Lack of secondary microthrombosis after
Journal of Thrombosis and Haemostasis, 12: 409–414
DOI: 10.1111/jth.12487
BRIEF REPORT
Lack of secondary microthrombosis after thrombin-induced
stroke in mice and non-human primates
M . G A U B E R T I , S . M A R T I N E Z D E L I Z A R R O N D O , C . O R S E T and D . V I V I E N
Inserm UMR-S U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, Inserm, Universite Caen Basse-Normandie, GIP
Cyceron, Caen, France
To cite this article: Gauberti M, Martinez de Lizarrondo S, Orset C, Vivien D. Lack of secondary microthrombosis after thrombin-induced
stroke in mice and non-human primates. J Thromb Haemost 2014; 12: 409–14.
Summary. Background: Secondary microthrombosis is a
major pathophysiologic mechanism leading to brain damage following transient mechanical vascular occlusion
(TMVO), the most widely used experimental stroke
model. Whether secondary microthrombosis also occurs
in non-TMVO stroke models represents an important
issue for clinical translation of antimicrothrombosis therapeutic strategies. Objectives: To assess the occurrence and
the pathogenic role of secondary microthrombosis in two
thrombin-induced stroke models in mice and non-human
primates (Macaca mulatta). Methods: Stroke was induced
in mice and non-human primates by intra-arterial administration of recombinant thrombin. This method induces
the formation of a fibrin-rich thrombus, which is spontaneously dissolved in the following hours by the endogenous fibrinolytic system. Perfusion-weighted imaging and
fluorescent-lectin microangiography were performed after
recanalization to detect secondary microthrombosis.
Moreover, to investigate its pathogenic role, thrombininduced stroke was induced in bradykinin receptor B1
(B1R) knockout mice, which are protected from the
thromboinflammation responsible for secondary microthrombosis in TMVO models. Results: Reperfusion was
stable and complete in all mice and non-human primates
tested, revealing no secondary decrease in cerebral blood
flow. No evidence of secondary microthrombosis was
found in the two models. Accordingly, deficiency in B1R
did not protect the mice from brain damage after thrombin-induced stroke. Conclusions: Our data demonstrate
that secondary microthrombosis does not occur after
thrombin-induced stroke. In view of this, the pathophysiologic roles of hematologic players promoting or protectCorrespondence: Denis Vivien, Inserm UMR-S U919, GIP Cyceron,
Bd H. Becquerel, 14074 Caen, France.
Tel.: +33 2 31 47 01 60; fax: +33 2 31 47 02 22.
E-mail: [email protected]
Received 28 October 2013
Manuscript handled by: M. Levi
Final decision: P. H. Reitsma, 29 November 2013
© 2013 International Society on Thrombosis and Haemostasis
ing against secondary microthrombosis (such as
factor XII, von Willebrand factor, and T cells) deserve to
be re-evaluated in non-TMVO stroke models.
Keywords: bradykinin; coagulation; inflammation; ischemia;
stroke; thrombosis.
Introduction
Numerous recent studies have demonstrated the involvement of secondary microthrombosis in the development
of cerebral ischemic lesions following transient mechanical
vascular occlusion (TMVO) in mice. Notably, inhibition
of microthrombosis with different strategies (including
factor XII inhibition [1], glycoprotein [GP]Iba blockade
[2], regulatory T-cell depletion [3], and kininogen/bradykinin blockade [4,5]) reduces ischemic lesion size by ~ 70%
in TMVO models, suggesting that secondary microthrombosis may constitute a promising therapeutic target for
acute stroke treatment.
Secondary microthrombosis occurs within minutes after
reperfusion in TMVO models, once the occluding filament has been removed. It induces delayed cerebral blood
flow (CBF) reduction, secondary ischemia, and lesion
growth, which is responsible for ~ 70% of the final ischemic lesion size [1]. These findings led to the concept of
stroke being a thromboinflammatory disorder [6]. However, clinical studies have demonstrated that secondary
ischemia and lesion growth are modest or non-existent
after reperfusion [7–9]. Moreover, serious concerns have
been raised about the clinical relevance of TMVO models
[10]. Therefore, whether secondary microthrombosis also
occurs in non-TMVO models represents a critical issue
for clinical translation of antimicrothrombosis strategies.
To address this question, we first investigated whether
microthrombosis occurs in other recently reported models
of acute ischemic stroke, induced by intra-arterial injection of recombinant thrombin in mice and non-human
primates [11,12]. Then, we studied the impact of
thromboinflammation blockade, by genetic deletion or
410 M. Gauberti et al
pharmacologic blockade of the bradykinin receptor B1
(B1R), on stroke outcome in the mouse model.
Material and methods
Mice
Male Swiss mice (28 animals weighing 30–35 g) were purchased from the CURB (Caen, France). Five male B1Rdeficient mice (B1R / , aged 8 weeks) from a C57BL6/J
background [13] were provided by INSERM U1048,
housed at the CURB, and compared with five male
C57BL6/J mice (aged 8 weeks; CURB). Mice were housed
with a 12-h light–dark cycle with free access to food and
water. We performed in situ thromboembolic occlusion in
anesthetized mice (2% isoflurane in 33 : 67 O2/N2O
[33% : 67%] with the rectal temperature maintained at
37 °C) by intra-arterial injection of thrombin, as previously
described [11]. The operator performing surgery was unaware of the genetic background and treatment group of the
mice. Pharmacologic inhibition of B1R was performed by
intravenous injection of R-715 (1 mg kg 1; Tocris, Bristol,
UK) 20 min after stroke onset [5].
Non-human primates
Experiments were performed in five male rhesus macaques
(Macaca mulatta) aged 5–6 years and with body weights
ranging from 7 kg to 11 kg. An experimental protocol was
submitted to the Regional Ethics Committee for Animal
Experimentation (Normandy), and approval was granted
to conduct the study (referral No. N/02-03-08/03/02-11).
Experiments were performed in accordance with French
ethical laws (act No. 87-848; Ministere de l’Agriculture et
de la For^et) and European Communities Council Directives (86/609/EEC and 2010/63/EU) guidelines for the care
and use of laboratory animals. We performed in situ
thromboembolic occlusion in anesthetized non-human primates as previously described [12].
Magnetic resonance imaging (MRI) analysis
Experiments in mice were carried out on a Pharmascan 7
T/12-cm system with surface coils (Br€
uker, Wissembourg,
France). T2-weighted images were acquired by use of a
multislice multiecho sequence: echo time (TE)/repetition
time (TR), 51/2500 ms; and spatial resolution,
70 9 70 9 500 lm3 [14]. Lesion sizes were quantified on
T2-weighted images with IMAGEJ software (v1.45r; National
Institute of Health, USA). For perfusion-weighted imaging, a gradient-echo fast imaging with steady-state precession sequence was used, with the following parameters:
TR/TE, 10/5 ms (Flip Angle = 8°); temporal resolution,
911 ms; and spatial resolution, 100 9 100 9 75 lm3 (halfscan). Mice were injected with 30 lL of a 0.5 M solution of
Dotarem (Guerbet, Aulnay-sous-Bois, France) during
repetitive image acquisitions. Then, DR2* images were generated with an in-house-created macro in IMAGEJ. CBF was
estimated by measurement of the ratio of ipsilateral and
contralateral DR2*, as described previously [15]. Experiments in non-human primates were performed as previously described [12]. All quantifications were performed
blind to the experimental data.
Fluorescein isothiocyanate (FITC)–lectin microangiography
Twenty-four hours after thrombin-induced middle cerebral artery (MCA) occlusion, mice were injected intravenously with FITC-conjugated lectin [16] (100 lg per
mouse). Two minutes after injection, mice were killed by
transcardiac perfusion with cold heparinized saline followed by paraformaldehyde and picric acid. Brains were
postfixed and cryoprotected before being frozen in Tissue-Tek. Cryostat sections (10 lm) were collected on
polylysine-coated slides before being analyzed with fluorescence microscopy. Sections were coincubated overnight
with goat anti-collagen type IV (1 : 1500; Southern
Biotech, Birmingham, AL, USA) or chicken anti-microtubule-associated protein 2 (1 : 8000; Abcam, Paris,
France). Primary antibodies were revealed with Fab′2
fragments of donkey anti-goat or anti-chicken IgG linked
to tetramethylrhodamine isothiocyanate (1 : 500; Jackson
ImmunoResearch, West Grove, PA, USA). Washed sections were coverslipped with antifade medium containing
4′,6′-diamidino-2-phenylindole, and images were digitally
captured with a Leica DM6000 microscope-coupled coolsnap camera and visualized with METAVUE 5.0 software
(Molecular Devices, Sunnyvale, CA, USA). All quantifications were performed blind to the experimental data.
Results and Discussion
We induced ischemic stroke in mice and in non-human
primates (M. mulatta) by in situ intra-arterial administration of thrombin, as previously described [11,12]. This
method induces the formation of a fibrin-rich thrombus
in the MCA, which is spontaneously dissolved in the following hours by the endogenous fibrinolytic system [17],
mimicking human pathology [18]. In these two models,
we measured CBF before and after arterial recanalization
with a bolus tracking method, using 7-T (mice) and 3-T
MRI (non-human primates). Our approach was similar to
the method previously performed by others to demonstrate secondary CBF reduction in the murine TMVO
model [1]. In our hands, after thrombin-induced thromboembolic stroke, reperfusion was stable and complete in all
mice (Fig. 1) and non-human primates (Fig. 2) tested,
revealing no secondary decrease in CBF in these conditions. Accordingly, microangiography with fluorescent
lectin (100 lg per mouse; Sigma-Aldrich, L’Isle-d’Abeau
France) failed to reveal any secondary obstructed microvessels 24 h after recanalization in mice (Fig. 1C,D),
© 2013 International Society on Thrombosis and Haemostasis
Lack of secondary microthrombosis 411
DWI
A
CBF
B
∆TTP
125
100 (AU)
25 s
0
+24 h
0
CBF (% of
contralateral side)
+20 min
*
75
50
25
0
20 min
0
Contra
D
FITC-lectin/Collagen IV/DAPI
50 µm
FITC-lectin/MAP-2/DAPI
100
50
*
0
Contralateral
50 µm
Ipsi
Ipsilateral
FITC-lectin-positive
vessels (% of total vessels)
FITC-lectin/Collagen IV/DAPI
+24 h
Ipsi
24 h
25 s
0
FITC-lectin-positive
vessels (% of total vessels)
FITC-lectin/Collagen IV/DAPI
100 (AU)
+20 min
C
100
100 µm
Ipsi
100 µm
100
50
0
Contralateral
Ipsilateral
Fig. 1. Lack of secondary cerebral blood flow (CBF) reduction after thrombin-induced stroke and reperfusion in mice. (A) Representative magnetic resonance images (7 T) of Swiss mice 20 min (top) and 24 h (bottom) after intra-arterial thrombin injection. The resulting ischemic lesion
is visualized on diffusion-weighted images (left, yellow arrow). In this model, CBF and time to peak (TTP) are altered 20 min after arterial
occlusion (red arrows), but are fully restored once the thrombus is dissolved by endogenous fibrinolysis. (B) Quantification of CBF, demonstrating full restoration after arterial recanalization (n = 5). (C) Representative fluorescent-lectin microangiographies demonstrating the lack of
perfusion in the ipsilateral middle cerebral artery territory 20 min after stroke onset. In contrast, 24 h after stroke onset, all the vessels are
reperfused (the images presented are representative of four animals per group). Microtubule-associated protein 2 (MAP-2) was used as a marker of neuronal viability to visualize the ischemic lesion (white dotted line). (D) Corresponding quantification of the percentage of fluorescein
isothiocyanate (FITC)–lectin-positive vessels in the ipsilateral (ischemic) and contralateral sides 20 min and 24 h after stroke onset. P < 0.05
was considered to indicate statistical significance. Data are presented as mean standard error of the mean. AU, arbitrary units; DAPI, 4′,6′diamidino-2-phenylindole; DWI, diffusion weighted imaging.
whereas 78% of the microvessels are occluded in TMVO
models at this time point [2]. These results demonstrated
that secondary microthrombosis does not occur after
thrombin-induced stroke.
We then wanted to investigate whether differences in
microthrombosis have therapeutic implications. As genetic
© 2013 International Society on Thrombosis and Haemostasis
deletion of B1R prevents the thromboinflammatory
reaction responsible for microthrombosis in TMVO
models [4–6], we investigated whether B1R / mice [13]
are protected from stroke in a thromboembolic model.
We chose to block B1R because it is not involved in the
initial thrombin-induced clot formation, in contrast to,
412 M. Gauberti et al
A
B
+24 h
+2 h
125
CBF
∆TTP
+2 h
DWI
CBF (% of contralateral side)
*
0
100 (AU)
0
15 s
100
75
50
25
+24 h
0
2h
0
100 (AU)
0
24 h
15 s
Fig. 2. Lack of secondary cerebral blood flow (CBF) reduction after thrombin-induced stroke and reperfusion in non-human primates. (A)
Representative magnetic resonance angiograms, demonstrating spontaneous recanalization 24 h after stroke onset in non-human primates subjected to thrombin-induced middle cerebral artery occlusion (top, yellow arrow). Representative magnetic resonance image (3 T) of non-human
primates 2 h and 24 h after intra-arterial thrombin injection, showing a cortical ischemic lesion (yellow arrows) and demonstrating full recovery
of CBF after spontaneous arterial recanalization (red arrows, n = 5). (B) Corresponding quantification of CBF. P < 0.05 was considered to
indicate statistical significance. Data are presented as mean standard error of the mean. AU, arbitrary units; DWI, diffusion weighted
imaging; TTP, time to peak.
C
R-715
B1R–/–
WT
Saline
A
B
D
NS
20
30
Lesion size (mm3)
Lesion size (mm3)
25
15
10
5
NS
25
20
15
10
5
0
0
WT
B1R–/–
Saline
R-715
Fig. 3. Bradykinin receptor B1 (B1R)-deficient mice are not protected from thrombin-induced ischemic stroke. (A) Representative T2-weighted
magnetic resonance image of wild-type (WT) and B1R / mice. (B) Quantification of the lesion sizes (n = 5) demonstrated the lack of protection of B1R / mice. P < 0.05 was considered to indicate statistical significance. (C) Representative T2-weighted magnetic resonance image of
Swiss mice treated with saline and R-715 (1 mg kg 1, a peptidic B1R antagonist) 20 min after stroke onset. (D) Quantification of the lesion
sizes (n = 5) demonstrated the lack of protection of R-715-treated mice. Data are presented as mean standard error of the mean. NS, not
significant.
© 2013 International Society on Thrombosis and Haemostasis
Lack of secondary microthrombosis 413
for example, FXII or platelet GPIba. We demonstrated
that wild-type and B1R / mice show similar ischemic
lesion sizes 24 h after stroke (Fig. 3A,B). Similarly, B1R
antagonist (R-715, 1 mg kg 1, 20 min after stroke
induction) failed to influence ischemic lesion size
(Fig. 3C,D).
Altogether, these data provide evidence that microthrombosis does not occur after thrombin-induced stroke
in either mice or non-human primates (Fig. 4). Accordingly, blockade of the thromboinflammatory reaction by
either genetic deletion or pharmacologic inhibition of
B1R failed to protect against stroke in the mouse model.
As blockade of the kininogen–bradykinin axis is also not
beneficial in a permanent ischemic stroke model [4], the
available data suggest that thromboinflammation blockade through B1R inhibition is ineffective in permanent
and thrombin-induced transient ischemic strokes, thus
questioning the potential impact of such therapeutic strategy in stroke patients.
In view of this, the pathophysiologic roles of hematologic players promoting or protecting against microthrombosis (such as FXII, von Willebrand factor, platelet
GPIba, and T-cells) deserve to be re-evaluated in non-
TMVO stroke models. Nevertheless, secondary microthrombosis may represent a potential therapeutic target
in patients treated with endovascular procedures, as late
secondary injury occurs in 18% of them [19]. Interestingly, these patients show transient reversal of the cytotoxic edema after reperfusion, a specific feature of TMVO
models [10]. As stroke is a polyetiologic disease, our
results cannot be extrapolated to all stroke subtypes. In
particular, the role of microthrombosis in lacunar stroke
pathogenesis was not assessed in the present study, and
remains to be investigated.
Addendum
M. Gauberti designed and performed research, and
wrote the manuscript. S. Martinez de Lizarrondo
and C. Orset provided scientific suggestions and
contributed to manuscript review. D. Vivien supervised
the study.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.
Hypoperfused area
Ischemic lesion
Thrombin
models
TMVO
models
–70%
Microthrombosis
blockade
Clot formation
Filament insertion
Spontaneous
reperfusion
Thrombin models
Filament removal
CBF
TMVO models
TMVO models with
microthrombosis blockade
MICROTHROMBOSIS
Time
Fig. 4. Working model of cerebral blood flow (CBF) and infarct development in thrombin and transient mechanical vascular occlusion
(TMVO) models.
© 2013 International Society on Thrombosis and Haemostasis
414 M. Gauberti et al
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© 2013 International Society on Thrombosis and Haemostasis
DOI: 10.1111/jth.12487
BRIEF REPORT
Lack of secondary microthrombosis after thrombin-induced
stroke in mice and non-human primates
M . G A U B E R T I , S . M A R T I N E Z D E L I Z A R R O N D O , C . O R S E T and D . V I V I E N
Inserm UMR-S U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, Inserm, Universite Caen Basse-Normandie, GIP
Cyceron, Caen, France
To cite this article: Gauberti M, Martinez de Lizarrondo S, Orset C, Vivien D. Lack of secondary microthrombosis after thrombin-induced
stroke in mice and non-human primates. J Thromb Haemost 2014; 12: 409–14.
Summary. Background: Secondary microthrombosis is a
major pathophysiologic mechanism leading to brain damage following transient mechanical vascular occlusion
(TMVO), the most widely used experimental stroke
model. Whether secondary microthrombosis also occurs
in non-TMVO stroke models represents an important
issue for clinical translation of antimicrothrombosis therapeutic strategies. Objectives: To assess the occurrence and
the pathogenic role of secondary microthrombosis in two
thrombin-induced stroke models in mice and non-human
primates (Macaca mulatta). Methods: Stroke was induced
in mice and non-human primates by intra-arterial administration of recombinant thrombin. This method induces
the formation of a fibrin-rich thrombus, which is spontaneously dissolved in the following hours by the endogenous fibrinolytic system. Perfusion-weighted imaging and
fluorescent-lectin microangiography were performed after
recanalization to detect secondary microthrombosis.
Moreover, to investigate its pathogenic role, thrombininduced stroke was induced in bradykinin receptor B1
(B1R) knockout mice, which are protected from the
thromboinflammation responsible for secondary microthrombosis in TMVO models. Results: Reperfusion was
stable and complete in all mice and non-human primates
tested, revealing no secondary decrease in cerebral blood
flow. No evidence of secondary microthrombosis was
found in the two models. Accordingly, deficiency in B1R
did not protect the mice from brain damage after thrombin-induced stroke. Conclusions: Our data demonstrate
that secondary microthrombosis does not occur after
thrombin-induced stroke. In view of this, the pathophysiologic roles of hematologic players promoting or protectCorrespondence: Denis Vivien, Inserm UMR-S U919, GIP Cyceron,
Bd H. Becquerel, 14074 Caen, France.
Tel.: +33 2 31 47 01 60; fax: +33 2 31 47 02 22.
E-mail: [email protected]
Received 28 October 2013
Manuscript handled by: M. Levi
Final decision: P. H. Reitsma, 29 November 2013
© 2013 International Society on Thrombosis and Haemostasis
ing against secondary microthrombosis (such as
factor XII, von Willebrand factor, and T cells) deserve to
be re-evaluated in non-TMVO stroke models.
Keywords: bradykinin; coagulation; inflammation; ischemia;
stroke; thrombosis.
Introduction
Numerous recent studies have demonstrated the involvement of secondary microthrombosis in the development
of cerebral ischemic lesions following transient mechanical
vascular occlusion (TMVO) in mice. Notably, inhibition
of microthrombosis with different strategies (including
factor XII inhibition [1], glycoprotein [GP]Iba blockade
[2], regulatory T-cell depletion [3], and kininogen/bradykinin blockade [4,5]) reduces ischemic lesion size by ~ 70%
in TMVO models, suggesting that secondary microthrombosis may constitute a promising therapeutic target for
acute stroke treatment.
Secondary microthrombosis occurs within minutes after
reperfusion in TMVO models, once the occluding filament has been removed. It induces delayed cerebral blood
flow (CBF) reduction, secondary ischemia, and lesion
growth, which is responsible for ~ 70% of the final ischemic lesion size [1]. These findings led to the concept of
stroke being a thromboinflammatory disorder [6]. However, clinical studies have demonstrated that secondary
ischemia and lesion growth are modest or non-existent
after reperfusion [7–9]. Moreover, serious concerns have
been raised about the clinical relevance of TMVO models
[10]. Therefore, whether secondary microthrombosis also
occurs in non-TMVO models represents a critical issue
for clinical translation of antimicrothrombosis strategies.
To address this question, we first investigated whether
microthrombosis occurs in other recently reported models
of acute ischemic stroke, induced by intra-arterial injection of recombinant thrombin in mice and non-human
primates [11,12]. Then, we studied the impact of
thromboinflammation blockade, by genetic deletion or
410 M. Gauberti et al
pharmacologic blockade of the bradykinin receptor B1
(B1R), on stroke outcome in the mouse model.
Material and methods
Mice
Male Swiss mice (28 animals weighing 30–35 g) were purchased from the CURB (Caen, France). Five male B1Rdeficient mice (B1R / , aged 8 weeks) from a C57BL6/J
background [13] were provided by INSERM U1048,
housed at the CURB, and compared with five male
C57BL6/J mice (aged 8 weeks; CURB). Mice were housed
with a 12-h light–dark cycle with free access to food and
water. We performed in situ thromboembolic occlusion in
anesthetized mice (2% isoflurane in 33 : 67 O2/N2O
[33% : 67%] with the rectal temperature maintained at
37 °C) by intra-arterial injection of thrombin, as previously
described [11]. The operator performing surgery was unaware of the genetic background and treatment group of the
mice. Pharmacologic inhibition of B1R was performed by
intravenous injection of R-715 (1 mg kg 1; Tocris, Bristol,
UK) 20 min after stroke onset [5].
Non-human primates
Experiments were performed in five male rhesus macaques
(Macaca mulatta) aged 5–6 years and with body weights
ranging from 7 kg to 11 kg. An experimental protocol was
submitted to the Regional Ethics Committee for Animal
Experimentation (Normandy), and approval was granted
to conduct the study (referral No. N/02-03-08/03/02-11).
Experiments were performed in accordance with French
ethical laws (act No. 87-848; Ministere de l’Agriculture et
de la For^et) and European Communities Council Directives (86/609/EEC and 2010/63/EU) guidelines for the care
and use of laboratory animals. We performed in situ
thromboembolic occlusion in anesthetized non-human primates as previously described [12].
Magnetic resonance imaging (MRI) analysis
Experiments in mice were carried out on a Pharmascan 7
T/12-cm system with surface coils (Br€
uker, Wissembourg,
France). T2-weighted images were acquired by use of a
multislice multiecho sequence: echo time (TE)/repetition
time (TR), 51/2500 ms; and spatial resolution,
70 9 70 9 500 lm3 [14]. Lesion sizes were quantified on
T2-weighted images with IMAGEJ software (v1.45r; National
Institute of Health, USA). For perfusion-weighted imaging, a gradient-echo fast imaging with steady-state precession sequence was used, with the following parameters:
TR/TE, 10/5 ms (Flip Angle = 8°); temporal resolution,
911 ms; and spatial resolution, 100 9 100 9 75 lm3 (halfscan). Mice were injected with 30 lL of a 0.5 M solution of
Dotarem (Guerbet, Aulnay-sous-Bois, France) during
repetitive image acquisitions. Then, DR2* images were generated with an in-house-created macro in IMAGEJ. CBF was
estimated by measurement of the ratio of ipsilateral and
contralateral DR2*, as described previously [15]. Experiments in non-human primates were performed as previously described [12]. All quantifications were performed
blind to the experimental data.
Fluorescein isothiocyanate (FITC)–lectin microangiography
Twenty-four hours after thrombin-induced middle cerebral artery (MCA) occlusion, mice were injected intravenously with FITC-conjugated lectin [16] (100 lg per
mouse). Two minutes after injection, mice were killed by
transcardiac perfusion with cold heparinized saline followed by paraformaldehyde and picric acid. Brains were
postfixed and cryoprotected before being frozen in Tissue-Tek. Cryostat sections (10 lm) were collected on
polylysine-coated slides before being analyzed with fluorescence microscopy. Sections were coincubated overnight
with goat anti-collagen type IV (1 : 1500; Southern
Biotech, Birmingham, AL, USA) or chicken anti-microtubule-associated protein 2 (1 : 8000; Abcam, Paris,
France). Primary antibodies were revealed with Fab′2
fragments of donkey anti-goat or anti-chicken IgG linked
to tetramethylrhodamine isothiocyanate (1 : 500; Jackson
ImmunoResearch, West Grove, PA, USA). Washed sections were coverslipped with antifade medium containing
4′,6′-diamidino-2-phenylindole, and images were digitally
captured with a Leica DM6000 microscope-coupled coolsnap camera and visualized with METAVUE 5.0 software
(Molecular Devices, Sunnyvale, CA, USA). All quantifications were performed blind to the experimental data.
Results and Discussion
We induced ischemic stroke in mice and in non-human
primates (M. mulatta) by in situ intra-arterial administration of thrombin, as previously described [11,12]. This
method induces the formation of a fibrin-rich thrombus
in the MCA, which is spontaneously dissolved in the following hours by the endogenous fibrinolytic system [17],
mimicking human pathology [18]. In these two models,
we measured CBF before and after arterial recanalization
with a bolus tracking method, using 7-T (mice) and 3-T
MRI (non-human primates). Our approach was similar to
the method previously performed by others to demonstrate secondary CBF reduction in the murine TMVO
model [1]. In our hands, after thrombin-induced thromboembolic stroke, reperfusion was stable and complete in all
mice (Fig. 1) and non-human primates (Fig. 2) tested,
revealing no secondary decrease in CBF in these conditions. Accordingly, microangiography with fluorescent
lectin (100 lg per mouse; Sigma-Aldrich, L’Isle-d’Abeau
France) failed to reveal any secondary obstructed microvessels 24 h after recanalization in mice (Fig. 1C,D),
© 2013 International Society on Thrombosis and Haemostasis
Lack of secondary microthrombosis 411
DWI
A
CBF
B
∆TTP
125
100 (AU)
25 s
0
+24 h
0
CBF (% of
contralateral side)
+20 min
*
75
50
25
0
20 min
0
Contra
D
FITC-lectin/Collagen IV/DAPI
50 µm
FITC-lectin/MAP-2/DAPI
100
50
*
0
Contralateral
50 µm
Ipsi
Ipsilateral
FITC-lectin-positive
vessels (% of total vessels)
FITC-lectin/Collagen IV/DAPI
+24 h
Ipsi
24 h
25 s
0
FITC-lectin-positive
vessels (% of total vessels)
FITC-lectin/Collagen IV/DAPI
100 (AU)
+20 min
C
100
100 µm
Ipsi
100 µm
100
50
0
Contralateral
Ipsilateral
Fig. 1. Lack of secondary cerebral blood flow (CBF) reduction after thrombin-induced stroke and reperfusion in mice. (A) Representative magnetic resonance images (7 T) of Swiss mice 20 min (top) and 24 h (bottom) after intra-arterial thrombin injection. The resulting ischemic lesion
is visualized on diffusion-weighted images (left, yellow arrow). In this model, CBF and time to peak (TTP) are altered 20 min after arterial
occlusion (red arrows), but are fully restored once the thrombus is dissolved by endogenous fibrinolysis. (B) Quantification of CBF, demonstrating full restoration after arterial recanalization (n = 5). (C) Representative fluorescent-lectin microangiographies demonstrating the lack of
perfusion in the ipsilateral middle cerebral artery territory 20 min after stroke onset. In contrast, 24 h after stroke onset, all the vessels are
reperfused (the images presented are representative of four animals per group). Microtubule-associated protein 2 (MAP-2) was used as a marker of neuronal viability to visualize the ischemic lesion (white dotted line). (D) Corresponding quantification of the percentage of fluorescein
isothiocyanate (FITC)–lectin-positive vessels in the ipsilateral (ischemic) and contralateral sides 20 min and 24 h after stroke onset. P < 0.05
was considered to indicate statistical significance. Data are presented as mean standard error of the mean. AU, arbitrary units; DAPI, 4′,6′diamidino-2-phenylindole; DWI, diffusion weighted imaging.
whereas 78% of the microvessels are occluded in TMVO
models at this time point [2]. These results demonstrated
that secondary microthrombosis does not occur after
thrombin-induced stroke.
We then wanted to investigate whether differences in
microthrombosis have therapeutic implications. As genetic
© 2013 International Society on Thrombosis and Haemostasis
deletion of B1R prevents the thromboinflammatory
reaction responsible for microthrombosis in TMVO
models [4–6], we investigated whether B1R / mice [13]
are protected from stroke in a thromboembolic model.
We chose to block B1R because it is not involved in the
initial thrombin-induced clot formation, in contrast to,
412 M. Gauberti et al
A
B
+24 h
+2 h
125
CBF
∆TTP
+2 h
DWI
CBF (% of contralateral side)
*
0
100 (AU)
0
15 s
100
75
50
25
+24 h
0
2h
0
100 (AU)
0
24 h
15 s
Fig. 2. Lack of secondary cerebral blood flow (CBF) reduction after thrombin-induced stroke and reperfusion in non-human primates. (A)
Representative magnetic resonance angiograms, demonstrating spontaneous recanalization 24 h after stroke onset in non-human primates subjected to thrombin-induced middle cerebral artery occlusion (top, yellow arrow). Representative magnetic resonance image (3 T) of non-human
primates 2 h and 24 h after intra-arterial thrombin injection, showing a cortical ischemic lesion (yellow arrows) and demonstrating full recovery
of CBF after spontaneous arterial recanalization (red arrows, n = 5). (B) Corresponding quantification of CBF. P < 0.05 was considered to
indicate statistical significance. Data are presented as mean standard error of the mean. AU, arbitrary units; DWI, diffusion weighted
imaging; TTP, time to peak.
C
R-715
B1R–/–
WT
Saline
A
B
D
NS
20
30
Lesion size (mm3)
Lesion size (mm3)
25
15
10
5
NS
25
20
15
10
5
0
0
WT
B1R–/–
Saline
R-715
Fig. 3. Bradykinin receptor B1 (B1R)-deficient mice are not protected from thrombin-induced ischemic stroke. (A) Representative T2-weighted
magnetic resonance image of wild-type (WT) and B1R / mice. (B) Quantification of the lesion sizes (n = 5) demonstrated the lack of protection of B1R / mice. P < 0.05 was considered to indicate statistical significance. (C) Representative T2-weighted magnetic resonance image of
Swiss mice treated with saline and R-715 (1 mg kg 1, a peptidic B1R antagonist) 20 min after stroke onset. (D) Quantification of the lesion
sizes (n = 5) demonstrated the lack of protection of R-715-treated mice. Data are presented as mean standard error of the mean. NS, not
significant.
© 2013 International Society on Thrombosis and Haemostasis
Lack of secondary microthrombosis 413
for example, FXII or platelet GPIba. We demonstrated
that wild-type and B1R / mice show similar ischemic
lesion sizes 24 h after stroke (Fig. 3A,B). Similarly, B1R
antagonist (R-715, 1 mg kg 1, 20 min after stroke
induction) failed to influence ischemic lesion size
(Fig. 3C,D).
Altogether, these data provide evidence that microthrombosis does not occur after thrombin-induced stroke
in either mice or non-human primates (Fig. 4). Accordingly, blockade of the thromboinflammatory reaction by
either genetic deletion or pharmacologic inhibition of
B1R failed to protect against stroke in the mouse model.
As blockade of the kininogen–bradykinin axis is also not
beneficial in a permanent ischemic stroke model [4], the
available data suggest that thromboinflammation blockade through B1R inhibition is ineffective in permanent
and thrombin-induced transient ischemic strokes, thus
questioning the potential impact of such therapeutic strategy in stroke patients.
In view of this, the pathophysiologic roles of hematologic players promoting or protecting against microthrombosis (such as FXII, von Willebrand factor, platelet
GPIba, and T-cells) deserve to be re-evaluated in non-
TMVO stroke models. Nevertheless, secondary microthrombosis may represent a potential therapeutic target
in patients treated with endovascular procedures, as late
secondary injury occurs in 18% of them [19]. Interestingly, these patients show transient reversal of the cytotoxic edema after reperfusion, a specific feature of TMVO
models [10]. As stroke is a polyetiologic disease, our
results cannot be extrapolated to all stroke subtypes. In
particular, the role of microthrombosis in lacunar stroke
pathogenesis was not assessed in the present study, and
remains to be investigated.
Addendum
M. Gauberti designed and performed research, and
wrote the manuscript. S. Martinez de Lizarrondo
and C. Orset provided scientific suggestions and
contributed to manuscript review. D. Vivien supervised
the study.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.
Hypoperfused area
Ischemic lesion
Thrombin
models
TMVO
models
–70%
Microthrombosis
blockade
Clot formation
Filament insertion
Spontaneous
reperfusion
Thrombin models
Filament removal
CBF
TMVO models
TMVO models with
microthrombosis blockade
MICROTHROMBOSIS
Time
Fig. 4. Working model of cerebral blood flow (CBF) and infarct development in thrombin and transient mechanical vascular occlusion
(TMVO) models.
© 2013 International Society on Thrombosis and Haemostasis
414 M. Gauberti et al
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