at 37°C. After blocking and washing, the sections were
incubated with mouse anti-BrdU, followed by a secondary antibody.
4⬘,6-Diamidino-2-phenylindole was used to label the nuclei (SigmaAldrich; 1:1000).
Sections were analyzed on a confocal laser scanning microscope
(Zeiss) with split panel and z-axis analysis to confirm marker
colocalization. VEGFR-2 expression was quantified in 3 high-power
fields in the stroke border zone at 2, 10, and 17 days after stroke. The
percent area in each high power field covered by VEGFR-2
immunoreactivity was measured using Image J.
Statistical Analysis
Quantitative data were expressed as mean⫾SD. Means were compared using 1-way analysis of variance and Student t test. P⬍0.05
was considered statistically significant.
Results
Stroke Morphology and Size
A group of 4 dMCAo rats underwent weekly T2-weighted
MRI, 18F-FDG, and 64Cu-DOTA-VEGF121 scans. A successful
stroke surgery and focal cortical stroke in the left hemisphere
was confirmed for all animals on T2-weighted MRI (Figure
1A). The stroke volume based on MRI was 244.7⫾33.5,
153.6⫾41.9, and 152.6⫾42.1 mm3 at days 1, 8, and 15,
respectively (Figure 1B; n⫽4). Typically, the stroke size is
overestimated on T2-weighted MRI early after surgery because of edema, which appears hyperintense in the images.
Hence, the lesion size significantly decreased from day 1 to
day 8 after surgery but remained stable thereafter, with the
true stroke size being ⬇150 mm3 (Figure 1B). Hematoxylin
and eosin staining confirmed the stroke presence (Figure 1C).
Sham-operated rats were also prepared and no stroke was
observed in their brains based on MRI.
18
F-FDG PET
Normal brain tissue has prominent uptake of 18F-FDG because of the constant need for glucose in the brain.24 A “cold
spot” was seen in the 18F-FDG PET of dMCAo rat brain,
which corresponds to the stroke area delineated by MRI
(Figure 1D). The contralateral brain uptake of 18F-FDG
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remained constant at ⬇1.0 to 1.1%ID/g, whereas that of the
stroke area was significantly lower at all time points examined (Figure 1E; P⬍0.05; n⫽4). The uptake in the stroke area
was ⬇0.5%ID/g at days 1, 15, and 22. At day 8, the stroke
area appeared to be smaller and the uptake was higher
(0.76⫾0.11%ID/g). Such phenomenon is likely attributable
to inflammation in the stroke area because 18F-FDG has been
well-established to be taken up by inflamed tissue (which
does not significantly affect the MR images).25,26
64
Cu-DOTA-VEGF121 PET
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The day after the MRI and 18F-FDG scans, the rats were
subjected to 64Cu-DOTA-VEGF121 PET. Uptake in the stroke
area could be clearly seen as early as day 2 (0.24⫾
0.02%ID/g; n⫽4; Figure 2A,B). The uptake in the stroke area
peaked at day 9 (0.46⫾0.11%ID/g) and remained elevated at
day 16 (0.29⫾0.10%ID/g). The uptake in the contralateral
brain was low at all time points (⬍0.10%ID/g). At day 23, the
uptake in the stroke area was no longer statistically significantly
different from the background (0.06⫾0.04%ID/g vs 0.03⫾
0.02%ID/g, respectively; n⫽4). The stroke-to-normal tissue
uptake ratio was 12.5⫾1.6, 9.0⫾1.8, 3.2⫾1.6, and 2.1⫾1.9 at
days 2, 9, 16, and 23, respectively (n⫽4; Figure 2C). The uptake
of 64Cu-DOTA-VEGF121 in the surgical wound was prominent in
most of the images because angiogenesis is also a key process
during wound healing.
To validate VEGFR expression in the stroke border zone,
125
I-VEGF165 autoradiography was performed on brain slices
at day 2 and day 9 after surgery because good stroke-tonormal tissue ratios were observed at these time points.
Clearly, VEGFR expression was seen at the proximity of the
stroke area but not in the contralateral brain or the normal
brain tissue (Figure 2D). The site of VEGFR overexpression
correlates to the area with 64Cu-DOTA-VEGF121 uptake in the
PET scans.
64
Cu-DOTA-VEGFmutant PET and
Sham-Operated Rats
Several control experiments were performed to confirm
VEGFR specificity of 64Cu-DOTA-VEGF121 uptake. The
VEGFmutant does not bind to VEGFR-2 with an IC50 value of
⬎10 ␮mol/L (2.8 nmol/L for VEGF121; Figure 3A). For
VEGFR-1, the IC50 values of VEGF121 and VEGFmutant were
5.4 nmol/L and 11.3 nmol/L, respectively (Figure 3B). A
group of 3 dMCAo rats (Figure 3C) were prepared and
subjected to 64Cu-DOTA-VEGFmutant scans at day 10 after
surgery (when the uptake of 64Cu-DOTA-VEGF121 was the
highest in the stroke border zone). The uptake of 64Cu-DOTAVEGFmutant in the stroke border zone was significantly lower
than that of 64Cu-DOTA-VEGF121 (0.20⫾0.08%ID/g vs
0.46⫾0.11%ID/g; P⬍0.05; Figure 3D).
Three sham-operated rats were prepared and both MRI and
18
F-FDG PET confirmed the lack of stroke in the rat brain
(Figure 4A). Subsequent 64Cu-DOTA-VEGF121 scans revealed
that the tracer uptake was in the wound, but not in the brain
(⬍0.10%ID/g at days 9 and 16; n⫽3; Figure 4B). The wound
uptake was the highest at day 2, and was significantly lower
at day 9 and day 16. Taken together, these 2 sets of control
Figure 4. Control experiments with sham-operated rats. A,
T2-weighted MRI and 18F-FDG PET both indicated the absence
of stroke in sham-operated rats. B, Axial and coronal PET
images of sham-operated rats at 2 hours postinjection of ⬇1
mCi of 64Cu-DOTA-VEGF121. Note that there is uptake in the surgical wound but not in the rat brain. C, Comparison of the PET
tracer uptake in dMCAo rats and sham-operated rats. n⫽3;
*P⬍0.05; **P⬍0.01.
experiments confirmed that the 64Cu-DOTA-VEGF121 uptake
in the stroke border zone was VEGFR-specific (Figure 4C).
Histology
Stroke morphology as seen on hematoxylin and eosin–stained
sections correlated well with that observed in MRI and PET
(Figure 1C). VEGFR-2 was expressed in the stroke border
zone as early as 2 days after surgery with predominantly
cellular expression (Figure 5A). At 10 days after surgery, a
mixed cellular and vascular expression pattern was noted
(Figure 5B,D), which decreased significantly at day 17
(Figure 5C). Phenotypic characterization showed colocalization of VEGFR-2 with astrocytes (GFAP; Figure 5E), neu-
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Figure 5. VEGFR-2 expression after stroke. VEGFR-2 staining at days 2 (A), 10 (B), and 17 (C) poststroke. At day 10 a mixed cellular
(arrow), but mainly vascular, expression (arrow heads) profile is observed. Green indicates VEGFR-2; blue indicates 4⬘,6-Diamidino-2phenylindole. Scale bar: 50 ␮m. D, At higher magnification the cellular (arrow) and vascular (arrowheads) expression of VEGFR-2 at day
10 is better-appreciated. Scale bar: 10 ␮m. Colocalization of VEGFR-2 (green) and GFAP (red; E), NeuN (red; F), and lectin (red; G) was
observed (single colors and overlays). Scale bar: 20 ␮m. H, Quantification of VEGFR-2 expression at 2, 10, and 17 days poststroke.
*P⬍0.001.
rons (NeuN; Figure 5F), and blood vessels (Lectin; Figure
5G) at day 10 after stroke. Quantification of VEGFR-2
expression in the stroke border zone confirmed the PET
findings with a peak expression at day 10 (Figure 5H). The
overall expression between day 2 and day 10 was not
significantly different, but there was markedly less vascular
expression at day 2 after stroke. VEGFR-2 expression in the
contralateral brain and normal brain tissue was minimal and
VEGFR-1 staining of the dMCAo rat brain slices was also
weak at all time points (data not shown). BrdU staining at day
10 demonstrated numerous BrdU-positive cells in the stroke
border zone (Figure 6A,B). Colocalization of lectin and BrdU
further substantiated the presence of poststroke angiogenesis
(Figure 6B,C).
Discussion
No treatment currently exists to restore the lost neurological
function after stroke. Increased vascularization in the stroke
border zone within a few days after stroke is associated with
neurological recovery.27 Stem cells,28 erythropoietin,29 chemokines,30 and gene therapy,31 among others, have been
shown to induce angiogenesis after stroke. Noninvasive
molecular imaging of poststroke angiogenesis, such as the
one demonstrated here, could be extremely valuable not only
Figure 6. Endogenous angiogenesis after stroke. A, At low magnification, multiple BrdU-positive cells (green) are observed in the stroke
border zone (arrows) at day 10. Scale bar: 100 ␮m. B, At higher magnification, several BrdU-positive cells are seen in close proximity
to (arrows) and overlapping (arrowheads) with blood vessels stained with lectin (red). Scale bar: 50 ␮m. C. Z-stack and orthogonal view
of confocal images demonstrate colocalization of BrdU and lectin, representing newly formed blood vessels in the stroke border zone.
Scale bar: 20 ␮m.
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as a prognostic factor but also as a measurement of success to
help guide proangiogenic therapies. Different scenarios can
be envisioned. To date we do not know the right timing or the
ideal place for cell or drug delivery in future clinical trials
with stroke patients. It is conceivable that molecular imaging
techniques will not only help us monitor the therapy effects
but also help us defining patient specific treatment strategies.
Moreover, it is also possible that early molecular and biological events (assessable by molecular imaging) might be
predictors of clinical outcome.
Upregulation of VEGFR was already found at 2 days
poststroke, indicating that signals to induce angiogenesis
appear very rapidly after stroke. The uptake of 64Cu-DOTAVEGF121 in the stroke border zone peaked at ⬇10 days after
surgery (significantly higher than the baseline uptake at days
2, 9 and 16, but not at day 23), confirmed by both histology
and autoradiography, which showed that VEGFR-2 expression peaked at ⬇10 days after stroke (Figures 2 and 5).
Previous studies have demonstrated peak VEGF expression
between 12 and 24 hours, preceding VEGFR-2 expression.32
Whereas VEGFR-1 mRNA level returned to baseline after 7
days, the VEGFR-2 mRNA level persisted for 7 days.32
64
Cu-DOTA-VEGF121 binds to both receptors;16 therefore, it
cannot differentiate between VEGFR-1 and VEGFR-2. However, the significantly lower uptake of 64Cu-DOTA-VEGFmutant,
which does not bind to VEGFR-2 but binds to VEGFR-1 with
lower affinity than 64Cu-DOTA-VEGF121 (Figure 3A,B), at
day 10 in the stroke border zone suggests that VEGFR-2 may
play a more dominant role over VEGFR-1 in poststroke
angiogenesis. In agreement with previous studies, VEGFR-2
expression was concentrated around the stroke border zone32
and was found to colocalize with the blood vessels, astrocytes,
and neurons (Figure 5).33,34 BrdU staining further confirmed the
active formation of new blood vessels (Figure 6).
Because there is a blood– brain barrier instability after
stroke, partially influenced by VEGF,34 it is possible that our
imaging not only represents endoluminal but also represents
some cellular expression of VEGFR. Recently, VEGFR
upregulation in the ventral premotor hand representation and
the M1 hind limb representation in squirrel monkeys 3 days
after stroke were reported.35 This underscores the potential
importance of VEGF/VEGFR pathway not only in acute
stroke but also in poststroke plasticity and perhaps long-term
rehabilitation.
Imaging VEGF and VEGFR expression are both important
for diagnosis and monitoring of angiogenesis-related treatment efficacy. Although increased expression of growth
factors and cytokines occurs in many stroke models, many of
the signaling pathways that are responsible for revascularization have only been subjectively proposed based on ex
vivo/in vitro studies. Much research will be needed to
develop blood– brain barrier-penetrating tracers for imaging
VEGF expression in the (stroke) brain. We envision that
examining the stroke brain in the same animals or patients
with both VEGF- and VEGFR-targeted tracers may give
important insights about the expression kinetics of VEGF and
VEGFR and may shed new light on the disease/recovery
mechanisms based on VEGF/VEGFR signaling. Generation
of VEGFR-1–specific or VEGFR-2–specific VEGFmutant with-
out any affinity to the other receptor may also help us better
understand poststroke angiogenesis in future studies. Besides
VEGF/VEGFR imaging, other angiogenesis-related targets
such as integrin ␣v␤3 should also be studied in the future.36
In this study, the wound in the rat skin is close to the stroke
area. In pilot studies, we also created a group of stroke rats
using a suture model in which the wound is not directly
adjacent to the stroke area. However, we found that the stroke
size varies to a great extent between animals, which led to
less reproducible results in MRI, 18F-FDG, and 64Cu-DOTAVEGF121 PET scans. Therefore, we chose the dMCAo model
in which the stroke size in each rat is quite consistent. Recent
development of hybrid PET/MR scanners may facilitate the
coregistration of the MR and PET images in future studies for
differentiation between stroke and normal brain tissue. Nonetheless, control experiments in sham-operated rats, the use of
a VEGFmutant, autoradiography, and histology all clearly demonstrated that the uptake in the stroke border zone is attributable to VEGFR overexpression in poststroke angiogenesis.
Summary
Multimodality imaging was used to assess the temporal
profile of the lesion size, metabolic activity, and angiogenesis
in the same animals after stroke. For the first time to our
knowledge, we successfully evaluated the time-course of
VEGFR expression noninvasively by PET in a rat stroke
model. The ability to visualize VEGFR expression level in
vivo could provide new opportunities to document angiogenesis after stroke, which may have significant clinical implications in guiding future therapies aimed at improving angiogenesis. Future studies of imaging VEGFR expression before,
during, and after new stroke therapies are warranted.
Sources of Funding
National Cancer Institute (P50 CA114747, R24 CA93862, R01
CA119053, R21 CA121842, and U54 CA119367) and a Benedict
Cassen Postdoctoral Fellowship.
Disclosure
None.
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Stroke. 2009;40:270-277; originally published online October 23, 2008;
doi: 10.1161/STROKEAHA.108.517474
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