Transient widespread blood-brain barrier alterations after cerebral photothrombosis as revealed by gadofluorine M-enhanced magnetic resonance imaging.

Transient widespread blood–brain barrier
alterations after cerebral photothrombosis as
revealed by gadofluorine M-enhanced magnetic
resonance imaging
Guido Stoll1,4, Christoph Kleinschnitz1,4, Sven G Meuth1, Stefan Braeuninger1,
Chi Wang Ip1, Carsten Wessig1, Ingo No¨lte2,5 and Martin Bendszus2,3,4
1Department of Neurology, University of Wuerzburg, Wuerzburg, Germany; 2Department of Neuroradiology,
University of Wuerzburg, Wuerzburg, Germany; 3Department of Neuroradiology, University of Heidelberg,
Heidelberg, Germany
Magnetic resonance imaging (MRI) is a powerful tool to assess brain lesions, but currently available
contrast agents are limited in the assessment of cellular and functional alterations. By use of the
novel MRI contrast agent gadofluorine M (Gf) we report on imaging of transient and widespread
changes of blood–brain barrier (BBB) properties as a consequence of focal photothrombotic brain
lesions in rats. After i.v. application, Gf led to bright contrast in the lesions, but also the entire
ipsilateral cortex on T1-weighted MRI. In contrast, enhancement after application of gadolinium
diethylenetriamine-pentaacetic acid (Gd-DTPA), a common clinical indicator of BBB leakage was
restricted to the lesions. Remote Gf enhancement was restricted in time to the first 24 h after
photothrombosis and corresponded to a transient breakdown of the BBB as revealed by
extravasation of the dye Evans blue. In conclusion, our study shows that Gf can visualize subtle
disturbances of the BBB in three dimensions not detectable by Gd-DTPA. Upon entry into the central
nervous system Gf most likely is locally trapped by interactions with extracellular matrix proteins.
The unique properties of Gf hold promise as a more sensitive contrast agent for monitoring BBB
disturbances in neurologic disorders, which appear more widespread than anticipated previously.
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 331–341; doi:10.1038/jcbfm.2008.129; published online
29 October 2008
Keywords: blood–brain barrier; gadofluorine; gadolinium-DTPA; magnetic resonance imaging; photothrombosis;
remote response
Introduction
There is increasing evidence that focal brain lesions
profoundly influence remote brain areas not affected
by the primary insult (reviewed in Witte et al, 2000).
Pathophysiologic alterations involve edema forma-
tion, cortical spreading depression (CSD), diaschisis,
and other adaptive responses. Magnetic resonance
imaging (MRI) is a powerful tool to assess focal brain
lesions, but structural and molecular alterations
remote from the initial insult are in general not
visualized by conventional MR sequences. Thus,
there is high interest in developing novel MR-
contrast agents for in vivo assessment of cellular
and functional responses in the brain (Artemov et al,
2004; Bulte and Kraitchman, 2004). In this study, we
report on in vivo MRI of transient and widespread
changes of blood–brain barrier (BBB) properties as a
consequence of focal photothrombotic brain lesions
by the use of a novel MR-contrast agent Gd-GlyMe-
DOTA-perfluoroocytyl-mannose-conjugate (gado-
fluorine M (Gf); Bayer Schering Pharma AG, Berlin,
Germany). Brain photothrombosis (PT) was intro-
duced as a simple model of focal cortical lesions by
Watson and Dietrich in the 1980s (Watson et al, 1985;
Dietrich et al, 1987a, b; reviewed in Stoll et al, 1998).
Photothrombosis can be induced in rodents by
systemic administration of a photosensitive dye such
as Bengal rose and subsequent focal illumination of
the brain through the intact skull by a light source
(Watson et al, 1985; Schroeter et al, 2002). Photo-
Received 30 June 2008; revised 7 October 2008; accepted 8 October
2008; published online 29 October 2008
Correspondence: Professor G Stoll, Department of Neurology,
University of Wu¨rzburg, Josef-Schneider-Str. 11, D-97080 Wu¨rz-
burg, Germany.
E-mail: stoll_g@klinik.uni-wuerzburg.de
This work was supported by the Deutsche Forschungsge-
meinschaft (SFB 688 TP B1).
4These authors contributed equally to this work.
5Current address: Department of Neuroradiology, University of
Heidelberg, Campus Mannheim, Mannheim, Germany.
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 331–341
& 2009 ISCBFM All rights reserved 0271-678X/09 $32.00
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thrombosis is minimally invasive and does not inflict
significant neurologic deficits assessed behaviorally
to animals, thereby allowing long-term observations.
Because of its focal nature PT facilitates the distinc-
tion between events within the lesion and remote
brain areas. Numerous studies have shown that PT
induces periinfarct depolarization closely related to
CSD (Dietrich et al, 1994; Jander et al, 2000) and
other neurophysiologic alterations in the ipsilateral
hemisphere such as increased spontaneous activity
of neurons (Schiene et al, 1996). Remote molecular
alterations encompass upregulation of proinflamma-
tory cytokines and growth factors (Jander et al, 2000;
Kleinschnitz et al, 2004), and rearrangement of
neurotransmitter receptors (Redecker et al, 2002).
Magnetic resonance imaging studies using the cont-
rast agent gadolinium diethylenetriamine-pentaacetic
acid (Gd-DTPA) as the conventional in vivo marker
for the BBB showed early and long-lasting break-
down of the BBB within the PT lesion (Lee et al,
1996; Schroeter et al, 2001; Kleinschnitz et al, 2003).
However, there were no remote contrast enhance-
ment or signal alterations on T2-weighted (T2-w)
images. By use of the novel MR-contrast agent Gf we
now provide in vivo evidence to confirm a wide-
spread remote alteration of the BBB after PT and a
novel MRI tool to visualize these profound and
transient BBB disturbances.
Methods
Induction of Photothrombotic Brain Lesions
All animal experiments were performed in accordance
with institutional guidelines and were approved by the
Regierung of Unterfranken. Focal cerebral lesions were
induced in male Wistar rats (200 to 250 g) by PT of cortical
microvessels under inhalation anesthesia with 2.5% iso-
flurane in a 2:1 nitrogen/oxygen atmosphere, as described
previously (Watson et al, 1985; Jander et al, 1995;
Buchkremer-Ratzmann et al, 1996; Kleinschnitz et al,
2003, 2005). During the operation procedure, rectal
temperature was kept between 36.51C and 37.51C by a
servo-controlled heating blanket. Briefly, a fiber optic
bundle of 4 mm diameter of a light source (150 W Philips
lamp type 6423, beam power 600 lm) was centered
stereotaxically 4 mm posterior and 4 mm lateral from
Bregma on the intact skull. Thereafter, 0.4 mL of a sterile-
filtered Rose Bengal solution (10 g/L; Sigma, Deisenhofen,
Germany) was administered by a femoral vein catheter, and
the brain was illuminated for 20 mins. With this setup used
in this and our numerous previous studies there was a
modest increase of 11C to 21C in the temperature of the
brain in the area of illumination (measured with digital
computer thermometer CTA 1220 and sensor EB01; Ebro,
Ingolstadt, Germany; Jander et al, 1995; Buchkremer-
Ratzmann et al, 1996). This procedure resulted in cone-
shaped cortical infarctions without neurologic symptoms.
At the end of the operation, subcutaneous fat and skin were
sutured in anatomic layers and animals were placed in
separate cages for recovery. Three animals underwent
sham operation and received Rose Bengal without irradia-
tion. Animals underwent MRI examination at different
stages of lesion development and in a subgroup vital brain
slice preparations were used to follow the spread of the
MR-contrast agent Gf ex vivo over time.
Contrast Agent and Magnetic Resonance Imaging
Gadofluorine M (Bayer Schering Pharma AG) is an
amphiphilic Gd complex with a molecular weight of
1,528 Da (Barkhausen et al, 2003; Misselwitz et al, 2004).
The contrast medium is synthesized by adding a perfluor-
octyl chain to a Gd-containing macrocycle. The complex
also contains a sugar moiety, which leads to increased
hydrophilicity. In vitro studies have shown that Gf forms
micelles in water (approximately 5.5 nm in diameter) and
binds reversibly to plasma albumin and matrix proteins.
After intravenous administration, Gf binds reversibly to
plasma proteins leading to macromolecular Gf–protein com-
plexes in the circulation. The plasma half-life T1/2b of these
Gf complexes is 15.6h (Raatschen et al, 2006). Gadofluorine
M has a high T1 relaxivity in plasma (15.9 L/mmol per sec per
Gd ion) and in water (15.8 L/mmol per sec; Misselwitz et al,
2004). For subsequent localization of Gf accumulation in
histologic sections, Gf was prelabeled with a carbocyanine
dye allowing detection of Gf by fluorescence.
All measurements were performed on a clinical 1.5 T
MRI unit (Magnetom Siemens Vison, Erlangen, Germany)
as described previously (Kleinschnitz et al, 2003, 2005).
Briefly, rats were anesthetized as described above. For all
MRI scans, animals were lying in supine position with
their heads fixed in a heatable custom-made dual channel
surface coil designed for investigations of the rat brain
(A063HACG; Rapid Biomedical, Wu¨rzburg, Germany). The
MR protocol included a scout sequence in three planes, a
coronal T1-weighted (T1-w) (repetition time 460 ms, echo
time 14 ms) and a coronal T2-w sequence (repetition time
2.500 ms, echo time 92 ms) with a slice thickness of 2 mm.
After a nonenhanced MR examination, Gd-DTPA
(0.2 mmol/kg body weight) or Gf (0.1 mmol/kg body
weight) was applied by the femoral vein. A second MRI
was performed always 20 mins after Gd-DTPA at the time
points 3, 6, 12, 24, and 72 h after PT in groups of animals.
In a first set of experiments MR measurements were
performed in separate groups of four animals each at 24,
48, and 72 h after PT (always 24 h after Gf application). In a
second set of experiments the spatiotemporal evolution of
Gf enhancement was studied: Gf was always given shortly
after induction of PT when irradiation was completed and
groups of animals underwent two MR examinations at 1
and 3 h (n = 3), 5 and 9 h (n = 3), or 18 and 26 h (n = 3) after
PT, respectively. Finally, three animals that had received Gf
at the time of PT underwent serial MR examinations at
24 h, 3 and 7 days after photothrombosis.
A subgroup of Gf-injected animals was killed at different
time points after PT in deep anesthesia and the brains were
quickly removed and snap frozen in isopentane for
fluorescence studies. Moreover, some rats were perfused
by 4% paraformaldehyde and the brains were embedded in
paraffin.
MRI of blood–brain barrier dysfunction
G Stoll et al
332
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 331–341

Assessment of Blood–Brain Barrier Patency
To study the patency of the BBB after PT using the Evans
blue method (Belayev et al, 1996) a separate group of rats
received 80 mg/kg of the dye Evans blue (solution 20 mg/mL)
i.v. immediately after cessation of the illumination for PT
induction, or with a delay of 48 h (for time point 72 h). Two
animals each were killed shortly thereafter (time point 0) or
1, 3, 6, 9, 12, 24, and 72 h after PT, respectively. Brains were
rapidly removed and fixed in 4% paraformaldehyde.
Photographs of the entire brain were taken to assess
Evans blue extravasation. Then the brains were cut
into 1 mm thick coronal slices for further morphologic
examination.
Brain Slice Preparations
For preparation of vital brain slices rats were deeply
anesthetized with isoflurane and decapitated. A block of
tissue containing the lesion was removed and transferred
into ice-chilled saline, containing (mmol/L): sucrose, 200;
PIPES, 20; KCl, 2.5; NaH2PO4, 1.25; MgSO4, 10; CaCl2, 0.5;
dextrose, 10; pH 7.35 with NaOH. Coronal sections
(300 mm) through the photothrombosed region were pre-
pared on a vibratome (Gala Instruments, Bad Schwalbach,
Germany). Before fluorescence microscopy slices were kept
submerged in standard artificial cerebrospinal fluid
(mmol/L): NaCl, 125; KCl, 2.5; NaH2PO4, 1.25; NaHCO3,
24; MgSO4, 2; CaCl2, 2; dextrose, 10; pH adjusted to 7.35 by
bubbling with a mixture of 95% O2 and 5% CO2. For serial
observations slices were transferred to standard cell
culture dishes and placed under the microscope (Axioskop
FS; Zeiss, Jena, Germany). Under these conditions slices
are alive for 6 to 8 h as indicated by previous electro-
physiologic recordings (Meuth et al, 2003, 2006).
In one set of experiments animals were killed 1.5 and 5 h
after PT and brain slices were analyzed by fluorescence
microscopy. In a second set of experiments brain slices
were obtained shortly after PT and application of Gf (1 min)
and Gf autofluorescence was repetitively followed for 3 h
by fluorescence microscopy. In a last set of experiments
slices were prepared 30 mins after PT induction and Gf
application, and slices were sequentially analyzed up to
2.5 h after the preparation.
N -Methyl-D -Aspartate-Receptor Blockage and
Lipopolysaccharide Application
To analyze whether the remote Gf enhancement was
mediated by N-methyl-D-aspartate (NMDA)-receptor sig-
naling, rats were injected i.v. with the noncompetitive
NMDA-receptor antagonist MK-801 (dizolcipine; Research
Biochemicals International, Natick, MA, USA) at a dosage
of 2 mg/kg body weight 30 mins before induction of PT.
Three MK-801-treated animals received Gf immediately
after PT induction and underwent MRI 24 h later.
Systemic application of the proinflammatory lipopoly-
saccharide (LPS) can alter permeability of the BBB (Xaio
et al, 2001) and, additionally, activate inflammatory path-
ways within the central nervous system (CNS; Tasaki et al,
1997). To address the potential role of these inflammatory
processes in the opening of the BBB (Blamire et al, 2000)
we aimed to mimic the remote Gf accumulation after PT by
systemic LPS application. Groups of three naive rats
received LPS (0.5 mg/kg body weight) intraperitoneally
and Gf 1 or 48 h thereafter i.v., and underwent MRI at 24 or
72 h, respectively.
Immunohistochemistry and Fluorescence
Thick paraffin (10mm) sections were cut and stained with
peroxidase-conjugated sheep immunoglobulin G antibo-
dies against rat albumin and 3040 diaminobenzidine as
chromogen (Cappel, Lot 37201). In addition, 10 mm thick
sections from snap-frozen brains were cut through the
lesion and analyzed for the presence of carbocyanine
labeled Gf by red fluorescence on a Zeiss Axiophot
microscope (Zeiss, Thornwoods, NY, USA).
Results
Gadolininium Diethylenetriamine-Pentaacetic
acid-Enhanced Magnetic Resonance Imaging of
Photothrombotic Lesions
After application of the conventional MR-contrast agent
Gd-DTPA, photothrombotic cortical lesions showed a
marked contrast enhancement within the lesions on T1-w
(Figure 1) and appeared hyperintense on T2-w MRI (Figure
2A) as described before (Schroeter et al, 2002; Kleinschnitz
et al, 2003). Importantly, no uptake of Gd-DTPA on T1-w
MRI was seen remote from the lesions in the ipsi- and
contralateral cortex at all time points examined, for example,
3, 6, 12, 24, and 72 h after PT. In these experiments Gd-DTPA
was applied according to clinical practice always 20 mins
before MRI, because the contrast agent is rapidly cleared
from the circulation resulting in a disappearance of tissue
enhancement.
Gadofluorine M-Enhanced Magnetic Resonance
Imaging after Photothrombosis
Application of Gf to normal- and sham-operated rats
showed contrast enhancement within large arterial and
venous vessels on T1-w MRI, but no parenchymal
enhancement (not shown). In animals after PT, Gf similarly
to Gd-DTPA led to contrast enhancement within the lesion
(Figure 2). This lesion-related Gf uptake was present at all
stages of lesion development. In contrast to Gd-DTPA,
which rapidly disappeared from the lesions again, Gf
enhancement persisted in the lesions for several days upon
repeated MR examinations (not shown). As our main
finding, Gf application led to an additional widespread
cortical enhancement involving mainly the entire ipsilat-
eral cortex remote from the lesion and to a lesser extent
also the contralateral cortex (Figures 2 and 3). The upper
layer of the cerebral cortex frontal, lateral, and occipital
from the PT lesion showed strong Gf enhancement at 24 h
(arrows in Figure 2B). Moreover the entire corpus callosum
exhibited marked Gf uptake (arrowheads in Figures 2B, 3E
MRI of blood–brain barrier dysfunction
G Stoll et al
333
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 331–341

and 3F). The spatiotemporal evolution of Gf enhancement
was further assessed in separate groups of animals in
which two subsequent MR examinations were performed
at 1 and 3 h, 5 and 9 h, or 18 and 26 h after PT, respectively
(Figure 3A–3F). Thereafter the interval between Gf
application and MRI varied between 1 and 26 h, which is
feasible because Gf has a much longer plasma clearance
time than Gd-DTPA (Misselwitz et al, 2004). At 1 h Gf
enhancement was restricted to the PT lesion and spared
the center because of restricted access of the contrast
medium by ongoing vessel occlusion (Kleinschnitz et al,
2005). At 3 h Gf enhancement extended into the upper
cortical layer close to the lesion. In the group of animals
studied between 5 and 9 h after PT, cortical enhancement
further spread ipsilaterally and now involved the frontal
and occipital cortex. Gadofluorine M uptake moreover
involved white matter tracts most prominently along
Figure 1 Gd-DTPA enhancement is restricted to photothrombotic
(PT) lesions. T1-w MRI of lesions at 6 h (A), 12h (B), and 24 h (C)
after PT after i.v. application of Gd-DTPA 20 min before MRI. Note
that Gd-DTPA enhancement is restricted to the lesions.
Figure 2 Gf enhancement in relation to tissue damage on T2-w
MRI at 24 h after induction of photothrombosis (PT). (A) T2-w
MRI and (B) corresponding T1-w MRI after Gf application at
lesion induction (24 h before MRI). The lesion appears
hyperintense on conventional T2-w images (A). Gf enhancement
in (B) by far exceeds the lesion limits and involves the remote
frontal, temporal, and parietal cortex (arrows) as well as the
corpus callosum (arrowheads). When given with a delay of 24 h
or later Gd-DTPA (C) as well as Gf enhancement (D) are
restricted to the lesion area and the Gf enhancement no longer
involves remote areas as shown for day 3 after PT.
MRI of blood–brain barrier dysfunction
G Stoll et al
334
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 331–341

Figure 3 Spatiotemporal evolution of Gf enhancement. (A) At 1 h Gf enhancement was restricted to the PT lesions sparing the center
because of ongoing vessel occlusion (Kleinschnitz et al, 2005). At 3 h the entire lesion showed breakdown of the BBB leading to
homogenous Gf uptake. As the main novel finding, in addition, small cortical areas adjacent to the lesion (arrow in B) showed Gf
enhancement. Within the next 23 h Gf enhancement further extended into cortical and subcortical areas outside the PT lesion and
involved fiber tracts underlying the lesion and the corpus callosum as shown at different stages in (C–F). Cryostat sections taken from
animals at 18 h revealed red fluorescence of prelabeled Gf at cortical and subcortical areas exactly corresponding to contrast
enhancement on T1-w MRI (G, H). (I) represents a phase contrast picture of the region showing labeled Gf fluorescence in (H). Bar in
(G) represents 50 mm, bar in (H) 25 mm for (H, I).
MRI of blood–brain barrier dysfunction
G Stoll et al
335
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 331–341