Hyperglycemia plays a more important role in the initiation and development of microangiopathy than in macroangiopathy among diabetes-induced complications. Platelet-endothelium interaction and thrombus formation might be accelerated in diabetic microcirculation. However, few in vivo studies have demonstrated thrombus formation in microcirculation of diabetic animal models. In this study, we conducted a photothrombotic vessel occlusion model in the mesenteric arterioles of normal rats or streptozotocin-induced diabetic rats. A photothrombosis, which is caused by production of active oxygen, was induced under the presence of a photosensitizing dye (Photofrin) and light irradiation. We measured the time from beginning of irradiation to initiation of platelet adhesion (Ti) and the time from beginning of irradiation to vessel occlusion (To). Then we conducted a photochemical platelet adhesion assay using platelets collected from healthy controls or diabetic patients with human cultured endothelial cells treated with normal glucose concentration, high glucose concentration or advanced glycation end products (AGE). We measured the area of platelet adhesion to endothelial cells. The in vivo experimental results showed that Ti and To in diabetic arterioles significantly decreased compared to those in normal arterioles. The in vitro results did not show any significant difference between adhesion of normal platelets and that of diabetic platelets to the cultured endothelial cells. However, platelet adhesion to the cultured endothelial cells treated with high glucose concentration and with AGE increased significantly compared to that with normal glucose concentration. From these results, it can be concluded that platelet-endothelium interaction and thrombus formation are accelerated in diabetic microcirculation.[MVRC 2 (1): 24-32,2008]

本研究では光化学反応によって産生される活性酸素作用で活性化された血管内皮細胞上に接着・粘着する血小板および白血球の定量観測に基づいて血栓生成前駆過程の内皮細胞と血球細胞の相互作用を定量評価するとともに,PDE阻害薬ibudilastの接着・粘着阻害作用の検討を行った.光化学反応による内皮細胞と血小板の接着についてibudilastは10μMより濃度依存的に接着を抑制したが,アスピリンには抑制作用は認められなかった.好中球の接着についてもibudilastは1μMより接着を抑制した.また,内皮細胞形態変化と内皮下露出の抑制効果を併せて検討したところ,その抑制効果が認められた.Ibudilastにはこれまでに報告されている抗血小板作用だけでなく,内皮細胞にも作用し,複合的な抗血栓作用を示す可能性が示唆された.今後は,作用接着分子の特定,内皮細胞由来接着促進因子についての検討などを行い,それらとibudilastの作用の関係を明らかにする必要があると考える.

Enhanced thrombogenesis in diabetes mellitus is mainly caused by endothelial injury due to excess ROS production induced by chronic hyperglycemia and AGEs subsequent to it.

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.

The advance of computerized image processing techniques has opened a new area in flow visualization and quantitative analysis of fluid dynamics. In this study, image analysis methods to measure blood velocity and flow profile in the microcirculation using a high speed video camera and image processing system are proposed. The image gradient method and image correlation method enable us to describe optical flow of blood flow dynamics; two dimensional velocity distribution and radial velocity profiles in the microscopic blood vessels. The results of image analysis indicate that the proposed system is very useful to measure the blood flow velocity with high temporal and spatial resolution.

To measure red blood cell (RBC) deformability in vitro, we used microchannels on a crystal substrate as a capillary model. We observed axisymmetry deformed RBCs to estimate their deformability and decided deformability index directly from RBCs themselves. The flowing RBCs in 200 folds through microscopy were recorded with an image-intensified high speed video camera system. This system allowed us to record individual RBC images clearly. We confirmed that the crystal microchannel is a valuable tool for RBC deformability measurement.Microangiopathy is a characteristic complication of diabetes mellitus. The decrease of RBC deformability is thought to cause this complication. In order to identify the deference in RBCs deformability between normal and diabetic RBCs. We could confirm that deformability of diabetic RBCs were much lower than normal ones, and this result suggests a possibility of serious damage in diabetic microcirculation.

Oxyradicaldependent chemiluminescence from granulocytes was visualized in rat mesenteric microvascular beds treated with platelet-activating factor (PAF), leukotriene B4 (LTB4) and bacterial chemotactic peptide (FMLP) by means of digital imaging photonic intensified microscopy. The topical application of PAF-acether (100 nM) caused remarkable granulocyte adherence on venular endothelial walls and the subsequent activation of a luminol-dependent photonic burst. Chemilumigenic sites clearly corresponded to the spatial distribution of sticking cells in post-capillary venules. LTB4 and FMLP, however, did not induce any significant photonic burst at concentrations which caused adherent changes between endothelial cells and granulocytes.These findings suggest that PAF may play a role in granulocyte-mediated oxidative stress on venular endothelial cells during acute inflammatory processes, and also provide the first evidence of an intravital oxidative burst of granulocytes in microcirculatory disturbances.

Neutrophil-mediated oxidative stress on the rat mesenteric microcirculation was studied in the experimental model of endotoxin-induced disseminated intravascular coagulation (DIC) by using an intravital fluorescent technique and luminol-dependent chemiluminescence (ChL) analysis. Leukocytes sticking to the venules were visualized by the injection of acridine orange, a fluorochrome tracer which shows high affinity to white cells. Endotoxin (E coli, O-111B4, Difco, USA) was infused intravenously at a dose of 2 mg/kg/hr. After starting the infusion of endotoxin, the number of sticking cells were gradually increased on the venular endothelium followed by a transient neutropenia. In order to investigate the distribution of infused endotoxin in the microvasculature, FITC-labeled endotoxin (Sigma, USA) was used. After administration of FITC-endotoxin, multiple patches of fluorescence along the venular walls were observed, while no fluorescent conjugates were found at the sticking neutrophils and along the arteriolar walls. ChL activities of neutrophils were also dramatically elevated, which may reflect the enhanced ability to generate oxyradical species. To investigate the inhibitory effects of heparin sodium and gabexate mesilate which was a synthetic protease inhibitor on locomotive and metabolic changes of neutrophils induced by endotoxemia, both agents were administered prior to endotoxin infusion. Gabexate mesilate attenuated these changes, but heparin sodium did not show any improving effects. It was concluded that endotoxin primarily affects the venular endothelial cells, resulting in the activation of neutrophils. Gabexate mesilate was more likely to attenuate neutrophil-mediated oxidative stress on microvasculature in endotoxin-induced DIC than heparin sodium.

We have investigated the effects of purified α-toxin (phospholipase C) of Clostridium perfringens (Yamakawa and Ohsaka, 1977) on the behavior of cellular components in the microcirculation, using cinematography on a microscopic level and electron microscopy. We demonstrated that after topical application of α-toxin to the mesentery of the rat, rolling of leucocytes along the vessel wall and sticking of leucocytes to the vessel wall occurred in venules but not in arterioles. Some of the leucocytes remained attached to the vessel wall. Thrombi were formed frequently in venules and capillaries, and at a later stage, in arterioles. With time, thrombi increased in number and size, leading eventually to stasis of the blood stream. Thrombi were also observed frequently in the mesenteric microcirculation when toxin was injected into the jugular vein of the rat. The experiments with adenosine pretreatment followed by topical application of α-toxin to the mesentery suggested that the formation of thrombi induced by this toxin does not involve the mediation of ADP. Electron-microscopic examination confirmed the formation of thrombi consisting solely of platelets. It was concluded that thrombosis must be involved as an early step in the pathogenesis of necrosis caused by α-toxin. The death of the animals injected intravenously with α-toxin may be due, at least in part, to thrombosis. It is possible that thrombosis induced by α-toxin may be one of the factors involved in the causation of toxemia often manifested in the late stage of gas gangrene.