Walberer et al. Experimental & Translational Stroke Medicine 2010, 2:22
http://www.etsmjournal.com/content/2/1/22
R E S E A R C H
Open Access
Dynamics of neuroinflammation in the
macrosphere model of arterio-arterial embolic
focal ischemia: an approximation to human
stroke patterns
Maureen Walberer1,2, Maria A Rueger1,2, Marie-Lune Simard1,2, Beata Emig1, Sebastian Jander4,
Gereon R Fink1,3*, Michael Schroeter1,2*
Abstract
Background: Neuroinflammation evolves as a multi-facetted response to focal cerebral ischemia. It involves
activation of resident glia cell populations, recruitment of blood-derived leucocytes as well as humoral responses.
Among these processes, phagocyte accumulation has been suggested to be a surrogate marker of
neuroinflammation. We previously assessed phagocyte accumulation in human stroke by MRI. We hypothesize that
phagocyte accumulation in the macrosphere model may resemble the temporal and spatial patterns observed in
human stroke.
Methods: In a rat model of permanent focal ischemia by embolisation of TiO2-spheres we assessed key features of
post-ischemic neuroinflammation by the means of histology, immunocytochemistry of glial activation and influx of
hematogeneous cells, and quantitative PCR of TNF-a, IL-1, IL-18, and iNOS mRNA.
Results: In the boundary zone of the infarct, a transition of ramified microglia into ameboid phagocytic microglia
was accompanied by an up-regulation of MHC class II on the cells after 3 days. By day 7, a hypercellular infiltrate
consisting of activated microglia and phagocytic cells formed a thick rim around the ischemic infarct core.
Interestingly, in the ischemic core microglia could only be observed at day 7. TNF-a was induced rapidly within
hours, IL-1b and iNOS peaked within days, and IL-18 later at around 1 week after ischemia.
Conclusions: The macrosphere model closely resembles the characteristical dynamics of postischemic
inflammation previously observed in human stroke. We therefore suggest that the macrosphere model is highly
appropriate for studying the pathophysiology of stroke in a translational approach from rodent to human.
Background
responses [2-4]. In the classical transient middle cerebral
Inflammation plays an important role in the cascade of
artery occlusion (tMCAO) model, hematogeneous cells
events following cerebral ischemia that may impact on
including polymorphonuclear neutrophils (PMN) and
the extent of tissue damage, infarct demarcation, tissue
macrophages rapidly infiltrate the ischemic region [5-7].
repair und functional recovery, and may hence act as a
Translating rodent research into the situation of
key target for therapeutic intervention [1,2].
human stroke, substantial progress has been made in
Animal research has characterized postischemic
visualizing aspects of postischemic inflammation in man.
inflammation as a multi-facetted response involving acti-
Starting with the first in vivo visualization of peripheral
vation of resident glia cells and recruitment of blood-
benzodiazepine receptor-expressing inflammatory cells
derived leucocytes as well as cascades of humoral
using Positron Emission Tomography (PET) and the
radiotracer [11C]PK11195 [8], postischemic inflammation
has been repeatedly characterized by PET [8-11], mag-
* Correspondence: g.r.fink@fz-juelich.de; michael.schroeter@uk-koeln.de
1Department of Neurology, University Hospital, Cologne, Germany
netic resonance imaging (MRI) [12], and cell specific
Full list of author information is available at the end of the article
© 2010 Walberer et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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contrast agents detected by MRI [13-15]. Early histo-
macrospheres (Ø 0.315-0.355 mm; BRACE, Alzenau,
pathological descriptions suggest a significant impact of
Germany). The macrospheres were advanced via ICA
permanent versus transient ischemia on the dynamics of
into the MCA by a slow injection of approximately 0.2
inflammation [6]. Additionally, in the classical tMCAO
ml saline (Figure 1A). In sham-operated animals, the
model, tissue damage and glia activation evolve in com-
same operation procedure was used without injection of
plex spatial and temporal dynamics that make it difficult
macrospheres (n = 4). Following operation, the animals
to interpret the results and to translate them into the
were transferred to their cages after they had fully
human situation [16,17]. Different dynamics of MRI sig-
recovered from anaesthesia. During the experiment all
natures in the tMCAO model and human stroke further
animals received an intensified care with subcutaneous
complicate a translational approach [15]. Accordingly, we
saline injections (5 ml 0.9%NaCl/day) and moistening of
searched for an experimental stroke model with particu-
food pellets. Animals in apparent clinical distress were
lar regard to the dynamics of postischemic inflammation
sacrificed, excluded from the study and replaced.
that resembles the human situation.
In the rat macrosphere model, permanent focal ische-
Histology and Immunocytochemistry
mia is induced by intra-arterial embolization of a
Groups of n = 3 animals each were allowed to survive for
defined number of TiO2 spheres into the middle cere-
24 hours, 3 days, and 7 days after MCA embolisation with
bral artery (MCA). With respect to parameters such as
TiO2 spheres before they were decapitated under deep
infarct development over time, final lesion size and clin-
anaesthesia with isoflurane. The brains were rapidly
ical outcome, this model is comparable to the estab-
removed, frozen in 2-methylbutane, and stored at -80°C
lished pMCAO (permanent MCAO) model using an
prior to further histological and immunocytochemical pro-
intraluminal thread [18,19]. However, in contrast to the
cessing. Adjacent serial coronal brain sections were cut at
permanent suture model, hypothalamic injury followed
500 μm intervals (slice thickness 10 μm) and stained with
by pathological hyperthermia is avoided in the macro-
hematoxylin and eosin (H&E) according to standard pro-
sphere model [20,21]. Moreover, macrosphere injection
tocols. Anti-NeuN (clone A60, dilution 1:1000, Millipore,
mimics arterio-arterial embolism of arteriosclerotic pla-
Billerica, USA, cat-# MAB377) was performed to recog-
que material as the leading etiology of human stroke,
nize the DNA-binding between neuron and the specific
while the thread occlusion model rather simulates a
protein NeuN, which is presented in most CNS/PNS neu-
thromboembolic event with subsequent thrombolysis
ronal cell types. For the identification of astrocytes, we
and large-vessel reperfusion [22]. Importantly, neuroin-
used mAb against Glial Fibrillary Acidic Protein (GFAP)
flammation has not been described in an arterio-arterial
(clone GFAP, dilution 1:1000, Millipore, Billerica, USA,
embolic stroke model to date.
cat-# MAB360) and Vimentin (VIM) (dilution 1:2000,
Hence, we investigated key features and dynamics of
Millipore, Billerica, USA, cat-# MAB3400). The mAb
postischemic inflammation in the macrosphere model
against the complement receptor 3/CD11b identified
and compared our findings to the human situation as
microglia/macrophages (clone OX42, dilution 1:1000,
revealed by previous PET and MRI Studies.
AbD Serotec, Oxford, UK, cat-# MCA275R). Microglia
activation was assessed by staining for MHC class II
Methods
(clone Ox6, dilution 1:400, AbD Serotec, Oxford, UK, cat-
Animals and Surgery
#MCA46G). Phagocytic cells were identified with mAb
All animal procedures were in accordance with the Ger-
ED1 (clone ED1, dilution 1:1000, AbD Serotec, Oxford,
man Laws for Animal Protection and were approved by
UK, cat-# MCA341). A double staining with anti-Iba1
the local animal care committee and local governmental
(dilution 1:1000, Wako, Neuss, Germany, cat-# 019-
authorities. Male Wistar rats (n = 33) weighing 270-340
19741) and anti-NeuN was performed additionally. Iba1 is
g were anesthetized with 5% isoflurane and maintained
up-regulated upon activation of microglia allowing the dis-
with 2.5% isoflurane in 65%/35% nitrous oxide/oxygen.
crimination between resting and activated microglia. For
Throughout the surgical procedure body temperature
visualization, the ABC Elite kit (Vector Laboratories,
was maintained at 37.0°C with a thermostatically con-
Burlingame, CA, USA) with diaminobenzidine (Sigma,
trolled heating pad. Ischemia was produced by intra-
Munich, Germany) or Vector SG substrate kit for peroxi-
arterial injection of 4 TiO2 spheres into MCA as
dase (Vector laboratories, Burlingame, CA, USA.) for anti-
described elsewhere [19]. Briefly, after exposure of the
NeuN staining as the final reaction product was used.
left common carotid artery (CCA), internal carotid
To quantify phagocytic cells at day 7, the area of ED1-
artery (ICA) and external carotid artery (ECA), the ECA
positive cells was measured by the analysis software
and the pterygopalatine branch of the ICA were ligated.
Image J (1.40G, National Institutes of Health, USA) and
PE-50 tubing was filled with saline and four TiO2
related to the ipsilateral hemisphere.

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Walberer et al. Experimental & Translational Stroke Medicine 2010, 2:22
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A
B
C
Figure 1 Characterization of the macrosphere stroke model. (A) Basal view of the rat brain displaying the cerebral arteries following
macrosphere-injection into the MCA. One macrosphere is located in the origin of the MCA and the distal ICA (arrow). Bar: 5000 μm.
(B) Schematic drawing of the rat brain (dorsal view) to localize tissue samples collected from the ischemic lesion [A] and contralateral cortex [B].
(C) Representative H&E staining of coronal brain sections demonstrating the extent of the ischemic lesion 7 days after ischemia induction.
Objective 1×; bar: 5000 μm.

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Quantitative real-time polymerase chain reaction (PCR)
Infarct demarcation
For PCR analysis, another group of animals were sacri-
One day after induction of stroke, the infarction is already
ficed 4 hours (n = 5), 24 hours (n = 5), 3 days (n = 5),
well demarcated from vital tissue (Figure 2A, D, G). H&E
and 7 days (n = 5) after ischemia induction. In addition,
staining showed eosinophilic coagulation necrosis in the
4 sham-operated animals without infarct served as con-
ischemic territory (Figure 2A) and loss of GFAP staining
trols. For RNA isolation, a sample of the cortex within
indicated cell loss and edema within the infarct core
the infarct core (sample A) and contralateral hemisphere
(Figure 2D). The infarct border is even more clear-cut 3
(sample B) were prepared along with non-ischemic cor-
days after macrosphere injection (Figure 2B, E, H) and the
tex from control brains (Figure 1B). Approximately 30
surrounding tissue appears to be slightly hypercellular.
to 50 mg tissue per tissue sample (wet weight) were
Around day 7, a sharp infarct demarcation with a sur-
obtained. Total RNA was prepared using the Trizol
rounding hypercellular infiltrate containing reactive astro-
reagent (Gibco BRL, Gaithersburg, MD) and quantified
cytes (revealed by GFAP) and phagocytic cells containing
spectrophotometrically. One microgramm RNA was
transformed microglia and hematogeneous macrophages
reversely transcribed using oligo (dT)19 (G/A/C) primers
(identified by ED1) developed (Figure 2C, F, I). The
and Superscript II reverse transcriptase (Gibco-BRL,
dynamics of infarct demarcation and astrogliosis did not
Gaithersburg, MD) according to manufacturer’s proto-
differ between subcortical and cortical regions at the
col. Quantitative assessment of TNF-a, IL-1b, IL-18 and
observation time-points, giving further evidence to the
iNOS gene expression levels was performed using a
notion that the infarcts developed promptly and in a syn-
7900HT Fast Real-Time PCR System (Applied Biosys-
chronous way in basal ganglia and cortical areas.
tems, Weiterstadt, Germany) and the Sybr Green
The mean ischemic lesion volume of all animals was
Universal Master Mix (Applied Biosystems) and gene-
161 mm3 ± 90.
specific primer pairs as described previously [23,24]. In
The contralateral hemisphere did not show any
all PCR analyses, glyceraldehyde 3-phosphate dehydro-
changes in immunoreactivity at any time point (Figure 2).
genase (GAPDH) [24] was used as the reference gene
since it exhibited constant expression levels under all
Cellular inflammatory response
tested conditions. Relative gene expression levels were
In the infarct core morphological signs of microglial
determined according to the manufacturer’s ΔΔCt
activation could be shown as early as 24 h after induc-
method. For quantification of PCR data, mRNA levels of
tion of focal ischemia by more intense Ox42-staining.
the ipsilateral hemisphere were compared to levels of
the contralateral hemisphere as well as to values of con-
trol animals.
day 1
day 3
day 7
Statistical analysis
H
The statistical analysis was performed with SigmaPlot 11
&
(Systat Software Inc, California, USA). Data are pre-
E
sented as mean ± SD. For the analysis of PCR results,
A
B
C
two-way analysis of variance (ANOVA) with a post hoc
G
test of Holm-Sidak was used. Statistical significance was
F
set at the less than 5% level (p < 0.05).
A
P
D
E
F
Results
Characterization of the macrosphere model
E
Four TiO
D
2 spheres were injected into the ICA after liga-
tion of the pterygopalatine artery, leading to a perma-
1
nent occlusion within the ICA/MCA vessel arborisation.
G
H
I
In all animals, one or more macrospheres directly
Figure 2 Infarct demarcation and cellular response. H&E (A-C),
blocked the proximal MCA (Figure 1A). H&E staining
GFAP (D-F) and ED1 (G-I) staining 24 h, 3 d, and 7 d after infarct
induction. The infarct is increasingly demarcated over time by
verified the infarct localization in the MCA territory of
reactive astrocytes that accumulate in the vital tissue bordering the
all animals (Figure 1C).
infarct. Scars circumscribe the infarcted tissue but GFAP-positive
All animals showed circling behavior 24 hours after
cells do not enter the infarct zone up to day 7. Phagocytic cells
infarct induction but recovered incompletely till
including transformed microglia and hematogeneous macrophages
72 hours. Animals in clinical distress were sacrificed,
accumulate at the infarct border in the necrotic tissue. Objective:
1×; bar: 200 μm.
excluded from the study and replaced (n = 4).

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Some amoeboid microglia with rounded cell bodies were
also present (Figure 3A, D, G). By day 3, microglia cells
H&E
N
N
eu
started to form a hypercellular rim around the infarct
core, exhibiting signs of activation by ED1-staining as an
indication for phagocytosis. MHC class II-positive cells
with stellate morphology were more numerous and
extended further into vital tissue compared to ED1-posi-
A
B
tive cells. (Figure 3B, E, H). At day 7, a dense cellular
ED1
Iba1/
wall of microglial cells surrounded the ischemic lesion
N
N
eu
(Figure 3C, F, I). In the core of the lesion, some - most
likely avital - cells exhibited a granular Ox42 immuno-
signal. There was abundant expression of ED1 and
MHC class II-positive cells constituting a rim around
VIM
the infarct border, with more scattered cells positive for
C
D
both markers at the infarct core. At day 7, the area of
GFAP
VIM
ED1-positive cells (54 mm3 ± 26) extended over 9 to
20% of the affected hemisphere.
The contralateral hemisphere did not show changes in
immunoreactivity at any time point.
E
F
Cellular interactions in the infarct demarcation zone
7 days after MCAO, eosinophilic coagulation necrosis
Figure 4 Cellular interaction in the infarct demarcation zone.
Infarct demarcation zone at day 7 after infarct induction (left:
(Figure 4A) and neuronal degeneration (Figure 4B)
healthy tissue; right: infarct zone) with different stainings: H&E (A),
could be observed in the infarct core. ED1+ phagocytes
NeuN (B), ED1 (C), anti NeuN antibody and anti-Iba1 antibody (D),
(Figure 4C) and activated microglia (Figure 4D, brown)
GFAP (E) and VIM (F). The infarct zone was clearly demarcated (A-F)
accumulated at the infarct border and interacted with
Eosinophilic coagulation (A) and neuronal degeneration (B) occurred
neurons (Figure 4D, red arrows). Astrocytic activation
at the infarct. ED1+ phagocytes (C) and activated microglia (D,
brown) are visible within the infarct. Interactions between neurons
(protoplasmic astrocytes) was clearly visible at the bor-
and activated microglia occurred (D, red arrows). GFAP-positive
der of the infarct, but not in the infarct core. A demar-
astrocytes (E) demarcate the lesion forming an astrocytic scar in the
cation of a scar-like formation could be observed at the
vital tissue adjacent to the infarct border. Vimentin
periphery of the infarct core (Figure 4E, F) and vimentin
immunoreactivity (F) depicts proliferating astrocytes within the scar
(black arrow) as well as other cell types within the infarct (green
arrow). Objective: 40×; bar: 50 μm.
day 1
day 3
day 7
immunoreactivity depicted immature astrocytes indicat-
E
ing proliferation of astrocytes.
D
1
Cytokine response
A
B
C
The proinflammatory cytokines TNF-a, IL-1b, iNOS
and IL-18 were quantified in the infarct core and homo-
O
topic contralateral regions over time (Figure 1B). Perma-
x
4
nent MCAO induced the early upregulation of TNF-a
2
D
E
F
mRNA within the first 4 hours after ischemia. TNF-a
stayed upregulated at significant levels for the entire
time period studied (Figure 5A). IL-1
O
b and iNOS were
x
upregulated at a slower rate and peaked 72 hours after
6
onset of ischemia (Figure 5B, C). IL-18 expression was
G
H
I
detectable at low levels within the first days and peaked
Figure 3 Cellular inflammatory response at the infarct border.
at 7 days after stroke (Figure 5D).
Phagocytic cells (ED1, A-C), microglia (Ox42, D-F) and activated
microglia (Ox6, G-I) are responding to focal cerebral ischemia. Over
Discussion
the course of one week after induction of ischemia, those cells
In the macrosphere model, the intra-arterial injection of
accumulated at the infarct border. Objective: 10×; bar: 100 μm.
4 TiO2 spheres leads to permanent occlusion of the

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A-infarct
A-infarct
80 TNF-
B-contralateral
30
IL1-
B-contralateral
++
++
**
25
**
v
el 60
v
el

le
l
e 20
A
+
+
N
**
NA
40
15
R
R
+
. m
.
m
l
l 10
20
re
re
5
++
+
+
+
++
0
0
ctr
4h
24h
72h
168h
168
ctr
4h
24h
24
72h
72
168h
16
A
time after ischemia
B
time after ischemia
A-infarct
iNOS
A-infarct
IL-18
A-infarct
80
B-contralateral
80
B-contralateral
++
++
60
e
v
e
l
**
v
e
l
**
+
60
*
le
NA l
A
40
R
N 40
R
20
r
e
l. m
20
+
r
e
l. m
++
0
+
+
++
0
ctr
4h
24h
72h
168h
ctr
4h
24h
72h
168h
time after ischemia
C
time after ischemia
D
Figure 5 Cytokine response. Quantitative real-time polymerase chain reaction (PCR) analyses of TNF-a (A), IL-1b (B), iNOS (C) and IL-18 (D)
mRNA levels shows upregulation of all cytokines within specific time windows. A rapid strong induction of TNF-a peaking at 4 h was
accompanied by a lower response of IL-1b and iNOS with a maximum at 72 h. IL-18 was induced very late around day 7. At each time point
after onset of infarct, n = 5 animals were studied, whereas 4 animals served as control. Bars represent mean ± SD; **p < 0.01; *p < 0.05
(compared to control animals); ++p < 0.01; +p < 0.05 (infarcted hemisphere compared to contralateral hemisphere).
MCA resulting in an ischemic lesion of the MCA-terri-
superparamagnetic iron oxide contrast agent (USPIO)
tory [20]. The interindividual variability of infarct
[14]. At 3-4 days after stroke onset, we detected subtle
volume caused by different occlusion sites of the macro-
contrast enhancement in only a minority of patients.
spheres is similar to other embolic stroke models. This
However, 6-8 days after onset of ischemia, we con-
technique mimics arterio-arterial embolism of “hard”
sistently found signs of macrophage infiltration in
atherosclerotic plaque material, the most frequent cause
subcortical as well as cortical areas. Macrophage infil-
of stroke in humans [22,25]. Moreover, in contrast to
tration heterogeneously affected subregions of the
the well-established suture model of permanent ische-
infarct with significant inter-individual differences.
mia, the macrosphere model avoids hypothalamic injury
This is in accordance with the dynamics of cellular
and subsequently hyperthermia [18,20,21], while
responses after permanent focal ischemia in primates
hypothalamic infarction is extremely rare in patients.
reported earlier [26]. Going back from “bedside to
Hyperthermia may be a confounding factor in therapeu-
bench”, two essential features of cellular responses in
tic studies and may influence CNS inflammatory
the macrosphere model are reminiscent of the human
responses as well. In those regards, the macrosphere
situation: (i) the slowly evolving macrophage infiltra-
model offers clear advantages compared to the com-
tion at the end of the first post-infarct week, and (ii)
monly used transient and permanent suture models, and
the heterogeneous distribution of the macrophage infil-
has therefore been suggested to represent a clinically
trate throughout the infarct.
relevant model to study the pathophysiology of stroke.
In rodent models of tMCAO, the temporal evolution
In a proof-of-principles study we previously visua-
of tissue damage has also been shown to be heteroge-
lized macrophage infiltration in human stroke by a
neously, extending in distinct patterns on a subcortical

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to cortical axis, which renders inflammatory responses
microglia activation could be detected by day 7, but not
complex and difficult to assess [16,17]. Reperfusion
at day 1, similar to the photothrombosis model
accelerates inflammation, facilitating the rapid infiltra-
[17,30,32,33]. In human PET studies using the periph-
tion of hematogeneous cells, to an early and diffuse
eral benzodiazepine ligand [11C]PK11195 as a surrogate
accumulation of PMNs throughout the infarct [5-7,27].
marker for inflammation to investigate the temporo-
In contrast, rodent models of permanent MCAO
spatial profile of microglia-activation and macrophage-
show rapid signs of tissue damage evolving in the
invasion [34-36] and MRI studies using ultrasmall
infarct core and succumbing to necrosis, while more
supermagnetic iron oxide (USPIO) to study the invasion
peripheral areas display a greater variability in the
of blood-borne macrophages into human brain [12,37],
severity of ischemia. After pMCAO, peripheral areas
microglia activation in human stroke have been detected
are usually associated with incomplete necrosis, while
to start as late as 3 days after onset of ischemia and
subcortical regions show only mild ischemic neuronal
reached its maximum within one week [12,34-37]. Inter-
damage without loss of neurons [28]. In a rat model
estingly, in the macrosphere model activated microglia
of pMCAO, direct surgical occlusion of the MCA led
and macrophages established a particularly dense cellu-
to an accumulation of PMN leukocytes with a maxi-
lar wall around the infarct core after 7 days, whereas
mum at 48-72 hours [16], which is just as late as the
reactive astrocytes had formed a ring already by day 3,
PMN infiltration seen after human stroke [7]. In a
similar to the situation observed in the photothrombosis
monkey model of pMCAO, PMNs within the reactive
model [29,31]. The time-course of cytokine release in
zone could be observed in modest numbers at
the macrosphere model differed slightly from the situa-
18 hours, and increased to 72 hours, while seven-day
tion in the photothrombosis model. While we found a
old lesion showed PMNs in the central infarcted zone
rapid induction of TNF-a similar to photothrombosis, a
only [16]. By comparing permanent and transient
much later upregulation of IL-1b and iNOS occurred
MCAO in spontaneously hypertensive rats, the infil-
peaking at day 3. TNF-a and IL-1b are synthesized by
tration of neutrophils was moderate in pMCAO.
PMNs [38] and therefore PMN accumulation may con-
Furthermore, the activity of the neutrophil marker
tribute to TNF-a and IL-1b production-besides resident
myeloperoxidase was 2 to 3-fold increased, depending
microglia as a principle source-peaking at 72 hours. The
of the reperfusion time in tMCAO compared to
induction of IL-18 occurred even later and increased up
pMCAO within the first day [27]. Those previous stu-
to day 7. This late induction of IL-18 is in good accor-
dies suggest that pMCAO models reflect the dynamics
dance with the situation previously observed in the
of human postischemic inflammation much better
photothrombosis model, where the maximum upregula-
than tMCAO models do.
tion was found at day 14, a time point not included in
In this study, the extent of ischemic damage obtained
our present study [23].
by macrosphere embolization corresponded to previous
By comparing macrosphere model, human stroke and
experiments and was comparable to other pMCAO
tMCAO, key features of post-ischemic neuroinflamma-
models [18-21]. In contrast, our pMCAO model was dif-
tion, e.g. microglia activation, macrophages infiltration
ferent from the previously described photochemically
throughout the infarct and phagocytic accumulation,
induced focal ischemia model that produces only a pure
showed a similar temporal appearance in the macro-
neocortical infarction [29,30]. While the infarcted area
sphere model in rats and human stroke, whereas
underwent necrosis, the surrounding tissue displayed an
tMCAO in rats leads to a rapid development of inflam-
increasing astrocyte reactivity over time as revealed by
mation (Table 1).
GFAP-staining, a phenomenon that was similar to
photothrombosis [30,31]. With respect to the cellular
Conclusions
inflammatory response, we observed a transition of
The macrosphere model as a model of focal cerebral
ramified into ameboid phagocytic microglia in the
ischemia resembles closely the dynamics of human
boundary zone of the infarct, with consecutive infarct
postischemic inflammation, imitating particularly the
demarcation as late as three days after the induction of
slow time course of human neuroinflammation. There-
ischemia, corresponding to the situation after photo-
fore we suggest that the macrosphere model mimics
thrombosis [29] as well as to that after human stroke
the clinical situation of human stroke better than the
[4]. Stoll et al. observed the accumulation of phagocytes
commonly used model of transient middle cerebral
in the border zone of human infarcts after 5 days [4].
artery occlusion. Accordingly the rodent macrosphere
This combined microglia/macrophage response was
model is regarded most relevant for studying the
accompanied by an up-regulation of MHC class II mole-
pathophysiology of stroke and possesses high clinical
cules. In the ischemic core, morphological signs of
relevance.

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Table 1 Cytokine response
Macrosphere model
Human stroke
tMCAO
Activation of microglia (maximum)
day 3 (day 7)
day 3 (day 7)37
6 h (day 3)17
Transformation from ramified to
from day 3 on
days?
22 h17
ameboid microglia in the infarct
border
Infiltration of macrophages/
day 7
day 6- day 814
6 h17
microglia in the infarct core
Spatial patterns of macrophages/
“wall-like” accumulation in the
“wall-like” accumulation in the
heterogeneous, extending in distinct
microglia accumulation
outer infarct, sparing the infarct
outer infarct, sparing the infarct
patterns on a subcortical to cortical
core
core14
axis17
Accumulation of phagozytes
day 7
day 5-84
day 317
Time-points of various signs of inflammation after focal cerebral ischemia in macrosphere model, human stroke and tMCAO.
Acknowledgements
occlusion and reperfusion in primates monitored by microdialysis and
GRF was supported by a grant from the Federal Ministry of Education and
sequential positron emission tomography. Stroke 2001, 32:1574-1580.
Research, Germany: “Brain Imaging Center West” (No. 01 GO 0517).
10.
Sette G, Baron JC, Young AR, Miyazawa H, Tillet I, Barré L, Travère JM,
We thank Birgit Blomenkamp for expert technical assistance for the PCR
Derlon JM, MacKenzie ET: In vivo mapping of brain benzodiazepine
experiments.
receptor changes by positron emission tomography after focal ischemia
in the anesthetized baboon. Stroke 1993, 24:2046-2057.
Author details
11.
Schroeter M, Dennin MA, Walberer M, Backes H, Neumaier B, Fink GR,
1Department of Neurology, University Hospital, Cologne, Germany. 2Max-
Graf R: Neuroinflammation extends brain tissue at risk to vital peri-
Planck-Institute for Neurological Research, Cologne, Germany. 3Institute of
infarct tissue: a double tracer [(11)C]PK11195- and [(18)F]FDG-PET study.
Neuroscience and Medicine (INM-3), Cognitive Neurology Section, Research
J Cereb Blood Flow 2009, 29:1216-1225.
Centre Juelich, Germany. 4Department of Neurology, Heinrich-Heine-
12.
Schroeter M, Franke C, Stoll G, Hoehn M: Dynamic changes of magnetic
University, Düsseldorf, Germany.
resonance imaging abnormalities in relation to inflammation and glial
responses after photothrombotic cerebral infarction in the rat brain.
Authors’ contributions
Acta Neuropathol 2001, 101:114-122.
MW made the conception and design of the study, carried out animals’
13.
Breckwoldt MO, Chen JW, Stangenberg L, Aikawa E, Rodriguez E, Qiu S,
surgery, participated in the acquisition of histological and PCR-data,
Moskowitz MA, Weissleder R: Tracking the inflammatory response in
performed statistical analysis of data and drafted the manuscript. MAD
stroke in vivo by sensing the enzyme myeloperoxidase. Proc Natl Acad
participated in the acquisition and interpretation of histological data and
Sci USA 2008, 105:18584-18589.
helped to draft the manuscript. MLS Simard participated in the acquisition
14.
Jander S, Schroeter M, Saleh A: Imaging inflammation in acute brain
of histological data. SJ participated in analysis and interpretation of PCR-
ischemia. Stroke 2007, 38:642-645.
data. GRF revised the manuscript and has given final approval of the version
15.
Jiang Q, Chopp M, Zhang ZG, Knight RA, Jacobs M, Windham JP, Peck D,
to be published. MS conceived the study, helped with the interpretation of
Ewing JR, Welch KM: The temporal evolution of MRI tissue signatures
histological data and revised the manuscript critically for important
after transient middle cerebral artery occlusion in rat. J Neurol Sci 1997,
intellectual content. All authors read and approved the final manuscript.
145:15-23.
16.
Garcia JH, Kamijyo Y: Cerebral infarction. Evolution of histopathological
Competing interests
changes after occlusion of a middle cerebral artery in primates. J
The authors declare that they have no competing interests.
Neuropathol Exp Neurol 1974, 33:408-421.
17.
Lehrmann E, Christensen T, Zimmer J, Diemer NH, Finsen B: Microglial and
Received: 27 July 2010 Accepted: 20 December 2010
macrophage reactions mark progressive changes and define the
Published: 20 December 2010
penumbra in the rat neocortex and striatum after transient middle
cerebral artery occlusion. J Comp Neurol 1997, 386:461-476.
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doi:10.1186/2040-7378-2-22
Cite this article as: Walberer et al.: Dynamics of neuroinflammation in
the macrosphere model of arterio-arterial embolic focal ischemia: an
approximation to human stroke patterns. Experimental & Translational
Stroke Medicine 2010 2:22.
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