Imaging of Structural Changes in Endothelial Cells and Thrombus Formation at the Site of FeCl<sub>3</sub>-Induced Injuries in Mice Cremasteric Arteries

Abstract

We investigated thrombus formation at the site of functional injury to endothelial cells by FeCl(3). After preparation of cremasteric arteries of mice, controlled endothelial injury was induced by application of FeCl(3). Endothelial cells were rendered fluorescent by addition of FITC (fluorescein isothiocyanate)-labeled isolectin B4. Circulating platelets and leukocytes were made fluorescent by rhodamine 6G. Three-dimensional (3D) growth of thrombi was visualized in real time. Effects of aspirin and clopidogrel pre-treatments on the growth of thrombi were investigated in vivo as well as in an ex vivo flow chamber system. Endothelial cells were tightly bound to each other to protect local thrombus formation. Platelets started to adhere to endothelial cells when FeCl(3) was applied. Three-dimensional growth of thrombi, which takes 10.6+/-7.5 minutes for complete occlusion in control, can be visualized with our imaging system. Aspirin pre-treatment at the dose tested did not influence either endothelial injury or platelet thrombus growth, while clopidogrel pretreatment significantly inhibited 3D growth and prolonged occlusion time up to 64.6+/-25.3 minutes (100 mg/kg). A similar inhibiting effect of clopidogrel was reproduced in ex vivo flow chamber experiments. We have developed an in vivo system to detect thrombus formation after functional damage to the endothelium.