Increased capillary vascularity in the femur of aged rats by exercise training.

During aging, bone loss occurs in association with alteration of blood perfusion in the tissue. A number of studies have shown that repaired blood perfusion in various organs was improved by regular exercise, but the effect of exercise on bone microcirculation has not been studied fully yet. This study aimed to investigate the effect of exercise training on capillary vascularity in rat femur by directly observing the bone microcirculation under a laser scanning confocal microscope. Male Wistar rats were divided into three groups: sedentary-young (aged 4-6 months), sedentary-aged (aged 20-22 months) and trained-aged (aged 20-22 months). The exercise program included swimming training 5 days/week for 8 weeks. Using our newly devised window chamber, we directly observed the femur microcirculation of each group under a laser scanning confocal microscopic system. Based on the fluorescent image of microvasculature recorded at the surface of the femur, bone capillary vascularity (CV) was measured using computer software. Liver malondialdehyde (MDA) level was also measured to examine the relationship between CV and oxidative stress in aged rats. In the sedentary-aged group, the CV significantly decreased, but the MDA level significantly increased, compared with sedentary-young group. In the trained-aged group, CV was significantly higher, whereas the MDA level was significantly lower, compared with the sedentary-aged group. In both sedentary-young and sedentary-aged rats, the CV was linearly correlated with the MDA level. In conclusion, the swimming exercise could attenuate aged-induced suppression of CV, closely related to exercise-ameliorated oxidative stress in aged.

The antioxidant Cerebralcare Granule attenuates cerebral microcirculatory disturbance during ischemia-reperfusion injury.

Cerebralcare Granule (CG) is a compound Chinese medicine used for treatment of headache and dizziness associated with cerebrovascular diseases. To clarify the mechanism underlying the clinical outcome of CG, this study investigated the effects of CG on the structure and function of cerebral microvasculature during I/R injury. A total of 138 Mongolian gerbils were included and divided into four groups, each composed of 36 or 30 animals, for evaluating various parameters of concern. A skull window was prepared for microcirculatory observation in animals, which were subjected to I/R with or without pretreatment with CG (0.4 or 0.8 g/kg). The velocity of red blood cells in the venules was observed by a high-speed video camera system, along with intravital confocal microscopic measurements of microvascular diameters, adherent leukocytes, and albumin leakage in the brain cortex. Changes in the fluorescence intensity of dihydrorhodamine 123 in cerebral microvessels and malondialdehyde level in the cortex were measured. The ultrastructure of the microvessels in the cerebral cortex was analyzed using both transmission and scanning electron microscopy. In addition, cerebral blood flow was monitored using the laser Doppler imaging technique. Pretreatment with CG (0.4 or 0.8 g/kg) significantly alleviated I/R injury-induced disorders in cerebral microvasculature, as evidenced by the data observed at 60 min of reperfusion wherein the values in CG (0.4 g/kg) pretreatment group, CG (0.8 g/kg) pretreatment group, and I/R group were 2.43 +/- 0.24, 2.28 +/- 0.18, and 6.00 +/- 0.35 for leukocyte adhesion, 2.51 +/- 0.40, 2.33 +/- 0.29, and 4.77 +/- 0.24 for albumin leakage, 7.06 +/- 0.81, 5.93 +/- 0.42, and 28.38 +/- 2.70 for dihydrorhodamine 123 fluorescence intensity in cerebral microvessels, 16.35 +/- 0.52, 14.34 +/- 0.68, and 21.46 +/- 0.71 for malondialdehyde level in the cortex, and 0.43 +/- 0.07, 0.46 +/- 0.02, and 0.17 +/- 0.08 for cerebral blood flow, respectively. I/R injury-elicited ultrastructural alterations in microvessels in cerebral cortex were also mitigated impressively by CG administration, manifested as attenuation of the reduced number of opening capillaries and the altered fine structures in endothelium, which were characterized by rough inner surface, increased intracellular vesicles, hypertrophy of digitations of intercellular contact, and swollen perivascular astroglial processes. Cerebralcare Granule is able to attenuate I/R injury-induced functional and structural changes in microvessels in the cerebral cortex of gerbils, an ability that is most likely correlated with its antioxidant potential.

Blood flow regulation in the cerebral microvasculature with an arcadal network: a numerical simulation.

Blood flow regulation in the cerebral microvasculature with an arcadal network was investigated using a numerical simulation. A mathematical model for blood flow in the arcadal network, based on in vivo data of cat cerebral microvasculature and flow velocity was developed. The network model consists of 45 vessel segments and 25 branching points. To simulate microvascular response to blood flow, non-reactive (solid), cerebral arteriole-like, or skeletal muscle arteriole-like responses to wall shear stress were taken into account. Numerical calculation was carried out in the flow condition where the inlet (arterial) pressure was changed from 60 to 120 mmHg. Flow-rate in each efferent vessel and the mean flow-rate over all efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The simulation demonstrated the wall shear stress-induced vasodilation in the arcadal network worked to maintain the blood flow at a constant level with pressure variable in a wide range. It is suggested that an individual microvessel (segment) should join in the regulatory process of flow, interacting with other microvessels (cooperative regulation).

Asian traditional medicine (ATM): recent progress based on scientific evidences.

Asian traditional medicine (ATM) (herbal medicine, acupuncture or moxibution) has gained some popularity among communities in Asia. Some therapies employed in ATM have been verified using modern techniques, but the significance of ATM has still remained unclear. This symposium was focused on experimental data obtained recently.

Effects of curcumin on tumor angiogenesis and biomarkers, COX-2 and VEGF, in hepatocellular carcinoma cell-implanted nude mice.

Anti-angiogenic activity of curcumin and effects of curcumin on angiogenic biomarkers, cycloxygenase (COX)-2 and vascular endothelial growth factor (VEGF) levels were investigated. One day after hepatocellular carcinoma cell (HepG2) cells (30 microl of 2 x 10(6) cells) were inoculated onto the upper layer of the skin-fold chamber (HepG2-group, n = 15), curcumin solutions of 300 and 3000 mg/kg BW were daily oral fed to HepG2-Cur-300 and HepG2-Cur-3000 groups (n = 30), respectively. Intravital fluorescence videomicroscopy was performed to monitor neocapillaries in the tumor on days 3, 7 and 14 post-tumor-inoculation, using RITC-dextran (0.1 ml of 0.5% injected intravenously). The tumor neocapillary density (NCD) was evaluated in correlation with the tumor area, using a digital image analysis. The results demonstrated that the NCD of HepG2-groups were significantly increased on day 7 and 14, compared to the aged-matched Sham-groups (p<0.001). The increased NCD on day 7 and 14 were attenuated significantly by daily treatment of curcumin solution (3000 mg/kg BW).The curcumin treatment reduced the tumor-induced over-expression of COX-2 and serum VEGF in HepG2 groups significantly (p<0.001), indicating that curcumin could inhibit tumor angiogenesis. This mechanism might be mediated through reduction of angiogenic biomarkers, COX-2 and VEGF.

Effects of Yahom on the regional cerebral blood flow in rat using fluorescence videomicroscopy.

This study was conducted to investigate effects of Yahom on the regional cerebral blood flow (rCBF) in rats using fluorescence videomicroscopy. Male Wistar Furth rats weighing 200-250 g were used, and divided into three groups: experimental I, II and control groups. The experimental I and II groups received single oral administration of Yahom 2 and 4 g/kg.bw, and topical application of Yahom on the pial cerebral cortex, respectively, while the control group received oral administration of 1 ml of 5% Tween. The rCBF was monitored using laser Doppler flowmetry at different periods (5-120 minutes) after the administration of Yahom or Tween. The mean arterial pressure (MAP) was measured through a femoral artery. The cerebral microcirculation was observed and recorded under an intravital fluorescence videomicroscopic system. The arteriolar diameter was measured based on the recorded videomicroscopic images. The MAP and rCBF increased after the oral administration of Yahom, while they did not alter in the control group. The maximum responses of MAP and rCBF were approximately 16% and 33% at 45 min and 30 min after the administration of Yahom 4 g/kg.bw, respectively. The topical application of Yahom induced vasodilation in the pial microvessels. In conclusion, single oral administration of Yahom may increase the regional cerebral blood flow under the effect of cerebral microvascular vasodilation.

Microembolic flow disturbances in the cerebral microvasculature with an arcadal network: a numerical simulation.

Flow disturbance due to microembolism in the cerebral microvasculature with an arcadal network was studied by a numerical simulation. A mathematical model for flow in the arcadal network was developed, based on in vivo data of cat cerebral microvasculature and flow velocity. The network model consisted of 45 vessel segments, and 25 branching points. To simulate microvascular responses to blood flow, the following three types of responses to wall shear stress were considered; non-reactive (solid-like), cerebral arteriole, and skeletal muscle arteriole-like responses. The numerical calculation was carried out in the condition where a feeding arteriole was occlused. Flow changes in efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The present simulation has demonstrated that blood flow in efferent vessels was influenced by the topology of the vascular network and the response pattern in single vessels. The arcadal structure of arterioles might be most effective in response to flow disturbances in efferent vessels.

Chronic changes of the iris microvasculature of streptozotocin-induced diabetic rats using fluorescence videomicroscopy.

This study was aimed to investigate chronic changes of the iris microvasculature in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar-Furth rats by intravenous injection of STZ (55 mg/kg.bw). The rats were divided into control (CON) and diabetic (STZ) groups. The experiments were performed at 8, 12, 24 and 36 weeks after the injection of STZ. The iris microcirculation was visualized under a fluorescence videomicroscope. Intraluminal diameters of microvessels were measured based on the FITC-dextran images. Leukocyte adhesion to the microvascular endothelium was evaluated by counting leukocytes (labeled with rhodamine 6G). The iris blood perfusion was measured using laser Doppler flowmetry. Tissue lipid peroxidation of the eye was evaluated. The results demonstrated that the lipid peroxidation increased significantly after the injection of STZ. Both the diameters of arterioles (or precapillaries) and the iris blood perfusion decreased significantly in STZ rats, compared to the control levels. Adherent leukocytes increased significantly at 8, 12, 24 and 36 week after the injection of STZ, compared with the control levels. This indicates that the increased in oxygen-derived free radicals may be a major contributor for iris vascular endothelial dysfunction in diabetes mellitus, including leukocyte adhesion and reducing the arteriolar diameter. The present model may be useful for assessing long-term effects of therapeutic agents on diabetic retinopathy.

Microvascular hemodynamic responses to arteriovenous shunting in rat limb.

Autologus veins have been used clinically as a bypass conduit for reconstruction of small arteries, but there are few data available for microvascular response to arteriovenous (AV) shunting. This study was aimed to evaluate microvascular hemodynamic changes induced by creating AV anastomosis in rat hindlimb. Using intravital fluorescence videomicroscopy, we measured velocities of red blood cells (RBCs) flowing in the microvascular network in the control state, in the occlusion state where the superficial femoral artery (SFA) was occluded, and in the AV shunting state where the AV anastomosis was opened after occlusion of SFA. RBC velocities were measured in 155 capillaries of 6 rats using a dual window method and a frame-by-frame technique. The mean velocity and the coefficient of variation were 0.61 mm/sec and 0.90 in the control state, 0.34 mm/sec and 1.30 in the occlusion state, 0.83 mm/sec and 1.24 in the AV shunting state, respectively. These indicated that hemodynamic heterogeneity among capillaries increased with decrease in mean velocity following the arterial occlusion, while the AV shunting augmented the heterogeneity with increase in mean velocity. Capillaries with low perfusion (<0.1 mm/sec) or high perfusion (>1.0 mm/sec) were 5.8% or 20.6%, 29.6 or 5.2%, and 22.6 or 30.3% out of all measured capillaries in the control, occlusion and AV shunting conditions, respectively. In conclusion, AV shunting increased capillary perfusion and also its spatial heterogeneity, preferentially inducing high velocity in the microvasculature.

Heterogeneity of capillary flow in the retrograde microcirculation induced in rat limb by arteriovenous shunting.

Arteriovenous (AV) fistulas have been used clinically for improving adjunctive bypass patency. Such AV shunting induces retrograde flow in the microvascular network, which may induce microvascular remodeling and angiogenesis at the chronic phase. This paper was aimed to examine heterogeneity of blood flow among capillaries in the retrograde microcirculation induced by AV shunting. An AV anastomosis was created in rat hind limb. Using a dual window method or frame-by-frame technique on the fluorescence microscopic video images, we measured velocities of red blood cells (RBCs) flowing in the capillary network in three flow conditions: control (normal flow), arterial occlusion, and AV shunting (retrograde flow). For each flow condition, RBC velocities were obtained in 155 capillaries of 6 rats. By classifying all the capillaries into four groups based on the levels of RBC velocity in the occlusion state, we evaluated the mean velocities, coefficient of variation (CV), and histograms for each group of capillaries. The mean velocity and CV in each group changed significantly from the control to AV shunting states. Especially, most significant changes appeared in capillary groups where the superficial femoral artery or its collateral arteries might have a direct influence. Though the AV shunting improved capillary perfusion in the mean level, major parts of capillaries still remained at low perfusion.

Cell morphological changes in venous remodeling induced by arteriovenous grafting in rat limb.

Vascular remodeling induced in rat limb by arteriovenous (AV) shunting was investigated by evaluating changes in vascular diameter and cell morphology. In Wistar rats, a vein graft was implanted in situ in the hind limb. Flow-rate in the grafted vein was assessed by measuring flow in the common femoral artery using an ultrasonic flowmeter. Nuclei and actin filaments of the venous wall were stained with propidium iodine and phalloidine-FITC, and the samples were observed using confocal laser microscopy. The grafted veins became circular in cross-section with increase in diameter during two weeks after AV shunting. Owing to the increase in diameter, the estimated wall shear stress was not increased so much as the flow-rate. The confocal laser microscopic observation showed that endothelial cells (ECs) and smooth muscle cells (SMCs) in the grafted veins were either aligned well (2 out of 8 samples), or ECs were denudated and SMCs were disrupted (in 6 out of 8 samples). The cell density of ECs was unchanged from the control level. In conclusion, the grafted vein was remodeled with morphological changes in ECs and SMCs during 2 weeks after AV shunting.

Capillary angiogenesis and remodeling induced in rat limb by arteriovenous shunting.

Capillary angiogenesis and remodeling induced by arteriovenous (AV) shunting in rat hind limb was investigated by evaluating changes in capillary density and diameter in the skeletal muscle subject to retrograde flow and high pressure. Wistar rats were used, and an AV anastomosis was created in the hind limb. Two weeks after AV shunting, the microvasculature in the limb was visualized by GS-lectine, and the samples were observed using confocal laser microscopy. The capillary density were increased by approximately 150% for small vessels (<13 microm in diameter) under retrograde flow condition, but no change appeared for large vessels (>13 microm in diameter). The capillary diameters were not significantly different between control and chronic condition. In conclusion, retrograde flow produced by AV shunting increased capillary density but it did not change the capillary diameter significantly.

Cerebral microvascular responses to air embolism-reperfusion in the cat using fluorescence videomicroscopy.

Cerebral microvascular changes due to air embolism-reperfusion in the cat were investigated. Air embolism-reperfusion was produced in the cerebral microvessels by an intra-carotid injection of air (0.2-0.3 ml). Air emboli in the cerebral arterioles were observed continuously from the air injection to reperfusion using fluorescence videomicroscopy. Arteriolar diameter was measured based on video images of arterioles filled with rhodamine-B isothiocyanate dextran, and red cell velocity was measured using a dual window technique with FITC-labeled red cells. Air bubbles ceased to move in the arterioles of 20-70 microm diameter and blood flow was almost stopped in distal arterioles. The air emboli were of cylindrical shape in the arterioles, with hemispherical end caps. The emboli progressed slowly at rates of 7-73 microm/sec and then flowed away. The air emboli induced ischemia-reperfusion with the ischemic duration of approximately 1 minute (10 sec-3 min) at the arteriolar level. Cerebral arterioles began to dilate after the formation of the air emboli and significantly dilated by approximately 50% after reperfusion. A transient increase in red cell velocity was observed after reperfusion. Arteriolar dilation and the increase in velocity led to a hyperemic response in arteriolar flow to ischemia-reperfusion.

Cerebral angiogenesis induced by growth factors: intravital microscopic studies using models.

Pharmacological induction of angiogenesis is a new treatment of cerebrovascular insufficiency without surgical treatment. It is an urgent task to investigate the dynamic process of angiogenesis and of the microvascular perfusion of the cerebral neoplastic tissue in vivo. The present study is concerned with microcirculatory aspects of cerebral neocapillaries in vivo. A novel model of cerebral angiogenesis was developed by inducing cerebral neocapillaries in mice using growth factors such as basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF). By a direct observation of the neocapillary microcirculation under a fluorescence videomicroscope, the neocapillary density, diameter and red cell velocity were measured to evaluate the development and remodeling of the neocapillaries with the number of days after incubation. The neocapillary response to topically applied acetylcholine (ACh) was examined by measuring changes in the diameter and red cell velocity. It was shown that PDGF-induced neocapillaries was dilated in response to ACh on day 28 after incubation, while bFGF-induced neocapillaries was not. Furthermore, the neocapillary pericytes were observed using confocal laser microscopy, based on the fluorescence immunohistochemical images of the neoplastic tissue. Several pericytes, stained with anti-NG_2, appeared in the neocapillaries. It was suggested that these pericytes might be recruited in the neocapillaries to regulate blood flow without vascular smooth muscle.

Asian traditional medicine (ATM) based on in vivo microcirculation evidence.

Asian traditional medicine (ATM) (herbal medicine, acupuncture or moxibution) has gained some popularity among communities in Asia, but there are still few evidences to prove the effectiveness of such therapeutic measures. A symposium was held with aim at the effectiveness of Asian traditional therapies in views of in vivo microcirculation. This report is concerned with the symposium, including Asian activities for ATM.

Cerebral endothelial dysfunction in diabetes: intravital microscopic analysis using streptozotocin-induced diabetic rats.

Endothelial function of cerebral microvessel in diabetes was evaluated using streptozotocin (STZ)-induced diabetic rats (blood glucose of >/=300 mg/dl). At 36 weeks after STZ injection, the rats were anesthetized with sodium pentobarbital. The cerebral microcirculation in control and STZ groups was directly observed, using intravital fluorescence videomicroscopy. To evaluate the endothelial function in vivo, the number of leukocytes adhering to postcapillary venules were counted, and cerebral arteriolar responses to acetylcholine (ACh) and nitroglycerine (NTG) were examined. The results showed that the leukocyte adhesion to cerebral postcapillary venular endothelium increased significantly in STZ-rats, compared with control rats. The vasodilatory responses of cerebral arterioles (20-30 microm) to ACh decreased significantly in STZ-rats, compared with control rats (p<0.01), but the responses to NTG did not alter in diabetes. These results indicate that the impaired responses should occur on the endothelial cell. In conclusion, endothelial dysfunction induced in diabetes are characterized by impaired endothelium-dependent vasodilation and increased leukocyte adhesion to endothelial cells.

Maturity of pericytes in cerebral neocapillaries induced by growth factors: fluorescence immuno-histochemical analysis using confocal laser microscopy.

The maturity of pericytes in cerebral neocapillaries induced by two different growth factors: basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF), was examined using an immunohistochemical staining technique. Cerebral angiogenesis was induced in mice by implanting a sandwich system of bFGF/PDGF gel and nylon-mesh over the exposed cortex. On 28th day after incubation, a small volume of cerebral tissue with the nylon-mesh was isolated and stained using tetramethyl rhodamine isothiocyanate (TRITC)-labeled secondary antibody to the primary antibody against NG_2 proteoglycan and fluorescein isothiocyanate (FITC)-labeled Griffonia simplicifolia (GS)-lectin. Using a confocal laser microscopic system, we observed the cerebral neocapillaries on the upper surface of the nylon-mesh and evaluated the maturity of pericytes stained with NG_2 based on the fluorescence immunohistological images. The pericyte appeared rich in neocapillaries induced by PDGF. It was suggested that pericytes might play a key role in the regulation of blood flow in neovessels.

Measurement of red cell velocity in microvessels using particle image velocimetry (PIV).

A new technique using particle image velocimetry (PIV) has been developed to evaluate the detailed velocity profiles of red cells flowing in microvessels. The microcirculation in rat mesentery was directly observed using high-speed videomicroscopy, and the images of red cells flowing in the mesenteric arterioles were recorded simultaneously with the arterial blood pressure. Based on the high-speed videomicroscopic images obtained, velocity vectors in single or branched arterioles were evaluated to obtain velocity profiles across the cross-section of arterioles. It was shown that in single and straight arterioles the velocity profile was blunt with a pit at the central region, and its pit was marked in bifurcation. The present technique enables us to analyze red cell velocity profiles up to 0.8 microm in the spatial resolution and 1 msec in the time interval.

The effect of long-term supplementation of vitamin C on leukocyte adhesion to the cerebral endothelium in STZ-induced diabetic rats.

The effect of long-term supplementation of vitamin C on leukocyte adhesion to the cerebral endothelium was investigated in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar Furth rats by intravenous injection of STZ. The vitamin C, ascorbic acid, was supplemented with drinking water (1 g/l). The rats were divided into control and diabetic groups without or with supplementation of vitamin C. The cerebral microcirculation was directly observed through a cranial window after different periods (12, 24 and 36 weeks) of vitamin C supplementation, using fluorescence videomicroscopy. Leukocyte adhesion to the venular endothelium was examined by labeling leukocytes with rhodamin 6G. The number density of adherent leukocytes in STZ-diabetic rats was increased significantly, compared with control rats. This increase in leukocyte adhesion was prevented by the long-term supplemented vitamin C. It was suggested that the antioxidant effect of vitamin C might be responsible for the prevention of leukocyte adhesion in diabetes mellitus.

Cerebral microcirculatory changes in rat with a cardiopulmonary bypass using fluorescence videomicroscopy.

Cerebral microcirculatory changes in rat with a cardiopulmonary bypass (CPB) at normothermia was investigated in relation to cerebrovascular disorders caused by surgical operation with CPB. The mean arterial pressure was changed from 50 to 200 mmHg by changing the pump flow-rate. A non-pulsatile flow model was developed by stopping the cardiac beat using a fibrillator. The pial microcirculation was visualized using fluorescence-labeled red cells and dextran, and was directly observed under a fluorescence videomicroscope during CPB. Based on the recorded videoimages, the arteriolar diameter and red cell velocity were measured, in which single arterioles with approximately 40 microm diameter were selected among the pial arterioles. It was shown that when the arterial pressure was changed: (1) arteriolar vasodilation or constriction appeared during pulsatile flow but it disappeared during non-pulsatile flow, and (2) the arteriolar red cell velocity increased or decreased linearly during non-pulsatile flow as well as pulsatile flow. The flow-rate was almost constant at a large range of the mean arterial pressure from 60 to 160 mmHg during pulsatile flow (autoregulation), but it increased or decreased during non-pulsatile flow with an increase or decrease in mean arterial pressure, respectively. It was suggested that pulsativity might be responsible for cerebral autoregulation.