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Supplementary Material to Sun et al. “Anticoagulation with
dabigatran does not increase secondary intracerebral
haemorrhage after thrombolysis in experimental cerebral
ischaemia” (Thromb Haemost 2013; 110.1)
Supplementary Methods
Preparation of clots in the thromboembolic model
To generate thrombi, 500 µL of fresh arterial blood from a donor rat were drawn into
an Eppendorf tube, mixed with 1.0 National Institutes of Health (NIH) unit of human
thrombin (Sigma Aldrich) and 5 µl of 1 mol/l CaCl2 for a final CaCl2 concentration of
10 mmol/l and allowed to coagulate for 2 h at 37°C. After incubation in deionised
water for 5 min before MCAO, the clot was placed into isotonic saline and cut into
thrombi -- each 0.35 mm in diameter and 1.5 mm in length -- under the microscope.
Neurological score
Neurological deficits were assessed using a behavioral score ranging from 0 to 6: 0=
no deficit, 1= forelimb flexion, 2= contralateral circling, 3=falling to contralateral side,
4=barrel rolling, 5=no spontaneous movement, and 6=dead.
Spectrophotometric Haemoglobin Assay
The brain tissue from corresponding ischaemic and non-ischaemic hemisphere were
homogenised in 1.0 ml of PBS on ice for 30 sec, insonated with pulse ultrasound for 1
min, and centrifuged at 13,000 rpm for 30 min. After the haemoglobin-containing
supernatant was collected, 120 µl of Drabkin's reagent (Sigma Diagnostics;
K3Fe(CN)6 200 mg/l, KCN 50 mg/l, NaHCO3 1 g/l, pH 8.6) was added to a 30-µl
aliquot and the mixture was allowed to stand for 15 min. The optical density was then
measured at a wavelength of 540 nm with a spectrophotometer (Synergy™ 2
Multi-Detection Microplate Reader, BioTec). To verify that the measured absorbance
reflected the amount of haemoglobin, blood was obtained from naive mice by cardiac
puncture after anesthesia. Incremental aliquots of this blood (1, 2, 4, 8, 16 and 20 µl)
were added to freshly homogenized brain tissue obtained from untreated mice to
generate a standard absorbance curve. This curve showed a linear relationship
between added blood volume and optical density (see Suppl. Figure 1A).
Evans blue extravasation
Briefly, 1% Evans blue in saline (w/v) was infused (4 ml/kg, i.v.) via the left femoral
vein 22 h after the onset of reperfusion (i.e. after 24 h MCAO). Two hours later, mice
were reanesthetised and transcardially perfused with PBS to remove the intravascular
dye. The brains were removed and divided into ischaemic and non-ischaemic
hemispheres. The ischemic hemispheres were frozen immediately and stored at
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−80°Cfor further analysis. Brain samples were homogenised in 1 ml of 50%
trichloroacetic acid and centrifuged (10,000 rpm, 20 min). The supernatant was
diluted four-fold with ethanol. A fluorescent plate reader (620 nm excitation, 680 nm
emission) was used to quantify dye concentrations. Calculations were based on
external standards (10–800 ng/ml) dissolved in the same solvent (1:3; 50%
trichloroacetic acid, ethanol). The amount of extravasated Evans blue was quantified
as nanograms per hemisphere.
Gelatin Zymography
Corresponding samples of ischaemic and non-ischaemic hemispheres were taken from
a series of 20 µm thick coronal cryosections with 460 µm distance and homogenised
in ice-cold lysis buffer (50 mmol/l Tris-HCl pH 7.5, 150 mmol/l NaCl, 5 mmol/l
CaCl2, 0.05% Brij-35,0.02% NaN3, and 1% Triton X-100). After centrifugation, the
supernatant was collected and protein concentration of each sample was determined in
triplicate using Bradford reagent (Bio-rad laboratories GmbH). Aliquots of lysates
containing 50 µg protein were subjected to electrophoresis on 8% sodium dodecyl
sulfate polyacrylamide gel copolymerised with 1 mg/ml gelatin (Sigma Aldrich)
under non-reducing conditions. After washing in 2.5% Triton-X 100 for 2 h, gels were
incubated in a developing buffer containing 50 mmol/l Tris-HCl, pH 7.5, 150 mmol/l
NaCl, 5 mmol/l CaCl2, 0.02% Brij-35, and 0.02% NaN3 for 60 h. Gels were then
stained with 0.125 % Coomassie blue R-250 in 10% acetic acid and 50% methanol for
30 min before they were destained in a solution containing 5% acetic acid and 25%
methanol until clear bands appeared on a dark blue background. After scanning
(MCID 7.0, InterFocus GmbH) densitometry of bands was performed using the public
domain Image J software (National Institutes of Health, USA). A mixture of murine
MMP-2 and -9 (AnaSpec Inc.), which were activated in 1 mM
p-Aminophenylmercuric acetate (APMA, Sigma Aldrich) at 37°C for 1 h, served as
positive controls.
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Supplementary Figures
A
B
Suppl. Figure 1: Standard curve of haemoglobin spectrophotometry to calibrate
for added blood volume (A) and blood volume in ischaemic hemisphere (B).
Suppl. Figure 2: Comparison of infarct size in control and anticoagulated mice.
Infarct size did not differ among groups 24 h after MCAO and reperfusion. A)
Thrombolysis was performed 2 h after transient filament MCAO in
non-anticoagulated control (NAC) and mice anticoagulated with either a single dose
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of DE (4.5 mg/kg or 9 mg/kg i.p.) or warfarin (INR 2-3). B) Thrombolysis was
performed 2h after transient filament MCAO in nonanticoagulated control (NAC),
mice pretreated with warfarin (INR 2-3) or mice pretreated with 5 injections of DE 9
mg/kg i.p. over 2 days. C) Secondary hemorrhage after prolonged 3 h MCAO with
only filament withdrawal without thrombolysis (i.e. only mechanical recanalisation).
Mice were either nonanticoagulated (NAC) or pretreated with a single dose of DE 9
mg/kg i.p. or warfarin (INR 2-3). D) Thrombolysis was performed after prolonged
duration of ischaemia for 3 h in non-anticoagulated control (NAC), mice pretreated
with warfarin (INR 2.0-3.0) or anticoagulated with a single dose of DE 9 mg/kg i.p. E)
Thrombolysis was performed 2 h after MCAO in a thromboembolic model in rats.
Suppl. Figure 3: Mice pretreated with warfarin showed worse neurological
outcomes compared to control and DE (*p<0.05, Mann-Whitney-U).
Thrombolysis was performed 2 h after transient filament MCAO in
non-anticoagulated control (NAC) and mice anticoagulated with either a single dose
of DE (4.5 mg/kg or 9 mg/kg i.p.) or warfarin (INR 2-3).
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Suppl. Figure 4: Effect of dabigatran-etexilate (DE) on systemic anticoagulation.
Ecarin clotting time (ECT) and plasma DE were measured 1, 4 and 24 h after i.p.
injection of 9 mg/kg DE (n=5).
Table 1: Physiological parameters in rats. N= 6 in each group.
Parameter
MAPB (mmHg)
10 min before MCAO
2.5 h after MCAO
PaO2
10 min before MCAO
2.5 h after MCAO
PaCO2
10 min before MCAO
2.5 h after MCAO
pH
10 min before MCAO
2.5 h after MCAO
Control
DE 9mg/KG
Warfarin
98 ± 13
101 ± 19
103 ± 11
99 ± 21
95 ± 14
97± 17
113.1 ± 17.3
103.9 ± 14.5
107.8 ±11.6
116.5 ± 16.4
121.4 ± 21.0
114.2 ± 19.3
47.4 ± 4.2
50.2 ± 5.0
48.5 ± 4.5
51. 7± 5,2
45.4 ± 4.1
47.1 ± 4.3
7.40 ± 0.09
7.36 ± 0.03
7.30 ± 0.04
7.33 ± 0.12
7.41 ± 0.06
7.35 ± 0.07
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