In vivo real-time two-photon microscopic imaging of platelet aggregation induced by selective laser irradiation to the endothelium created in the beta-actin-green fluorescent protein transgenic mice

Abstract

Although thrombus formation in vivo has recently been reported, all previous laser induced thrombus models have been associated with vessel wall disruption. This study aimed to evaluate in vivo real-time platelet aggregation after selective endothelial injury, and to visualize thrombus formation without disruption and swelling of the arterial intima induced by two-photon laser irradiation. Cecal arteriole thrombi were created in beta-actin-green fluorescent protein transgenic mice by selective endothelial injury using two-photon laser-scanning microscopy (TPLSM). The in vivo real-time process of thrombus formation was assessed. Anticoagulant drug efficiency for thrombi was also analyzed in detail. TPLSM allowed visualization of microvessel components from the arterial smooth muscle to the intimal layer. Immediately after selective laser irradiation of the intimal layer, platelet adhesion and aggregation were seen only at the area of injury of the intimal layer after forming linear adhesions downstream of the injured area. When shear stress was overcome, thrombus formation began at the downstream edge of the injured area. Thrombus volume plateaued approximately 60min after laser irradiation. The thrombolytic effects of anticoagulant drugs were precisely assessed; therefore, our model appears the most advanced model in point of real-time imaging of pathophysiological processes in vivo currently reported. In vivo real-time imaging of thrombus formation can be achieved using TPLSM in combination with an organ stabilizing system. The high magnification and resolution of TPLSM allows investigation of the mechanisms of thrombus formation along with assessment of antithrombotic drug efficacy with little interexperimental variation.