In vivo visualization of vascular leakage in photochemically induced cortical infarction.

The photothrombotic model for stroke was originally described as a focal cortical infarction resulting from occlusive thrombosis. However, subsequent studies have shown evidence for extravasation of water and proteins using ex vivo techniques. The aim of the present study was to determine the role of vascular leakage in the pathophysiology of photochemically induced infarction in vivo. At several times points after infarct induction, analysis of blood flow and vascular leakage was performed using intravital microscopy and fluorescent labelling of blood plasma. In the first hour following infarct induction, massive vascular leakage of the plasma label occurred inside the lesion core that was destined to become infarcted. Flow had stopped completely in this area at 4h after illumination. On the day following infarct induction substantial leakage was still present in the penumbral area, defined as the area immediately surrounding the lesion core where reduced flow velocities were observed. Thus, together with the formation of occlusive thrombi, vascular leakage is an important factor in the pathophysiology of photothrombotic stroke.

Both ADP and thrombin regulate arteriolar thrombus stabilization and embolization, but are not involved in initial hemostasis as induced by micropuncture.

OBJECTIVE: Thrombosis and embolization are main causes of morbidity and mortality. Up to now, the relative importance of mediators involved is only partly known. It was the aim of this study to investigate the involvement of ADP and thrombin in subsequent phases of arteriolar hemostasis and thromboembolism in vivo. METHODS: Rabbit mesenteric arterioles were punctured, which induced bleeding, hemostasis, and subsequent thromboembolism. This reaction as well as the activation state of platelets involved ([Ca(2+)](i)), was monitored in real time by intravital (fluorescence) microscopy. RESULTS: Neither inhibition of thrombin formation or thrombin activity nor blockade of platelet ADP receptors P2Y(1) and P2Y(12) influenced the initial hemostatic reaction: in all experiments initial bleeding was stopped by a primary thrombus within 2-3 s. On the other hand, both thrombin inhibition and P2Y(1) blockade increased rebleeding frequency, which indicates reduced thrombus stability in the long term. Finally, inhibition of either thrombin or ADP (via both receptors) reduced aggregate formation during the embolization phase by at least 90%. While most participating platelets exhibited a transient increase in [Ca(2+)](i) during embolization, an increased percentage of platelets showed no calcium response at all during P2Y(1) blockade, which was accompanied by reduced platelet-platelet interaction strength. CONCLUSIONS: Whereas thrombin and ADP are not involved in the initial hemostatic reaction, both substances appear to be essential to prevent rebleedings in the long term. During subsequent embolization, ADP (via both receptors) and small amounts of thrombin are involved in platelet activation.

Real-time detection of activation patterns in individual platelets during thromboembolism in vivo: differences between thrombus growth and embolus formation.

Knowledge on single platelet behavior and intracellular mechanisms during thromboembolism in vivo is scarce. In the present study, we used a new method that enables real-time detection and quantification of activation of individual platelets participating in a thromboembolic process in vivo, using their intracellular free Ca(2+) concentration ([Ca(2+)](i)) as a marker of activation. Isolated platelets were labeled with the Ca(2+)-sensitive fluorescent probe fluo-3 and injected into anesthetized rabbits so that 0.5-1% of their circulating platelets were labeled. Wall puncture of mesenteric arterioles resulted in thrombus formation followed by embolization. Fluorescence intensity changes of labeled platelets participating in this process were quantified. Within 30 min after injection, labeled platelets behaved similarly to native platelets, and fluorescence intensity was not influenced by dye leakage. Upon adherence to the stationary thrombus, platelets exhibited a prolonged [Ca(2+)](i) increase, accompanied by shape change and degranulation, which is consistent with a role for strong platelet agonists like collagen. In contrast, when platelets adhered to a growing embolus their [Ca(2+)](i) rise was transient, and they hardly showed shape change and degranulation, suggesting the involvement of weaker agonists like ADP. These results show, for the first time, the relation between single platelet activation patterns, which are different during thrombus growth and embolus formation, and their behavior in a thromboembolic process in vivo.