Laser-induced cremaster arteriole thrombosis model

Male C57BL/6J mice (n = 3) were intravenously dosed with the following treatments: (i) saline control (equivalent volume), (ii) ML355 (1.5 mg/kg), (iii) sHDL (50 mg/kg), or (iv) ML355-sHDL (sHDL at 50 mg/kg and ML355 at 1.5 mg/kg). Twenty-four hours after administration, mice were anesthetized and surgically prepared as above described. DyLight 647–conjugated rat anti-mouse platelet GP1bβ antibody (0.1 μg/g; X649, EMFRET Analytics) was administered by jugular vein cannula before vascular injury. Multiple independent thrombi (8 to 10) were induced in the arterioles (30 to 50 μm diameter) in each mouse (three mice per group) with a laser ablation system. All captured images were analyzed for change in fluorescence intensity over time of thrombus formation by subtracting fluorescent background defined on an uninjured section of the vessel using the SlideBook program. To monitor and compare dynamic thrombus formation among different treatment groups, the relative fluorescence intensity of Alexa Flour 647–labeled platelets (recruited within thrombus) was plotted using the mean fluorescence at each time point. Data were evaluated for significance with two-way analysis of variance (ANOVA) and Mann-Whitney test for nonparametric data using Prism 6 software (GraphPad, La Jolla, CA, USA).