In vivo imaging of stepwise vessel occlusion in cerebral photothrombosis of mice by 19F MRI.

In Vivo Imaging of Stepwise Vessel Occlusion in Cerebral
Photothrombosis of Mice by 19F MRI
Gesa Weise1,3., Thomas C. Basse-Lu¨sebrink1,2,3., Christoph Kleinschnitz1, Thomas Kampf2, Peter M.
Jakob2,3, Guido Stoll1,3*
1Department of Neurology, University of Wu¨rzburg, Wu¨rzburg, Germany, 2Department of Physics, EPV, University of Wu¨rzburg, Wu¨rzburg, Germany, 3 Interdisciplinary
Center for Clinical Research Wu¨rzburg, University of Wu¨rzburg, Wu¨rzburg, Germany
Abstract
Background: 19F magnetic resonance imaging (MRI) was recently introduced as a promising technique for in vivo cell
tracking. In the present study we compared 19F MRI with iron-enhanced MRI in mice with photothrombosis (PT) at 7 Tesla.
PT represents a model of focal cerebral ischemia exhibiting acute vessel occlusion and delayed neuroinflammation.
Methods/Principal Findings: Perfluorocarbons (PFC) or superparamagnetic iron oxide particles (SPIO) were injected
intravenously at different time points after photothrombotic infarction. While administration of PFC directly after PT
induction led to a strong 19F signal throughout the entire lesion, two hours delayed application resulted in a rim-like 19F
signal at the outer edge of the lesion. These findings closely resembled the distribution of signal loss on T2-weighted MRI
seen after SPIO injection reflecting intravascular accumulation of iron particles trapped in vessel thrombi as confirmed
histologically. By sequential administration of two chemically shifted PFC compounds 0 and 2 hours after illumination the
different spatial distribution of the 19F markers (infarct core/rim) could be visualized in the same animal. When PFC were
applied at day 6 the fluorine marker was only detected after long acquisition times ex vivo. SPIO-enhanced MRI showed
slight signal loss in vivo which was much more prominent ex vivo indicative for neuroinflammation at this late lesion stage.
Conclusion: Our study shows that vessel occlusion can be followed in vivo by 19F and SPIO-enhanced high-field MRI while in
vivo imaging of neuroinflammation remains challenging. The timing of contrast agent application was the major
determinant of the underlying processes depicted by both imaging techniques. Importantly, sequential application of
different PFC compounds allowed depiction of ongoing vessel occlusion from the core to the margin of the ischemic lesions
in a single MRI measurement.
Citation: Weise G, Basse-Lu¨sebrink TC, Kleinschnitz C, Kampf T, Jakob PM, et al. (2011) In Vivo Imaging of Stepwise Vessel Occlusion in Cerebral Photothrombosis
of Mice by 19F MRI. PLoS ONE 6(12): e28143. doi:10.1371/journal.pone.0028143
Editor: Cesario V. Borlongan, University of South Florida, United States of America
Received May 22, 2011; Accepted November 2, 2011; Published December 15, 2011
Copyright: � 2011 Weise et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access
Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: stoll_g@klinik.uni-wuerzburg.de
. These authors contributed equally to this work.
Introduction
Among the non invasive imaging modalities MRI provides high
resolution imaging with excellent soft tissue contrast allowing in
vivo follow up of pathological processes. Commonly, small or
ultrasmall superparamagnetic iron oxide (SPIO, USPIO) nano-
particles are used for tracking of labeled cells. Accumulation of
these cells in tissues leads to focal signal loss on T2- and T2*-w
MRI. However, although relatively low numbers of iron-laden
cells can give a strong MRI signal void [1] confounding factors
such as blood pool effects and bleedings limit the strength of this
technique. Moreover, endogenous iron-laden macrophages can
give rise to signal loss even in the absence of contrast agents
especially at high field strength [2,3]. In 2005 19F MRI was
introduced as a novel imaging technique for in vivo cell tracking
after injection of ex vivo labeled cells [4]. In contrast to iron contrast
agents, 19F markers exhibit a unique MRI signal that can be
detected directly [5]. Due to the lack of 19F background signal in
the host’s tissue 19F MRI is extremely selective for the labeled cells.
However, 19F MRI requires high numbers of 19F spins to
accumulate in order to generate sufficient signal-to-noise ratio
(SNR). Previous studies have shown that systemic intravenous
injection of perfluorocarbons (PFC) leads to significant and
spontaneous PFC uptake by cells of the macrophage/monocyte
system [6,7]. By applying in vivo 19F MRI areas with macrophage
infiltration could be visualized in mice in myocardial and cerebral
infarctions [7] and in rodent models of acute allograft rejection
[8]. We could recently depict neuroinflammation in rats within
peripheral nerve lesions by 19F MRI [9].
In the present study we applied 19F MRI at different stages of
cerebral photothrombosis (PT) at high field strength of 7 Tesla (T).
In 1985, Watson et al. introduced brain PT as a simple model of
focal cerebral ischemia [10]. To achieve thrombosis a photosen-
sitive dye is injected systemically. Subsequent illumination through
the intact skull leads to local activation of the dye with free radical
formation and photoperoxidation of the endothelium. Endothelial
injury is then supposed to mediate clot formation in illuminated
vessels. Thus, the pathophysiology of PT lesion development
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encompasses endothelial damage, breakdown of the blood brain
barrier (BBB) and rapid edema formation, but also occlusion of
vessels thereby partly mimicking cerebral ischemia [11]. More-
over, PT lesions elicit a strong, but delayed inflammatory reaction
[12]. While several factors such as the little ischemic penumbra
limit the strength of PT as a model for human stroke [13] it was
chosen in this study because lesions show a predictable pattern of
macrophage infiltration.
MRI studies at 1.5 T using SPIO in rats showed that in early
stages of PT lesion development ongoing vessel occlusion can be
visualized by iron-enhanced MRI [14], while delayed application
of SPIO depicted neuroinflammation [15]. To elucidate which
processes (acute damage versus late neuroinflammation) are
visualized in mice with PT we compared lesion development at
7 T after PFC/SPIO injection by 19F or T2-w MRI. Our results
suggest that 19F imaging and iron-enhanced MRI depict vessel
occlusion with high sensitivity in the early phase of photothrom-
botic infarction while at later stages the inflammatory response is
covered. The use of two different PFC markers, moreover, allowed
detection of stepwise microvascular occlusion in a single PT lesion.
In conclusion, the timing of contrast agent application was the
major determinant of the underlying processes depicted by these
two imaging techniques.
Materials and Methods
Photothrombotic infarction
Animal experiments were performed in accordance with
institutional guidelines and were approved by the institutional
ethics committee for animal welfare of the University of
Wu¨rzburg, Germany (permit number 62.1-2531.01-23/04; 55.2-
2531.01-17/10). Focal cerebral ischemia was induced in 44 adult
C57/BL6-mice (25–30 g) by PT of cortical microvessels under
inhalation anesthesia with enflurane in a 2:1 nitrogen/oxygen
atmosphere, as described previously [15,16]. Briefly, a fiber optic
bundle of a cold light source was centered stereotactically 2 mm
posterior and 2.4 mm lateral from Bregma on the skull exposed
via a dorsal midline incision of the skin. 0.2 ml of a sterile-filtered
rose Bengal solution were given by intraperitoneal injection and
the brain was illuminated for 20 min. Subsequently the skin was
sutured and the mice were allowed to recover. This procedure
resulted in cone-shaped cortical infarctions. For MRI measure-
ments mice were anesthetized using 1.5% isoflurane in a 2 l/min
oxygen atmosphere.
Contrast agents/markers
PFC markers. A perfluoro-15-crown-5-ether emulsion (10%
wt/wt, Fluorochem Ltd. Glossop, UK) was used in experiments in
which only one PFC emulsion was applied. Production and
properties of are described in detail elsewhere [7].
For experiments applying two chemically shifted PFC com-
pounds multi-resonant perfluorooctlybromid (PFCA) was used.
The nanoemulsion contained 40% wt/wt PFCA (Chempur;
Karlsruhe, Germany) and 2.4% wt/wt purified soybean lecithin
S75 (Lipoid; Ludwigshafen, Germany) in an isotonic buffer (7 mM
Na2HPO4 - 2 H2O, 3 mM NaH2PO4 - 2 H2O, 2.5% glycerol,
pH 7.4) and was prepared as previously reported [7]. High
pressure homogenization (75 MPa, 10 cycles) with Emulsiflex C5
(Avestin; Mannheim, Germany) resulted in a particle size of
<240 nm as determined by dynamic light scattering (average of at
least ten runs measured at the stationary level) using ZetatracTM
(Particle Metrix; Meerbusch, Germany). The nanoemulsion was
sterilized by autoclavation and stored at 6uC until application.
Besides PFCA the single-resonant perfluoro-15-crown-5-ether
(PFCB) was used. The 30% v/v PFCB emulsion was obtained
from Celsense Inc. (Pittsburgh, PA, USA).
SPIO contrast agents. SPIO particles (ResovistTM, Bayer
Schering Pharma AG, Berlin, Germany) were used in a dosage of
0.2 mmol Fe/kg body weight as described in previous studies at
1.5 T [15,17].
Hardware
All measurements were performed on a 7 T Bruker Biospec
System (Bruker BioSpin GmbH, Reinstetten, Germany) at room
temperature. For in vivo 1H and 19F imaging a home-built surface
coil adjustable to both frequencies was used. Ex vivo imaging of the
fixed mouse brains was performed with a home-built solenoid coil.
Additionally, a home-built, actively decoupled 19F birdcage coil in
combination with an actively decoupled 19F receive-only surface
coil was used for the experiments with two PFC compounds. Even
though the solenoid and the birdcage with integrated surface coil
were optimized for the 19F resonance frequency, their perfor-
mance was still sufficient to acquire 1H anatomical background
images.
In vivo 1H/19F imaging
Experiments in the acute phase of lesion
development. In order to visualize and quantify different
stages of acute ‘‘ischemic’’ damage PFC emulsion was applied
via the tail vein in a dosage of 250 ml either directly after the end of
illumination (n= 8) or two hours later (n = 8). Two mice with
cortical infarctions that obtained 250 ml of 0.9% sodium chloride
intravenously instead of PFC served as negative controls.
MRI scans were performed 24 hours after PFC injection. In
n= 8 animals (0 h/2 h= 4/4) 2D single slice experiments were
performed. In all experiments the slice was located axially through
the middle of the infarction using information obtained from scout
scans. For 1H reference images, a turbo spin echo (TSE) sequence
was used (echo time (TE)/repetition time (TR): 40 ms/5000 ms;
inter-echo time: 10 ms; turbo factor: 8; field-of-view (FOV):
25625 mm; matrix: 2566256; slice thickness (SI): 2 mm; number
of averages (NA): 1). Regarding 19F imaging 2D steady-state free
precession CSI (SSFP-CSI) sequences [18] were performed with
the same geometry as the TSE scans (pulse shape: hermite; pulse
bandwidth: 5400 Hz; acquisition time(TAQ)/TR: 10.3 ms/
13.6 ms; FOV: 25625 mm; spectral points: 512; matrix: 41641;
SI: 2 mm; NA: 158). The overall protocol time was ,1.5 hours.
To be able to quantify the lesion volume 3D experiments were
performed in additional n= 8 mice (0 h/2 h=4/4). Thus, 3D
acquisition weighted 19F SSFP-CSI data were acquired [19]. The
FOV was set to 25625620 mm, the matrix and the NA were
adjusted to allow the same nominal spatial resolution and total
imaging time as a 40640620 and 8 times averaged scan with full
coverage of the k-space. To eliminate banding artifacts an
additional 3D 19F SSFP-CSI scan with a 180u phase shift
alternation of the excitation pulse was performed. Additional
multislice (SI = 1 mm) 1H TSE reference scans were performed
(otherwise same parameters as described above). For the
quantification study the overall protocol time was <2.5 hours.
Lesion maturation experiments. In order to follow the 19F
signal during lesion maturation qualitatively n= 2 (2 h) mice
underwent additional 2D single slice MRI scans 3, 8 and 10 days
after PT as described above.
Different stages of acute ‘‘ischemic’’ damage visualized
in a single experiment. To visualize ongoing vessel occlusion
in the acute ‘‘ischemic’’ stage n= 3 mice received 125 ml PFCA
emulsion intravenously directly after the end of illumination. Two
hours later 125 ml of PFCB was applied into the same animals.
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1H and 19F scans were performed at days three and eight after
PT. Multislice 1H TSE datasets of the mouse brain were acquired
for anatomical references (slices: 28; SI: 1 mm; FOV: 25625 mm;
matrix: 2566256). 3D 19F SSFP-CSI datasets without slice
selection were acquired using the same geometry as the 1H TSE
datasets (pulse shape: hard pulse; pulse bandwidth: 50000 Hz;
FOV: 25625628 mm; matrix: 41641614; NA: 6). The pulse
bandwidth was increased to 50000 Hz to excite the complete
spectrum of the PFCA compound. Otherwise the same parameters
were used as described above. The protocol time was ,1 hour.
Neuroinflammation experiments in the late stage of
PT. In n= 8 mice the PFC emulsion was administered at day
6 when secondary invasion of hematogenous macrophages is
supposed to happen [12]. MRI measurement was carried 48 hours
later at day 8 to allow labeled macrophages more time to invade
into the photothrombotic lesion. The same scans were performed
as described for the volume quantification experiments. The
protocol time was ,2.5 hours.
Ex vivo 1H/19F imaging
In vivo MRI of mice injected at day 6 did not exhibit fluorine
signal within the lesion. To clarify whether a lack of signal led to
insufficient sensitivity of in vivo imaging post mortem scans of the
isolated brain were performed. Mice were sacrificed after the in
vivo MRI measurement at day 8 by perfusion with 0.9% sodium
chloride followed by 4% paraformaldehyde in deep anesthesia.
The brains were removed in toto and fixed overnight in 4%
paraformaldehyde.
For 1H reference images, a multislice TSE sequence was used
(FOV: 15615 mm; matrix: 1286128; SI: 1 mm; 36 slices, otherwise
same parameters as in vivo 1H/19F imaging). Regarding 19F imaging
3D acquisition weighted SSFP-CSI scans were acquired with the
same geometry as the TSE scans. The matrix and the number of
averages were adjusted to allow the same nominal spatial resolution
and total imaging time as a 30630618 and 256 times averaged scan
with full coverage of the k-space. Otherwise same imaging
parameters as described in the in vivo 1H/19F imaging section were
applied. The overall ex vivo protocol time was 16 hours.
In vivo SPIO-enhanced MRI
In accordance with the PFC injection protocol a parallel group of
mice (n= 15) received SPIO intravenously immediately after the
end of illumination (n= 4), two hours later (n= 4) or at day 6 (n= 7).
MRI measurements were performed 24 hours after systemic
administration of the contrast agent. For MRI T2-w imaging the
same single slice TSE sequences as for the anatomical reference in
the 19F in vivo experiments were used. Furthermore, multislice
(SI= 1 mm) TSE measurements were performed. The total
protocol time for the in vivo SPIO measurements was,30 minutes.
Ex vivo SPIO-enhanced MRI
Mice with delayed application of SPIO at day 6 were sacrificed
after the in vivo MRI scan by perfusion with 0.9% sodium chloride
followed by 4% paraformaldehyde in deep anesthesia. The brains
were removed in toto and fixed overnight in 4% paraformalde-
hyde. Two post mortem T2*-w 3D fast low angle shot (FLASH)
scans of the brain were obtained (TE/TR: (4 ms/20 ms)/50 ms;
FOV: 15615636 mm; matrix: 15061506360; NA: 4). Each ex
vivo FLASH scan lasted three hours.
Post Processing
All post processing was done in MATLAB (The MathWorks
Inc., Natick, USA). Thereby semi-automated, home-written
algorithms were used for quantification experiments as described
below. Quantification was performed independently by three
different persons.
19F imaging. To generate 19F images only the signal of the
19F peak was integrated. Furthermore, a threshold was applied to
the 19F data and afterwards overlaid with the 1H TSE data. For
each overlay image the threshold was adjusted individually.
Regarding quantification, sum-of-squares (SOS) reconstructions
were generated of the data with and without alternating phase to
minimize banding artifacts in a first step. In a second step the signal
of the 19F peak was integrated to generate 19F images. Afterwards
the 3D 19F dataset was zerofilled to the size of the 1H reference
dataset (2566256620). For all 19F datasets, the volume of the infarct
containing 19F signal was evaluated by only counting pixels with 19F
signal greater than a threshold value set by the investigator.
Thereby, only the pixels of the 19F dataset in the infarct area were
regarded. The infarct area was manually segmented by the
investigator using the contrast of the 1H reference dataset (positive
T2 contrast) prior to the
19F quantification.
SPIO-enhanced MRI. For quantification, the infarct area
was manually segmented by the investigator using the contrast of
the 1H reference dataset (positive/negative T2 contrast). In a
second step the volume of the infarct showing signal voids was
calculated by only counting pixels with SNR lower than a
threshold value defined by the investigator.
Histology
After the ex vivo MRI PFA-fixed brains were washed in
phosphate-buffered saline (PBS) and embedded in paraffin.
Subsequently 5 mm thick coronal sections were cut at multiple
levels through the infarction, deparaffinized with xylene, rehy-
drated and washed in water and PBS. For iron detection tissue
sections were rinsed in deionized water and immersed in Perl’s
solution containing 2% potassium ferrocyanide and 2% HCl at a
1:1 concentration for 30 minutes. Sections were then rinsed in
deionized water and either dehydrated, counter stained with
hematoxylin-eosin and coverslipped or further processed for
immunohistochemistry. Immunohistochemistry was performed
using von Willebrand Factor (vWF) (polyclonal rabbit anti-mouse
IgG, ab6994, 1:800; Abcam, USA), an endothelial cell marker, as
primary antibody upon the same sections. Enzymatic antigen
retrieval was performed with 0.1% pronase for 10 min at 35uC.
Binding of the antibodies to cells was visualized by a biotinylated
goat anti-rabbit IgG secondary antibody (1:100, Vector laborato-
ries, USA). For negative controls, the primary antibody was
omitted from the diluent.
Results
19F and iron-enhanced MRI in early stages of infarct
development
In a first set of experiments mice received either PFC emulsion
or SPIO directly after the end of illumination of the brain or with
two hours delay. Control animals with cortical infarctions but no
PFC injections did not exhibit fluorine signal under inhalation
anesthesia with isoflurane (data not shown).
Administration of PFC directly after illumination led to a bright
19F signal throughout the entire cortical infarction as shown on the
in vivo MRI scan 24 hours later (Fig. 1A). When PFC were injected
with two hours delay lesions displayed a ring-like fluorine signal at
the lesion margins (Fig. 1A). The rim-like fluorine signal seen at
day 1 persisted when individual 2 h-animals were scanned again
during further lesion maturation at days 3, 8 and 10 (Fig. 2). On
1H-TSE images the lesions initially appeared hyperintense. Within
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the first week after PT induction signal changes decreased leading
to an almost isointense tissue signal at day 8 (Fig. 2B). The
semilunar 19F signal appeared more intense with shrinkage of the
lesion over time (Fig. 2B).
The distribution of the signal loss on T2-w MRI after SPIO
injection closely resembled the 19F signal following PFC
administration directly after illumination or with a delay of
2 hours. Whereas in animals which obtained SPIO immediately
Figure 1. In vivo imaging patterns of 19F and SPIO-enhanced MRI in the acute stage of cerebral photothrombosis. (A) All images were
acquired 24 hours after PT induction. The 1H/19F overlay shows fluorine signal within the entire cortical infarction when PFC were applied directly
after the end of illumination (left, upper image). Delayed injection two hours later led to a rim-like signal in the boundary zone (left, lower image).
Note the similar distribution pattern of signal loss on T2-w MRI when SPIO were administered immediately after illumination (right, upper image) and
with two hours delay (right, lower image). The relative volume of iron-induced signal loss and 19F signal in proportion to the total infarct volume
decreased from 0.8560.13/0.6160.13 to 0.5960.03/0.2960.08 (PFC/SPIO) within the first two hours after illumination (B) (both cases p,0.05). When
corresponding time points of SPIO/19F marker injection were compared a significant difference was found between the volume fraction showing 19F
signal and the signal voids in SPIO-enhanced MRI (p,0.05).
doi:10.1371/journal.pone.0028143.g001
Figure 2. Development of the 19F signal during lesion maturation. Individual animals that received PFC two hours after photothrombotic
infarction were scanned sequentially on days 1, 3, 8 and 10 (A). On 1H-TSE lesions initially appeared hyperintense due to increased T2 values caused
by tissue damage and edema formation. Over time proton signal changes decrease leading to an almost isointense tissue signal at day 8 (B). The rim-
like fluorine signal seen one day after PT induction persisted until day 10 and appeared even more intense with shrinkage of the lesion over time. An
additional fluorine signal could be observed at the site of skin incision (arrows).
doi:10.1371/journal.pone.0028143.g002
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after completion of PT markedly hypointense cortical lesions were
depicted on T2-w MRI, injection with 2 hours delay led to a
hyperintense infarct core with a hypointense ring at the lesion
margins (Fig. 1A). Furthermore, using statistical t-tests the
quantification of each of the three independent researchers
revealed a significant difference between the 19F volume fractions
of the lesions when the PFC emulsion was injected 0 h or 2 h after
the illumination (p,0.05). Similarly, the volume fractions showing
signal voids in SPIO-enhanced MRI 0 h and 2 h after the
illumination significantly differed (p,0.05). However, when
corresponding time points of SPIO/PFC injection were compared
a significant difference was found between the volume fraction
showing 19F signal and the signal voids in SPIO-enhanced MRI
(both cases: p,0.05). Thus, the mean volume fraction and mean
error of the lesion exhibiting 19F signal was 0.8560.13/0.5960.03
(0 h/2 h) and 0.6160.13/0.2960.08 (0 h/2 h) for the iron-
enhanced experiments (Fig. 1B). A relative measurement of the
volume fraction was chosen due to a certain interindividual
variance of the stroke volumes. Therefore, total quantification
could have generated misleading results with high standard
deviations.
In vivo visualization of different stages of acute
‘‘ischemic’’ damage. Additional 3D CSI experiments with
two chemically shifted PFC compounds were performed which
allowed differentiation of both markers and thus visualization of
ongoing vessel occlusion in a single MRI measurement (Fig. 3).
Administration of the first emulsion (PFCA) directly after induction
of PT led to a fluorine signal throughout the cortical infarction
(Fig. 3C). The second 19F marker (PFCB) that was applied two
hours later, however, accumulated at the outer margins sparing
the center of the infarcted zone (Fig. 3C). By merging the signals
on a 1H background image the different spatial distribution of the
compounds could be identified in a single measurement (Fig. 3C).
19F and iron-enhanced MRI in late stages of infarct
development: Visualization of neuroinflammation
When the PFC emulsion was administered with a delay of 6
days after PT and animals were scanned at day 8 no fluorine signal
was detectable within the lesions on in vivo 19F MRI scans (Fig. 4A).
The 19F marker was only visible at the site of the skin incision on
the skull (Fig. 4A, arrow). However, when mice were sacrificed
after the in vivo measurement and post mortem scans of the isolated
brain with long acquisition times and the use of a more sensitive
solenoid coil were performed a fluorine signal in the ischemic
lesion was regularly detectable (Fig. 4B). After SPIO application at
day 6 a small rim of signal loss was regularly visible on in vivo T2-w
MRI (Fig. 4C) as shown previously for rats at 1.5 T [15]. The ring-
like hypointensity was even more prominent on ex vivo T2*-w MRI
scans of the removed brain (Fig. 4D). Proton signal changes in PT
decrease during the first week leading to an almost isointense
infarct signal at day 8 so that quantification of the 19F signal in
relation to the total infarct volume was impossible at this late stage
of lesion development.
Histological analysis
Macroscopically the circular PT lesions could be identified at
the frontoparietal cortex as shown previously [20]. Prussian blue
staining on coronal sections cut through the infarction of mice
sacrificed one day after PT induction revealed entrapment of iron
particles throughout the infarct core when SPIO were applied
directly after the end of the illumination (Fig. 5A). The
intravascular location of the iron deposits was confirmed by co-
staining of identical sections with antibodies against vWF, an
endothelial cell marker (Fig. 5A–C). Iron particles could only be
detected within the vessel lumina in the infarct region, but not in
the brain parenchyma or remote cortex. After two hours delay the
iron nanoparticles were trapped in the cortical vessels at the outer
margin of the infarction while vessel thrombi in the center of the
lesion were devoid of iron particles (Fig. 5B). This finding supports
our MRI results depicting ongoing vessel occlusion despite
cessation of the illumination exclusively in the periphery of the
photothrombotic lesion. At late lesion stages eight days after PT
induction accumulation of blue iron particles was no longer found
in the vessel lumina but intracellular within the infarcted zone
(Fig. 5D). These findings correspond to previous results obtained
in rats [14].
Discussion
As principal finding we show that 19F MRI can visualize
ongoing vessel occlusion and cellular infiltration in cortical PT, a
model of focal cerebral ischemia. The timing of contrast agent
application was the major determinant of the underlying processes
depicted by 19F MRI.
19F and iron-enhanced MRI in early stages of infarct
development
When the PFC emulsion was injected directly after PT
induction the fluorine signal was displayed throughout the entire
lesion. Two hours delay led to a fluorine rim at the periphery of
the infarction. Both changes were present during the acute phase
of lesion formation in which hematogenous macrophages are not
yet significantly involved [12,15]. Signal alterations visualized by
19F imaging during acute PT closely resembled the pattern of
signal loss on T2-w MRI after application of SPIO. However, the
relative volume fractions showing a 19F signal or signal voids in
SPIO-enhanced MRI at corresponding injection time points
differed significantly. This might on one hand be due to partial
volume effects leading to an overestimation of the 19F signal as
only a relatively low spatial in-plane resolution could be obtained.
On the other hand, PFC emulsions and SPIO have different blood
clearance times. The PFC emulsion which was used in the present
study for quantification experiments was shown to circulate in the
blood up to two days [7]. In contrast, SPIO concentrations
significantly decline in the circulation within the first hour after
application due to uptake of the iron particles in the reticuloen-
dothelial system of the liver [21]. On tissue sections iron deposits
could be exclusively found in vessels of the infarct core when SPIO
were applied directly after the end of the illumination while SPIO
injection two hours later lead to intravascular entrapment of the
iron particles mainly at the outer margins of the lesion indicating
ongoing vessel occlusion despite cessation of the illumination. The
same mechanism very likely underlies the fluorine signal observed
in the early phase of photothrombotic stroke in our study.
19F and iron-enhanced MRI in late stages of infarct
development
Unlike the in vivo 19F MRI in the early stage of infarct
development no intracranial fluorine signal could be detected in
vivo when the PFC emulsion was applied with a delay of six days
after PT. However, ex vivo 19F MRI with long acquisition times and
use of a more sensitive coil disclosed a fluorine signal within the PT
lesions at this late lesion stage most likely reflecting the infiltration
of fluorine-labeled macrophages from the blood.
Several groups have already shown the uptake of PFC in
activated macrophages [6,7,8]. In their work, Flo¨gel and
colleagues [7] focused on cellular infiltration after myocardial
infarction, but also studied 19F MRI in the PT model. In contrast
In Vivo 19F-Imaging of Stepwise Vessel Occlusion
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