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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 1 −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. 2 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 3 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). 4 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 5