Amyloid-? (A?) deposition in the cerebral vasculature is accompanied by remodeling which has a profound influence on vascular
arcA? model of cerebral amyloidosis. To estimate the density of the cortical microvasculature in vivo, we used contrast-enhanced
magnetic resonance microangiography (CE-?MRA). Three-dimensional gradient echo datasets with 60 ?m isotropic resolution were
mated algorithm was applied for assessing the number and size distribution of intracortical vessels. With CE-?MRA, cerebral arteries
age-matched wt mice, whereas there was no difference between transgenic and wt mice of 4 months of age. Immunohistochemistry
Cerebral amyloid angiopathy (CAA) results from the failure to
eliminate amyloid-? (A?) and other amyloid peptides from the
disease (AD; Glenner et al., 1981; Weller et al., 2008, 2009). A?
illaries and arterioles (Fischer at al., 1990; Buee et al., 1994; Miao
cerebral blood supply, reduced vascular reactivity, and increased
susceptibility to cerebral ischemia (Gilbert and Vinters, 1983;
Cadavid et al., 2000; Haglund et al., 2006; Ryu and McLarnon,
term structural changes known as vascular remodeling. Blood
brane is altered (de la Torre, 1997; Moody et al., 1997), and the
overall vascular density decreases (Lee et al., 2005; Bouras et al.,
2006). Furthermore, changes in the proteolytic microenviron-
and the occurrence of cerebral microbleeds (CMBs) and intrace-
rebral hemorrhage (Gilbert and Vinters, 1983; Franceschi et al.,
1995; Ryu and McLarnon, 2009). There is increasing evidence
that vascular remodeling might not only reflect an adaptive pro-
cess, but also constitutes an important mechanism in the patho-
physiology of AD and CAA (Weller et al., 2009; Marchesi, 2011).
Cerebrovascular A? deposition can be mimicked in mouse
models, which, therefore, have become central for studying the
pathophysiology of CAA and evaluating novel therapeutic ap-
proaches (Calhoun et al., 1999; Herzig et al., 2004; Miao et al.,
2005; Kouznetsova et al., 2006). Recently, the arcA? mouse line
dysfunction as reflected by BBB impairment, neurovascular un-
coupling (Merlini et al., 2011), decreased vascular reactivity
(Princz-Kranz et al., 2010), and the occurrence of cerebral mi-
J.K., C.B., R.M.N., and M.R. designed research; J.K., C.B., F.P.-K., and I.K. performed research; C.B., D.R., and
M.R. and 310030-132629 to I.K.). We gratefully acknowledge Daniel Schuppli (Division of Psychiatry Research,
of Zurich and ETH, AIC–ETH HCI D426, Wolfgang-Pauli-Strasse 10, CH-8093 Zu ¨rich, Switzerland. E-mail:
TheJournalofNeuroscience,February1,2012 • 32(5):1705–1713 • 1705
related remodeling of the cerebral microvasculature. We used
high-resolution contrast-enhanced magnetic resonance angiog-
matched wild-type (wt) control mice to assess age-dependent
as detected with conventional time-of-flight MRA (TOF-MRA),
immunohistochemistry revealing that A? and fibrinogen are pre-
larger cerebral vessels. Hence, the reduced density of intracortical
vessels observed in advanced disease may be attributed to obstruc-
tion of perfusion as a result of abnormal fibrinolysis. In murine
models of cerebral amyloidosis, the remodeling of these vascular
Animals. All experiments were performed in accordance with the Swiss
Federal Act on Animal Protection. We used arcA? transgenic mice of
either sex, which express the human APP 695 containing both the Swed-
ish and the Arctic mutation under the control of the prion protein pro-
moter as described previously (Knobloch et al., 2007). Age- and gender-
matched wt littermates served as controls. Mice at the age of 4 months
(arcA?, n ? 4; wt, n ? 4) and 24 months (arcA?, n ? 6; wt, n ? 5) were
used for the study. Animals were kept at standard housing conditions
(temperature, 20–24°C; relative humidity, minimum 40%; light/dark
cycle, 12 h) providing water and food ad libitum.
BioSpec 94/30 (Bruker BioSpin) small animal MR system operating at
400 MHz with a gradient system, capable of a maximum gradient
surface coil (Bruker BioSpin) was used for RF signal transmission and
(Abbott) in a 4:1 air/oxygen mixture and maintained with 1.2% isoflu-
rane. Mice were endotracheally intubated and mechanically ventilated
with 90 breaths/min, applying a respiration cycle of 25% inhalation and
75% exhalation (MRI-1 Volume Ventilator, CWI). Temperature was
kept at 36.0 ? 0.5°C using a warm-water circuit integrated into the
animal support (Bruker BioSpin). Body temperature was monitored
with a rectal temperature probe (MLT415, ADInstruments). The tail
veins of the mice were cannulated for administration of contrast agents.
Tri-pilot scans were used for accurate positioning of the animal head
inside the magnet. For high-resolution structural imaging, we used a 2D
FLASH sequence with the parameter TE/TR? 8/345 ms; RF pulse angle,
? ? 30°; bandwidth ? 50 kHz; number of averages (NA) ? 1. Fifteen
0.5-mm-thick slices with an interslice distance of 0.6 mm were acquired
with a field of view (FOV) of 20 ? 20 mm2and matrix dimension of
384 ? 384, resulting in a spatial resolution of 52 ? 52 ?m2. For TOF-
MRA a 3D FLASH sequence with the parameters TE/TR? 2.5/15 ms,
? ? 30°, bandwidth ? 98 kHz, NA ? 2 was used. A horizontal slab of
19.8 ? 15.9 ? 8 mm3was recorded using a matrix dimension of 248 ?
199 ? 100, resulting in isotropic voxel dimensions of 80 ?m3. The ac-
quisition time was 7 min and 27 s. For CE-?MRA, precontrast and
postcontrast images were acquired using a 3D FLASH sequence with
TE/TR? 2.9/150 ms, ? ? 20°, bandwidth ? 82 kHz, NA ? 2. A slab of
15 ? 12 ? 2.2 mm3was recorded with matrix dimensions of 248 ?
199 ? 36 with a spatial resolution of 60 ? 60 ? 61 ?m3. Animals were
injected intravenously with 50 ?l of an iron-oxide contrast agent (corre-
sponding to 0.52 mg of Fe; Endorem, Guerbet) as a bolus into the tail
Determination of contrast-to-noise ratios. Regions of interest (ROIs)
were drawn in vessels and adjacent tissue of precontrast images. A noise
ROI was placed outside the head of the animal. Signal-to-noise (SNR)
the SD of the noise ROI. SNR values were calculated for four regions
separately and then averaged.
Reconstruction of angiograms. For TOF-MRA datasets, angiograms
were generated by maximum intensity projections (MIPs) using Paravi-
sion 5.0 (Bruker BioSpin MRI). All angiograms were evaluated by inves-
tigators blinded to the genotype of the mouse. MIPs of TOF-MRA were
the signal intensities in an identified blood vessel reached background
the anterior cerebral artery (Fig. 1C,D). Distality count, i.e., the number
of hierarchical levels of vascular branches detected, was only estimated
was not covered in the acquisition.
the postcontrast image from the precontrast image before MIP genera-
tion using home-written software based on IDL (Interactive Data Lan-
guage, version 6.4; ITT Visual Information Solutions).
Assessment of vessel number and vessel radius. For segmentation of the
cerebral cortex, ROIs were drawn for each animal on each 61-?m-thick
High-resolution 3D TOF-MRA of the intracranial and extracranial vasculature of a 24-month-old wt control mouse (A) and an age-matched arcA? mouse (B). The representative
1706 • J.Neurosci.,February1,2012 • 32(5):1705–1713Klohsetal.•DecreaseofVesselDensityinCerebralAmyloidosis
slice of the 3D dataset. The ROIs were chosen to encompass both hemi-
spheres of the neocortex over a depth of 430 ?m. To exclude meningeal
surface were not considered in the ROI analysis. A noise ROI was placed
outside the brain. The selected ROIs were combined to define a volume
of interest (VOI) required for the subsequent 3D vessel analysis. In this
algorithm, vessel structures were identified according to the following
the signal of the voxel was higher than the signal threshold (SThreshold) as
to or higher than the connectivity threshold. SThresholdwas defined ac-
SThreshold? S?ROI,Noise? tNoise? SD?SROI,Noise),(1)
where S?ROI,Noiseand SD(SROI,Noise) define the average and the SD, respec-
(in a corner of the field-of-view). The parameter tNoisecould be chosen
for which the number of connected voxels exceeded the connectivity
threshold set to 3, 6, and 9 were considered as vessels.
taken by the vascular compartment. Correspondingly, evaluation of
thus an upper limit for the vessel dimensions. For this purpose we as-
sumed a perpendicular orientation of the vessel with respect to the im-
aging slices. For transcortical vessels oriented radially with regard to the
cortical surface, this approximation appears reasonable. Within each
slice the voxel with the highest signal intensity Sk,maxin the difference
images was assumed to comprise only the vascular compartment. Ac-
slice was calculated as
Sk,max? ? AVoxel
running over all voxels v in slice k contributing to vessel i. The average
displaying the vessel.
Immunohistochemistry. All animals were deeply anesthetized by intra-
peritoneal injection of ketamine/xylazine (100/20 mg/kg body weight)
and perfused transcardially with PBS (pH 7.4), followed by 4% parafor-
in paraffin and cut into 10-?m-thick horizontal sections. The sections
were pretreated in 85°C Na-Citrate buffer (0.1 M, pH 4.5, in the micro-
wave), followed by a 5 min submersion in 95% formic acid. Blocking of
Klohsetal.•DecreaseofVesselDensityinCerebralAmyloidosis J.Neurosci.,February1,2012 • 32(5):1705–1713 • 1707
nonspecific binding was done in a mixture of 4% BSA, 5% normal goat
serum, and 5% horse serum in PBS for 1 h at room temperature. The
sections were then incubated overnight at 4°C in the primary antibody
solution containing mouse antifibrinogen antibodies (Abcam, catalog
catalog #NCL-vWFp, 1:500) in PBS containing 2% normal goat serum
and 0.2% Triton X-100. After three washes in PBS, tissue sections were
incubated for 30 min at room temperature in corresponding secondary
antibodies coupled to Cy3 (diluted 1:500; Jackson ImmunoResearch).
Thioflavin S counterstaining involved 10 min incubation in filtered 1%
lowed by two washes for 5 min in 80% EtOH, 5 min wash in 95% EtOH,
and three 5 min washes in distilled water. Brain sections were then air-
dried in the dark and mounted with aqueous permanent mounting me-
dium containing DAPI for nuclear counterstainings (DAKO).
Analysis of immunohistochemical stainings. Qualitative evaluation of
the immunofluorescence labeling was done with a confocal microscope
(LSM-710, Zeiss) using the 40? (NA1.3) objective. Double immunoflu-
1708 • J.Neurosci.,February1,2012 • 32(5):1705–1713Klohsetal.•DecreaseofVesselDensityinCerebralAmyloidosis
orescence staining was visualized using sequential acquisition of each
channel. The pinhole aperture was set to 1.0 Airy unit for each channel.
in z) were acquired. For visual display, Z-sections of both channels were
projected in the z-dimension (maximal intensity), and merged using the
image analysis software Imaris (Bitplane). Cropping of images and ad-
justments of brightness and contrast were identical for each labeling and
done using Adobe Photoshop (Adobe Systems).
Quantitative analyses of the vWF-immunoreactivity associated with
the endothelial cells of the cerebral vasculature were done on three
different horizontal brain sections obtained from 5 mice per age and
genotype. Two digital images per brain hemisphere were acquired using
was quantified using a threshold algorithm of ImageJ software. The bi-
nary images were segmented and the individual vessels outlined. The
by the anti-vWF immunoreactivity. The different measures were aver-
aged per animal and included in the statistical analysis.
For the quantitative evaluation of the fibrinogen accumulation and
colocalization with thioflavin S associated with vascular plaques, three
different brain sections per animal (n ? 5 per age and genotype) were
and green channels were individually segmented and then analyzed with
regard to pixel overlap within outlined vessels using a colocalization
tive distribution and frequency calculated as a function of their size.
Statistical analysis. Data are presented as mean ? SD. Student’s t test,
1-way ANOVA, or repeated-measures ANOVA followed by Tukey’s test
(SigmaStat 3.0, Systat Software) were used for statistical comparison,
where appropriate. The statistical significance level was set to p ? 0.05.
4 months (n ? 4 each) and 24 months (n ? 6 each) of age. In
entered the imaging volume and reached the respective vascular
need of administering a contrast agent (Reese et al., 1999). MIPs
displayed in different orientations allow clear identification of
major intracranial arteries as illustrated for a 24-month-old wt
and arcA? mouse (Fig. 1A,B). No apparent flow abnormalities
could be detected in the vessels of wt control and arcA? mice at
both ages. The distality count did not reveal any trait-specific
for 4-month-old wt and arcA? mice and 3.5 ? 0.4 and 3.5 ? 0.5
for 24-month-old wt and arcA? mice, respectively. The distality
value tended to be slightly lower in aged animals, though the
difference was not significant (Fig. 1C,D). Interestingly, flow
atine arteries of wt and arcA? mice at both ages (for 24 months,
Fig. 1A,B, white arrows). Again, no difference was observed be-
tween wt and arcA? mice.
Visualization of vascular structures can be enhanced using
intravascular contrast agents such as iron oxide nanoparticles,
which, due to their large net positive magnetic moments, induce
spin dephasing resulting in a net signal loss (Boxerman et al.,
1995; Bolan et al., 2006). Figure 2 displays representative hori-
zontal images of the brain of a 24-month-old wt control and
an arcA? mouse. Following the administration of Endorem,
transcortical blood vessels became visible as focal signal voids
that could not be discerned on the precontrast image (Fig. 2B,E)
variations in the signal intensity of the vascular structures in the
difference images. This is attributed to partial volume effects.
This intensity information can, in turn, be used to estimate the
size of the vascular compartment within a voxel, from which a
maximum vessel diameter might be deduced (assuming that the
Several large circular hypointensities (mean diameter of 242.5 ?
108.1 ?m) have been observed in the brains of 24-month-old
arcA? mice already before the administration of the contrast
agent (Fig. 2D, white arrow). Such structures have not been ob-
served in the brains of 4-month-old arcA? mice and 4- and 24-
thresholds corresponding to 3, 6, and 9 pixels, corresponding to minimal length of a vessel
their estimated vessel radius when the connectivity threshold was set to 3. The algorithm
Klohsetal.•DecreaseofVesselDensityinCerebralAmyloidosis J.Neurosci.,February1,2012 • 32(5):1705–1713 • 1709
iron-containing A? plaques (Benveniste et al., 1999) or cerebral
microbleeds (Klohs et al., 2011). These structures disappear in
in all animals few foci of reduced signal intensity have been de-
tected in the precontrast images. These are attributed to venous
structures for which the relatively high concentration of para-
magnetic deoxyhemoglobin leads to decreased blood T2* values
already in the absence of an exogenous contrast agent (Ogawa et
al., 1990; Reichenbach et al., 1997).
For illustration purposes, 3D stacks of difference images have
been constructed for computing of MIPs (Fig. 3). Clearly, these
MIPs provide significantly more detail about the vascular anat-
omy than those obtained from TOF angiograms, which essen-
tially reveal only large arterial structures. While CE-MIPs are
dominated by large vessels running at the brain surface, such as
sels become apparent (Fig. 3A,B). These can be highlighted by
visualizing a cortical subcube of the full 3D dataset (Fig. 3C–E).
An algorithm was developed which assumes that blood vessels
ence images were counted as vessels provided they fulfilled the
connectivity criterion, which states that the vessel can be ob-
served over multiple slices within the 3D dataset. The strength of
this criterion could be adjusted. The number of vessels was esti-
mated for different values of vessel connectivity i.e., 3, 6, and 9
pixels, corresponding to minimal length of a vessel segment of
ual datasets have been analyzed in a semiautomated fashion. The
quantitative results on vessel counts are illustrated in Figure 4.
wt and arcA? mice (Fig. 4A). However, there was a significant
transgenic compared with wt animals in a volume of ?11 mm3
(*p ? 0.05). Vessels were categorized according to their vessel
radius as estimated based on its signal intensity, which is gov-
4B). There were no significant differences between the numbers
of vessels in each category among the four groups, though the
mean number of small (20–40 ?m) and medium sized (40–60
?m) cortical vessels in the group of 24-month-old arcA? mice
tended to be smaller than in the respective group of wt animals.
Immunohistochemical assessment of vascular density, A? and
fibrinogen accumulation was performed on brain sections of
tified endothelial cells (Fig. 5A–C). Additional vWF immunore-
activity was revealed in thrombus-like structures in microvessels
of 24-month-old arcA? mice only (Fig. 5C, white arrow). Quan-
titative analysis of vWF immunoreactivity demonstrated no dif-
ferences between the four groups investigated.
Thioflavin S-positive A? accumulation was apparent in cere-
5B) nor in 4-month-old transgenic (Fig. 6A) and wt mice (data
not shown). Investigation of different anatomical regions re-
areas of 24-month-old arcA? mice, while vessels in the thalamus
showed almost no A? deposition (results not shown). Autofluo-
rescence of pigment granules were observed in 24-month-old wt
1710 • J.Neurosci.,February1,2012 • 32(5):1705–1713 Klohsetal.•DecreaseofVesselDensityinCerebralAmyloidosis
and arcA? mice (Fig. 6B,C, white arrows). Such intracellular
lipofuscin aggregates were not observed in 4-month-old wt and
transgenic mice and are known to be a sign of the aging of neu-
rons (Dowson and Harris, 1981).
Fibrinogen accumulation was observed in vessels of 24-
month-old arcA? mice (Fig. 6C). The fibrinogen accumulation
was always colocalized by thioflavin S-positive signal (Fig. 6G).
thioflavin S/fibrinogen-immunoreactivity clearly differed be-
tween vessel categories. In small- and medium-sized arteries,
thioflavin S/fibrinogen-positive signal was highly abundant,
of the small vessels were filled by the fibrinogen deposition, indi-
cating vessel stenosis (Fig. 6D). Quantitative assessment of im-
munoreactivity showed highest thioflavin S/fibrinogen-positive
Apart from the formation of parenchymal plaques, cerebral am-
age-dependent aberrations in the structure and function of the
cerebrovasculature have been reported (Princz-Kranz et al.,
2010; Klohs et al., 2011; Merlini et al., 2011). We used MRA
techniques to noninvasively assess cerebral vasculature. While
TOF-MRA did not display any pathological changes, CE-?MRA
arcA? mouse during advanced disease state.
els of the vascular tree. In TOF-MRA the signal depends on the
blood flow (Lin et al., 1997), confining the method to the visual-
resolution TOF-MRA has been applied to transgenic mouse
models of AD and CAA, reporting prominent flow voids in the
major arteries of aged APP overexpressing transgenic animals
(Beckmann et al., 2003; Krucker et al., 2004; Thal et al., 2009; El
Tannir El Tayara et al., 2010). In contrast, we did not detect flow
voids in arcA? mice. This is not surprising, because immunohis-
tochemical analysis revealed that A? and fibrinogen deposition
prevails in small- and medium-sized cerebral vessels, but effec-
tively spares larger cerebral vessels. There was a trend toward a
Klohsetal.•DecreaseofVesselDensityinCerebralAmyloidosis J.Neurosci.,February1,2012 • 32(5):1705–1713 • 1711
reduced distality count in TOF MRI of 24-month-old compared
trol mice. This effect can be attributed to a decline in blood flow
et al., 2003; Krucker et al., 2004; Thal et al., 2009; El Tannir El
nial vessels, such as the pterygopalatine artery, in APP overex-
pressing mice. As the transgene is expressed under the control of
the Thy-1 promoter, A? deposition should be confined to intra-
cerebral vessels. In the current study we observed similar flow
voids, however, both in arcA? and wt mice of both age groups.
This finding is consistent with earlier reports in APP/PS1 and
age-matched wt mice (El Tannir El Tayara et al., 2010) and indi-
cates that the flow voids observed are neither linked to the A?
pathology nor the genetic trait. Instead, their occurrence might
be attributed to susceptibility effects caused by the proximity of
the tympanic bulba to the petrygopalatine artery (El Tannir El
Tayara et al., 2010). Hence, in TOF-MRA the detectability of a
vessel may be compromised either by reduced blood flow rates
(Lin et al., 1997) or by local flow disturbances (Underwood and
Mohiaddin, 1993). The technique appears of limited value for
potential abnormalities in flow pattern.
Therefore, we exploited the use of CE-?MRA, which is inde-
pendent of blood flow rates (Haacke et al., 1994; Lin et al., 1997;
Bolan et al., 2006). As contrast agent we intravenously injected
Endorem, which has a biphasic clearance kinetic (Majumdar et
sition. Hence, contrast-agent filled blood vessels (both arteries and
By using CE-?MRA, blood vessels smaller than the nominal
image resolution can be detected, because the area affected by
extravascular dephasing caused by the susceptibility difference
between vessel and stationary tissue exceeds the vessel dimen-
sions (Ogawa and Lee, 1990). Thus, significant changes in signal
only a fraction of the voxel volume. We have used an algorithm
that counted the vessels in the differences images when they ful-
filled the criteria of Eq. 1. With this method, we found a signifi-
cantly reduced number of intracortical vessels (radii of 20–80
?m) in a mouse model of cerebral amyloidosis, indicating that
noninvasive CE-?MRA is sensitive in detecting vascular pathol-
ogy affecting the microvasculature. Yet, the resolution of the
method is not sufficient in resolving the status of capillaries with
a diameter of ?7–10 ?m. Information about capillary density
may be inferred using stochastic approaches such as vessel size
imaging (Tropre `s et al., 2001).
nificant reduction in the number of intracortical vessels in 24-
whereas there was no difference between transgenic and wt mice
of 4 month of age. For verification of these findings, we investi-
gated the density of intracortical vessels in corresponding brain
no differences in expression levels of vWF among all groups,
indicating that intracortical vessels in the 24-month-old arcA?
mice have not been lost. However, as the vessels of these mice
show some additional vWF immunoreactivity in nonendothelial
structures, we cannot rule out that this increase in vWF expres-
sion level has masked a potential decrease in immunoreactivity
due to vessel loss.
Vascular accumulation of A? has been associated with the
deposition of fibrin which leads to the formation of clots that are
al., 2007; Cortes-Canteli et al., 2010). Therefore, we used antifi-
brinogen immunohistochemistry to investigate whether the ves-
sels in the 24-month-old arcA? mice were functionally altered.
Aggregation of fibrin(ogen) was evident in cortical vessels of 24-
month-old arcA? mice, but not in 4-month-old arcA? mice and
in none of the age-matched wt controls. Some of the smaller
blood vessels showed signs of stenosis due to the fibrin(ogen)
noreactivity was observed in thrombus-like structures in cross-
sectioned blood vessels. As vWF displays also thrombogenic
functions (Ruggeri, 2007) this indicates that the vessels of aged
arA? are prone to thrombus formation.
The aggregation of fibrin(ogen) was always coincident with
the vascular deposition of A?, as indicated by the strong colocal-
devoid of fibrinogen. Furthermore, A?/fibrinogen deposition
seems to preferentially occur in vessels of a radius of 10–40 ?m.
Despite the fact that the absolute vessel diameter estimates of
vessel diameter differ by factor 2–4 between the two methods
(MRI and histology) due to tissue shrinkage during the histolog-
ical preparation (Ratering et al., 2011), these vessels correspond
to the same vessel category, which were found to be significantly
reduced in number in the CE-?MRA data. Together, these data
mice is due to a lack of perfusion of existing blood vessels as a
In summary, high resolution CE-?MRA revealed an age-
dependent reduction in the density of functional intracortical
vessels (radii of 20–80 ?m) in the arcA? mouse. This class of
and seems to be prone to the formation of fibrin clots leading
ultimately to vessel stenosis. These results suggest that the mech-
slowing the progression of A? pathology. Due to its noninvasive
ics of cerebrovascular remodeling in the course of the disease
els of AD and CAA.
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