Background and Purpose— Dual antiplatelet treatment poses a risk for increased hemorrhagic transformation (HT) following intravenous thrombolysis and mechanical thrombectomy. The aim of this study was to implement a model of experimental stroke with tissue-type plasminogen activator (tPA)-associated HT in mice on dual antiplatelet treatment to enable mechanistic studies and also to allow for an initial assessment of therapeutic approaches to limit HT. Methods— Male C57BL6 mice were fed with Aspirin and Clopidogrel via drinking water for 3 days. Subsequently, mice were subjected to 2-hour transient middle cerebral artery occlusion, and tPA was infused when indicated. HT was quantified by measuring hemorrhaged areas in brain sections with ImageJ. TTC staining was used to determine infarct size. Platelet function was tested in vitro using flow cytometry and in vivo with standard tail bleeding tests. Results— Both flow cytometry and tail bleeding volumes indicated significantly reduced platelet function following Aspirin and Clopidogrel treatment. While tPA administered 2 hours after onset of middle cerebral artery occlusion did not cause bleeding in control mice (0.51±0.13 mm2), HT significantly increased by 18.9±5.4 mm2 ( P =0.0045) in Aspirin and Clopidogrel mice treated with tPA. HT in aspirin and clopidogrel mice not treated with tPA was nonsignificantly elevated by 8.0±4.6 mm 2 ( P =0.3784) compared with controls. Infarct sizes did not differ between groups. The HT persisted when the tPA dosage was reduced. Conclusions— We successfully established a translational stroke model of tPA treatment under dual antiplatelet treatment. The impaired platelet function led to an increased risk for HT in tPA-treated mice. Reducing the dosage of tPA did not prevent this hemorrhagic complication.
Dual antiplatelet treatment (DAPT) increases the risk of tPA-associated hemorrhagic transformation (HT) in ischemic stroke. To investigate the effects of DAPT in rodents, reliable indicators of platelet function utilizing a minimally invasive procedure are required. We here established a fluorescence-based assay to monitor DAPT efficiency in a mouse model of ischemic stroke with HT. Male C57/BL6 mice were fed with aspirin and clopidogrel (ASA+CPG). Venous blood was collected, stimulated with thrombin, labeled with anti-CD41-FITC and anti-CD62P-PE, and analyzed by flow cytometry. Subsequently, animals were subjected to experimental stroke and tail bleeding tests. HT was quantified using NIH ImageJ software. In ASA+CPG mice, the platelet activation marker CD62P was reduced by 40.6 ± 4.2% (p < 0.0001) compared to controls. In vitro platelet function correlated inversely with tail bleeding tests (r = −0.8, p = 0.0033, n = 12). Twenty-four hours after drug withdrawal, platelet activation rates in ASA+CPG mice were still reduced by 20.2 ± 4.1% (p = 0.0026) compared to controls, while tail bleeding volumes were increased by 4.0 ± 1.4 µl (p = 0.004). Conventional tests using light transmission aggregometry require large amounts of blood and thus cannot be used in experimental stroke studies. In contrast, flow cytometry is a highly sensitive method that utilizes small volumes and can easily be incorporated into the experimental stroke workflow. Our test can be used to monitor the inhibitory effects of DAPT in mice. Reduced platelet activation is indicative of an increased risk for tPA-associated cerebral hemorrhage following experimental stroke. The test can be applied to individual animals and implemented flexibly prior and subsequent to experimental stroke.
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