Two-photon microscopy on vital corotid arteries: imaging the relation between collagen and inflammatory cells in atherosclerotic plaques

We used two-photon laser scanning microscopy (TPLSM) to demonstrate for the first time its potential in studying relational details at the cellular level of atherogenesis in intact, viable mouse carotid arteries. Isolated and mounted arteries of ApoE-/-mice, aged 15 or 21 weeks (7 and 13 weeks on western diet), were imaged after labeling with specific fluorescent markers for cell nuclei, inflammatory cells, collagen, and lipids. Data were compared with C57BL6/J mice fed a chow diet. Control vessels had intact endothelium without adhering blood cells or significant intimal collagen labeling. In ApoE-/-mice already at 15 weeks, inflammatory cells adhered to the endothelium and increased labeling of collagen was observed in tunica intima at both lesion-prone and non-lesion-prone sites, indicating endothelium activation. In plaques, internalized inflammatory cell density increased with age and plaque progression in tunicae adventitia and intima, but not media. In the whole plaque, aging or plaque progression did not alter the direct relationship between inflammatory cells and collagen. However, within the fibrous caps specifically, direct contact between inflammatory cells and collagen increased with age. This study demonstrates the potential of TPLSM in determining detailed information regarding the complex relationship between inflammatory cells and collagen during atherogenesi

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 Ca2+ concentration ([Ca2+](i)) as a marker of activation. Isolated platelets were labeled with the Ca2+-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 [Ca2+](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 [Ca2+](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. Copyright (C) 2002 S. Karger AG, Base

Tumor cell plasticity in Ewing sarcoma, an alternative circulatory system stimulated by hypoxia

A striking feature of Ewing sarcoma is the presence of blood lakes lined by tumor cells. The significance of these structures, if any, is unknown. Here, we report that the extent of blood lakes correlates with poor clinical outcomes, whereas variables of angiogenesis do not. We also show that Ewing sarcoma cells form vessel-like tubes in vitro and express genes associated with vasculogenic mimicry. In tumor models, we show that there is blood flow through the blood lakes, suggesting that these structures in Ewing sarcoma contribute to the circulation. Furthermore, we present evidence that reduced oxygen tension may be instrumental in tube formation by plastic tumor cells. The abundant presence of these vasculogenic structures, in contrast to other tumor types, makes Ewing sarcoma the ideal model system to study these phenomena. The results suggest that optimal tumor treatment may require targeting of these structures in combination with prevention of angiogenesis

In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy

In vivo (molecular) imaging of the vessel wall of large arteries at subcellular resolution is crucial for unraveling vascular pathophysiology. We previously showed the applicability of two-photon laser scanning microscopy (TPLSM) in mounted arteries ex vivo. However, in vivo TPLSM has thus far suffered from in-frame and between-frame motion artifacts due to arterial movement with cardiac and respiratory activity. Now, motion artifacts are suppressed by accelerated image acquisition triggered on cardiac and respiratory activity. In vivo TPLSM is performed on rat renal and mouse carotid arteries, both surgically exposed and labeled fluorescently (cell nuclei, elastin, and collagen). The use of short acquisition times consistently limit in-frame motion artifacts. Additionally, triggered imaging reduces between-frame artifacts. Indeed, structures in the vessel wall (cell nuclei, elastic laminae) can be imaged at subcellular resolution. In mechanically damaged carotid arteries, even the subendothelial collagen sheet (~1 µm) is visualized using collagen-targeted quantum dots. We demonstrate stable in vivo imaging of large arteries at subcellular resolution using TPLSM triggered on cardiac and respiratory cycles. This creates great opportunities for studying (diseased) arteries in vivo or immediate validation of in vivo molecular imaging techniques such as magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET)