Red blood cell velocity and oxygen tension measurement in cerebral microvessels by double-wavelength photoexcitation.

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.

Optical bioimaging: from living tissue to a single molecule: imaging and functional analysis of blood flow in organic microcirculation.

Activity of blood cells, erythrocytes, leucocytes, and platelets, in microcirculation was observed by using an intravital microscope and confocal laser scanning microscope connected with an image processing system including fluorescence and phosphorescence emission methods. Dynamic functions of the blood flow were mainly observed in mesentery, brain, and liver tissues of rats. The results are summarized as follows: Deformability of diabetic erythrocytes was significantly lower than that of healthy controls, particularly at high shear rate. The spring constant and Young's modulus of diabetic erythrocytes obviously stiffened, making them hard to deform in the capillary. During hemorrhagic shock and thrombosis, flow velocity and oxygen partial pressure of blood decreased in the brain and liver tissues that can be visualized by using FITC stained erythrocytes and Pd-porphyrin derivative as a pO(2) probe. Platelet adhesion and thrombus formation in the micro-vessels accelerated under the photodynamic reaction; diabetic platelets showed augmented adhesion and aggregation on the vessel wall which was followed by acute thromboembolism. Active oxygen radicals take part in thrombus formation, accompanied with adhesion of the activated leucocytes. Fluorescent dye probes, rhodamine G and acridine orange, are quite useful for visualization of the flow behavior of platelets and leucocytes, respectively.

Platelet adhesion and arteriolar dilation in the photothrombosis: observation with the rat closed cranial and spinal windows.

The mechanism of cerebral infarction, in which thrombus formation and platelet-endothelium interaction play an important part, have not yet been clearly elucidated in vivo. The aim of this study was to observe rolling and adherent platelets and to analyze adherent leukocytes and vessel diameter change in vivo using a photothrombotic vessel occlusion model.A photothrombosis, which is mediated by free radicals, was induced in male Wistar rats in the presence of a photosensitizing dye (Photofrin II) and exposure to a filtered light. Rhodamine 6G-labeled platelets and leukocytes were visualized with intravital fluorescence videomicroscopy through a closed cranial or spinal window. The vessel diameter, photothrombosis and leukocyte adhesion were analyzed. Rolling and adherent platelets were observed during irradiation through the cerebral and spinal window. Before the platelets were recognized, the irradiated arteriole dilated significantly. After the photochemical occlusion of an arteriole, other arterioles also dilated and the adherent leukocytes increased in the venules. The photothrombosis were almost completely composed of platelets according to electron microscopic analysis. The arteriolar dilation rate and the number of adherent leukocytes in the cerebrum were greater than those in the spinal cord. By combining the photochemical thrombus formation and the fluorescence microscope techniques, we were able for the first time to observe rolling and adherent platelets and microvascular responses during photothrombosis in the cerebral and spinal microvasculature. It is suggested that free radicals, which can lead to platelet aggregation, play an important role as a cerebral vasodilator. This model is useful for cerebral and spinal microcirculatory analysis to investigate the platelet-endothelium interaction, the platelet aggregation and the effect of free radicals on cerebral and spinal microcirculation.