Imaging of Structural Changes in Endothelial Cells and Thrombus Formation at the Site of FeCl₃-Induced Injuries in Mice Cremasteric Arteries

Abstract

Aim: We investigated thrombus formation at the site of functional injury to endothelial cells by FeCl₃.
Methods: After preparation of cremasteric arteries of mice, controlled endothelial injury was induced by application of FeCl₃. 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.
Results: Endothelial cells were tightly bound to each other to protect local thrombus formation. Platelets started to adhere to endothelial cells when FeCl₃ 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.
Conclusions: We have developed an in vivo system to detect thrombus formation after functional damage to the endothelium.