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Optical coherence tomography (OCT) has a comparatively high spatial resolution among tomographic bioimaging techniques and is less affected by changes in physiological conditions such as temperature, blood pressure, and osmolytes in the tissue. OCT detects changes in the refractive index of tissues, which is a function of the tissue glucose concentration (TGC). OCT signal intensity generally decreases with tissue depth, and its slope is expected to show a negative correlation with TGC in the interstitial fluid, reflecting blood glucose concentration (BGC). The currently applied OCT system for measuring TGC does not satisfy the accuracy for clinical demand, mainly because of the temporal and spatial variations of living tissues. In this paper, we propose a low magnification OCT (LM-OCT) for noninvasive monitoring of TGC by reducing the spatial resolution and show that LM-OCT significantly improves the accuracy of glucose measurement. The continuous development of LM-OCT in this direction is expected to offer a reliable and noninvasive tool for the clinical monitoring of TGC.

Background In the microcirculation, red blood cells (RBCs) were observed to be confined to an axial stream surrounded by a marginal RBC depleted layer. This axial accumulation of RBCs is considered to arise from the RBC deformability. Objective To quantitatively evaluate the effect of RBC deformability on their axial accumulation at a flow condition comparable to that in arterioles by developing a new observation system for accurate measurements of radial RBC positions in the cross section of capillary tubes. Methods The cross-sectional distributions of normal and hardened RBCs as well as softened RBCs suspended in capillary tube flows were measured with high spatial resolution. A new observation system was developed in which enface views of the cross-section of the tube were obtained at small distances upstream of the outlet at various longitudinal positions in the tube. Results The radial positions of individual RBCs were detected within 1 μm accuracy. It was found that normal and softened RBCs rapidly migrated away from the wall towards the tube axis, whereas glutaraldehyde-hardened RBCs were dispersed widely over the tube cross-section, depending on the concentration of glutaraldehyde solution. Conclusions The newly devised observation system revealed quantitatively the essential role of RBC deformability in their axial accumulation.

Purpose: 11.7 Tesla MRI was examined to detect Virchow–Robin spaces (VRSs) smaller than 100 μm in the rat brain. The effects of maternal exposure to lipopolysaccharide (LPS) were evaluated on basis of the number of dilated VRSs in the offspring rat brain. Methods: T2-weighted MRI with an in-plane resolution up to 78 μm (repetition time = 5000 ms, echo time = 35 ms, slice thickness = 250 μm, imaging plane, coronal) was applied to identify VRSs. The dilated VRSs were counted in the rat brain at 5 and 10 weeks of age. The dams of half the number in each group were treated with LPS during pregnancy, and the remaining half was employed as control. LPS injection in gestation period was used to simulate maternal infections, the method of which was widely accepted as a rat model inducing neuropsychiatric disorders in the offspring. Effects of LPS exposure on the offspring rat brain were statistically investigated. Results: VRSs as small as 78 μm were successfully detected by the ultra high-field MRI. All dilated VRSs were observed within lacunosum molecular layer of hippocampus, and molecular and granular layers of dentate gyrus around hippocampal fissure. In juvenile rats (5 weeks of age), the number of dilated VRSs was significantly increased in the prenatal LPS exposed rat brain (12.9 ± 2.4, n = 7) than in the control (5.3 ± 1.5, n = 7, P < 0.05), while in young adult rats (10 weeks of age), there was no significant difference in the number between the prenatal LPS exposed rat brain (3.6 ± 0.9, n = 5) and the control (2.6 ± 0.4, n = 5). Conclusion: The results of the present study suggested that maternal infection might cause dilatation of VRSs through neural damages especially in the dentate gyrus of the offspring rats. Thus, ultra high-field MRI can offer a promising diagnostic tool capable of determining the location of neonatal brain damage caused by maternal infections.

Three types of focusing positions over a tube cross section are experimentally and numerically found for spherical particles suspended in laminar square tube flows. The focusing patterns are classified into four regimes, depending on the Reynolds number for the particle-to-tube-size ratio ~ 0.1.

Using 11.7T ultra high-field T2-weighted MRI, the present study aimed to investigate pathological changes of primary and secondary trigeminal pathways following neonatal transection of infraorbital nerve in rats. The trigeminal pathways consist of spinal trigeminal tract, trigeminal sensory nuclear complex, medial lemniscus, ventromedial portion of external medullary lamina and ventral posterior nucleus of thalamus. By selecting optimum parameters of MRI such as repetition time, echo time, and slice orientation, this study visualized the trigeminal pathways in rats without any contrast agents. Pathological changes due to the nerve transection were found at 8 weeks of age as a marked reduction of the areas of the trigeminal pathways connecting from the injured nerve. In addition, T2-weighted MR images of the trigeminal nerve trunk and the spinal trigeminal tract suggest a communication of CSF through the trigeminal nerve between the inside and outside of the brain stem. These results support the utility of ultra high-field MRI system for noninvasive assessment of effects of trigeminal nerve injury upon the trigeminal pathways.

The present study aims to validate the applicability of infrared (IR) thermal imaging for the study of brain function through experiments on the rat barrel cortex. Regional changes in neural activity within the brain produce alterations in local thermal equilibrium via increases in metabolic activity and blood flow. We studied the relationship between temperature change and neural activity in anesthetized rats using IR imaging to visualize stimulus-induced changes in the somatosensory cortex of the brain. Sensory stimulation of the vibrissae (whiskers) was given for 10 s using an oscillating whisker vibrator (5-mm deflection at 10, 5, and 1 Hz). The brain temperature in the observational region continued to increase significantly with whisker stimulation. The mean peak recorded temperature changes were 0.048 ± 0.028, 0.054 ± 0.036, and 0.097 ± 0.015°C at 10, 5, and 1 Hz, respectively. We also observed that the temperature increase occurred in a focal spot, radiating to encompass a larger region within the contralateral barrel cortex region during single-whisker stimulation. Whisker stimulation also produced ipsilateral cortex temperature increases, which were localized in the same region as the pial arterioles. Temperature increase in the barrel cortex was also observed in rats treated with a calcium channel blocker (nimodipine), which acts to suppress the hemodynamic response to neural activity. Thus the location and area of temperature increase were found to change in accordance with the region of neural activation. These results indicate that IR thermal imaging is viable as a functional quantitative neuroimaging technique.

Pneumatically driven, diaphragm-type left ventricular assist devices (LVADs) were implanted into 8 goats with profound induced infarction to the left ventricle by using multiple ligations of the left anterior descending (LAD) coronary artery as well as small arteries in the LAD distribution area. Left ventricular diameters, regional myocardial segment lengths, and wall thicknesses were measured by sono-micrometers. After left ventricular function seemed to be recovered, the goats were weaned off the LVADs after a gradual decrease of pump bypass flow over several days. Thereafter, hemodynamic and cardiac parameters were observed for about 1 month more. Three animals recovered successfully owing to the LVAD pumping. Before starting pump-weaning procedures, the bulk mechanical work (BMW) done by the left ventricle during LVAD pumping and under temporary pump-off conditions was 0.08 +/- 0.01 (mean +/- SE) and 0.22 +/- 0.01 W/100 g left ventricular weight (LVW), respectively, while the regional mechanical work done by the normal myocardium (RMWn) was 1.5 +/- 0.4 and 4.3 +/- 0.9 mW/cm3 during pumping and under temporary pump-off conditions, respectively. BMW and RMWn values obtained under pump-on conditions both increased gradually during the weaning process. Even after pump removal, they continued to increase and reached constant values of about 0.3 W/100 g LVW and 10 mW/cm3, respectively, around 2 weeks after pump removal. Although the myocardium in the infarction area did no work for the first several days after surgery, it recovered to do some external work with the aid of LVAD pumping. However, recovery of left ventricular function owed more to compensatory increases in pumping ability of the remaining normal myocardium than to recovery of the damaged myocardium. The LVAD could salvage severely damaged hearts unless the infarction area exceeded 50% of the left ventricular wall.

Fluorescent probes that emit in the near-infrared (NIR, 700-1,300 nm) region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs) have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH)-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs) were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell) QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4). The GSH-QDs (800 nm emission) were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer), and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM), the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIR-fluorescence imaging of a lymph node in a mouse is presented.

The current study was aimed to investigate effects of long-term supplementation of vitamin C on the iris microcirculation in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar-Furth rats by intravenous injection of STZ (55 mg/kg b.w.). The rats were divided into three groups: control rats (CON), STZ-induced diabetic rats (STZ), and STZ rats supplemented with vitamin C (STZ-vitC). For supplementation of vitamin C, ascorbic acid (1 g/l) was added into the drinking water. The experiments were performed at different periods (8, 12, 24 and 36 weeks) after injection of STZ. Blood glucose, tissue lipid peroxidation and plasma vitamin C were measured. To examine the endothelial function, leukocyte adhesion to the venular endothelium was evaluated in the iris post-capillaries by means of counting the number of leukocytes labeled with rhodamine 6G. Blood flow perfusion in the iris was monitored using a laser Doppler flow meter. In the STZ rats, hyperglycemia was induced with an increase in HbA(1c) and lipid peroxidation but with a decrease in the plasma vitamin C level which improved by vitamin C supplementation. The number of adherent leukocytes increased significantly, associated with reduction in the iris blood flow perfusion, at 8, 12, 24 and 36 weeks after injection of STZ. In the STZ-vitC rats, the iris blood flow perfusion was significantly increased in comparison with the STZ rats, while the leukocyte adhesion was decreased at 24 and 36 weeks. The statistical analysis shows that the leukocyte adhesion decreased with increase in the iris blood flow perfusion in STZ and STZ-vitC rats. In conclusion, vitamin supplementation suppressed leukocyte adhesion and thus endothelial dysfunction, associated with increase in iris blood flow perfusion in diabetes. The antioxidant vitamin C may be a therapeutic agent for preventing diabetic retinopathy.

We examined neural plasticity in mice in vivo using optical coherence tomography (OCT) of primary somatosensory (S1) and motor (M1) cortices of mice under the influence of sciatic nerve chronic constriction injury (CCI), a model of neuropathic pain widely utilized in rats. The OCT system used in this study provided cross-sectional images of the cortical tissue of mice up to a depth of about 1 mm with longitudinal resolution up to 11 μm. This is the first study to evaluate neural plasticity in vivo using OCT. CCI mice exhibited cold allodynia and spontaneuos pain behaviors, which are signs of neuropathic pain, 30 days after sciatic nerve ligation, when OCT observation of S1 and M1 cortices was carried out. The scattering intensity of near-infrared light within the hind paw area of S1 and M1 regions in the contralateral hemisphere was significantly higher than in the ipsilateral hemisphere. These CCI-induced increases in scattering intensity within cortical regions associated with the hind paw probably reflect elevated neural activity associated with neuropathic pain. Synapses and mitochondria are believed to have high light scattering coefficients, since they contain remarkably high concentrations of proteins and complicated membrane structure. Number densities of mitochondria and synapses are known to increase in parallel with increases in neural activity. Our findings thus suggest that neuropathic pain gives rise to neural plasticity within the hind paw area of S1 and M1 contralateral to the ligated sciatic nerve.

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.

Female Wistar/ST rats were ovariectomized at 14 weeks of age. Ethynylestradiol, 17β-estradiol (E 2 β), medroxyprogesterone acetate (MPA) and 4-hydroxy-tamoxifen were administered subcutaneously every other day for 3 weeks from 1 week after ovariectomy. Closed cranial windows were created and the thrombotic potential was measured using the He–Ne laser-induced thrombosis method. The mean red cell velocity was measured with a fiber-optic laser-Doppler anemometer microscope. The plasma concentrations of nitrite/nitrate (nitric oxide (NO) metabolites) were determined using the Griess reagent. The diameters mean red cell velocity and blood flow in pial arterioles were reduced after ovariectomy, however, the thrombotic tendency was increased after ovariectomy. These parameters were reversed after E 2 β treatment. In contrast, MPA had the opposite effects. The plasma concentrations of nitrite/nitrate were significantly decreased following ovariectomy and were increased after treatment with E 2 β. MPA did not affect the concentration of NO metabolites. These results strongly indicated that estrogen in the female rat mediated beneficial effects on the cerebral microcirculation and moderated cerebral thrombotic mechanisms by enhancing the plasma levels of NO. The findings suggest that increased blood flow and inhibition of thrombosis might contribute to the prevention of cerebrovascular disease in a normal female.

SUMMARY 1. The effects of oestrogen on thrombogenesis and the cerebral microcirculation of the female rat were studied during the oestrous cycle and after ovariectomy. 2. Serum levels of oestradiol (E2) and plasma concentrations of nitric oxide (NO) metabolites were significantly greater at pro-oestrus than at dioestrus. Blood vessel diameter, mean red cell velocity, wall shear rate and blood flow at pro-oestrus were significantly higher than at dioestrus. Thrombotic tendency, assessed using a He–Ne laser-induced thrombosis model, was significantly decreased at pro-oestrus compared with dioestrus. 3. The long-term deprivation of oestrogen by ovariectomy significantly depressed serum levels of E2 and plasma concentrations of NO metabolites. Thrombotic tendency was significantly increased 4 weeks after ovariectomy. Vessel diameter, mean red cell velocity, wall shear rate and blood flow in pial arterioles were significantly reduced after ovariectomy. 4. Exogenous administration of oestrogen (17β-oestradiol) after surgery reversed the increased thrombotic tendency mediated by ovariectomy. 5. These results strongly indicate that oestrogen mediates beneficial effects on the cerebral microcirculation and moderates cerebral thrombotic mechanisms in the female rat.

The preventive effects of sesamin, a lignan from sesame oil, and vitamin E on hypertension and thrombosis were examined using stroke-prone spontaneously hypertensive rats (SHRSP). At 5 weeks of age the animals were separated into four groups: (i) a control group; (ii) a vitamin E group, which was given a 1,000 mg alpha-tocopherol/kg diet; (iii) a sesamin group, given a 1,000 mg sesamin/kg diet; and (iv) a vitamin E plus sesamin group, given a 1,000 mg alpha-tocopherol plus 1,000 mg sesamin/kg diet for 5 weeks from 5 to 10 weeks of age. Resting blood pressure was measured by the tail-cuff method once weekly. A closed cranial window was created and platelet-rich thrombi were induced in vivo using a helium-neon laser technique. The number of laser pulses required for formation of an occlusive thrombus was used as an index of thrombotic tendency. In control rats, systolic blood pressure and the amount of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) became significantly elevated with age. However, the elevation in blood pressure and 8-OHdG were significantly suppressed in rats administrated vitamin E, sesamin, or vitamin E plus sesamin. At 10 weeks, the number of laser pulses required to induce an occlusive thrombus in arterioles of the control group was significantly lower than in the other groups (p

We assessed the effects of active site-directed, fibrinogen recognition exosite (FRE)-directed and bifunctional thrombin inhibitors, on shear-induced platelet reactivity (adhesion/aggregation) and dynamic coagulation (coagulation of flowing blood). An in vitro test for shear-induced haemostatic plug formation and dynamic coagulation (haemostatometry) was employed using non-anticoagulated rat blood. The active site-directed inhibitors (argatroban, P891, P899) caused inhibition of platelet reactivity and coagulation at 1-, 100- and 100-microM concentrations, respectively. Bifunctional inhibitors (P553, P1053) exerted inhibitory effects at 0.1 microM. A dimeric bifunctional inhibitor P824 caused significant inhibition at 1 microM. The FRE-directed inhibitor (P960) inhibited shear-induced platelet reactivity at 10 microM but the dynamic coagulation at 1 microM. Combination of active site-directed argatroban and FRE-directed P960 did not show any synergistic effect. The most potent inhibition was observed in monomeric bifunctional inhibitors. The inhibitory effects were compared with the K(i) values against human thrombin and with the IC(50) values against fibrin clot formation. The minimum effective concentrations on shear-induced platelet reactivity and dynamic coagulation were comparable with the IC(50) values, but not with the K(i) values.

1. The preventive effects of exercise and L-arginine intake on hypertension and thrombosis in stroke-prone spontaneously hypertensive rats (SHRSP) were studied. 2. Stroke-prone spontaneously hypertensive rats were divided into three groups: (i) the control, sedentary group; (ii) the exercise group, which was allowed to run voluntarily on running wheels; and (iii) the L-arginine intake group, which was given 2.25% L-arginine solution for 8 weeks from 4 to 12 weeks of age. In the control group, one rat died from stroke and symptoms of stroke were observed in the remaining animals. Similar symptoms were recorded in one rat of the exercise group, but not in the L-arginine group. 3. Blood pressure increased in the control group and this increase was suppressed significantly in the exercise and L-arginine groups. Thrombotic potential in cerebral vessels was the lowest at 4 weeks in all groups and was increased significantly at 12 weeks in the control group, but not in the exercise and L-arginine groups. Plasma concentrations of NO2/NO3 were lower in all animals at 12 weeks compared with those at 4 weeks. This reduction was significantly less marked in the L-arginine group. Cerebral arterioles in control rats at 12 weeks of age were significantly smaller in diameter than those at 4 weeks and these changes were less pronounced in the exercise and L-arginine groups. 4. The results provide clear evidence for the beneficial effects of L-arginine intake and voluntary exercise in mechanisms related to hypertension, thrombosis and stroke.

Clopidogrel is a new thienopyridine derivative similar to ticlopidine, which inhibits adenosine diphosphate-induced platelet aggregation. The in vitro effects of clopidogrel on shear-induced platelet activation and coagulation were assessed after oral administration to rats, by subjecting non-anticoagulated blood to haemostatometry. Clopidogrel significantly inhibited shear-induced platelet activation and coagulation 2 h after administration at doses of 7.5 and 15 mg/kg. Both ticlopidine (200 mg/kg) and aspirin (200 mg/kg) inhibited shear-induced platelet activation, but not coagulation. The peak inhibition of plaetelet activation by clopidogrel occurred 2 h after oral administration, but significant inhibition persisted even after 24 h. These results suggest that clopidogrel could be a more potent antithrombotic agent than ticlopidine or aspirin, and also that ADP plays an important role in shear-induced platelet activation.

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

Pneumatically driven, diaphragm type left ventricular assist devices (LVADs) were implanted into 2 goats with normal hearts for approximately 1 month to study the effects of long-term pumping of LVAD on the cardiac mechanics. One sham-operated goat was used to obtain control data. Diameters and myocardial segment lengths of the left ventricle were measured with an ultrasonic displacement meter to calculate the bulk mechanical work (BMW) and regional myocardial mechanical work (RMW), respectively. The LVAD was pumped in the 2:1 drive mode (one counterpulsated pumping in every two cardiac cycles), and was temporarily driven in the 1:1 mode (one pumping in every cardiac cycle) or stopped to obtain the data under these conditions. During the second half of the post-operative period while the animal condition was stable, the BMW in the 2:1 and 1:1 modes were approximately 59% and 72% of that observed under the temporary pump-off condition (0.22 W/(100 g)), respectively. The RMW in the 2:1 and 1:1 modes were 69% and 74% of that obtained during pump-off (6.2 mW/cm3), respectively. The myocyte diameter in the subendocardial layer was reduced by unloading effect of 1-month pumping, whereas those in middle and subepicardial layer showed little change.

Tetrahydrobiopterin (BH4) is an important cofactor for endothelial nitric oxide synthase activity. The relationship between endothelial function in vivo and aortic BH4 level is not fully understood, however. In the present study, we aimed to clarify whether reduction of aortic BH4 levels contributes to endothelial dysfunction in vivo using spontaneously hyperlipidemic mice. To estimate endothelial function in vivo and in real-time state, we developed a flow-mediated vasodilation (FMV) method in mice, which measured changes in the diameter of the femoral artery in response to increased blood flow. C57BL/6 mice and apoE/low-density lipoprotein receptor double knock-out mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 12 weeks from 6 weeks of age. HFD feeding impaired FMV in double knock-out mice, but not in C57BL/6 mice. Furthermore, HFD feeding reduced plasma NOx concentration and aortic BH4 level in double knock-out mice. Conversely, exogenous injection of BH4 (2 mg/kg) markedly increased aortic BH4 levels and restored endothelial function. In conclusion, we demonstrated that HFD feeding impaired nitric oxide-mediated endothelial function and reduced BH4 level in vivo, and that acute augmentation of aortic BH4 levels improved endothelial function. These findings indicate that BH4 is a critical determinant of nitric oxide-mediated endothelial function in hypercholesterolemia.

Gd(3+)-functionalized near-infrared emitting quantum dots were synthesized as a dual modal contrast agent for in vivo fluorescence imaging and magnetic resonance imaging.

Vasoconstriction is known to occur in cerebral arterioles during ischemia and considered to be distinct from vasospasm seen after subarachnoid hemorrhage. To elucidate the mechanism and functional significance underlying ischemic vasoconstriction, we investigated the relationship between arteriolar constriction and tissue energy metabolism during bilateral common carotid artery occlusion in gerbils. Using video microscopy and microspectroscopy, the arteriolar caliber, the total hemoglobin (Hb) content, and the redox state of cytochrome oxidase (cyt.aa3) were monitored in the cerebral cortex in vivo. After in situ freezing of the brain, adenine nucleotides, creatine phosphate (P-Cr), and lactate levels were analyzed using high-performance liquid chromatography in vitro. Tissue damage was also assessed immunohistochemically using antibodies against microtubule-associated proteins. There was a slight reduction of the diameter of pial arterioles during the initial 1 min of ischemia. A rapid decline of total Hb and reduction of cyt.aa3 were observed with rapid decreases of P-Cr and ATP in the cortical tissue during the initial 0.5 min, but all of them showed tendencies to return toward preischemic levels at 0.5-1 min. Beyond 1.5 min, extensive vasoconstriction occurred together with further decline of total Hb, reduction of cyt.aa3, and decreases of ATP and P-Cr. Neuronal damage developed in the cerebral cortex immunohistochemically beyond 3 min. The present investigation demonstrated two phases of vasoconstriction with the possibilities that the immediate vasoconstriction likely contributed to transient improvement of cortical oxygen/energy metabolism, and the second extensive vasoconstriction was an index of tissue energy failure and imminent neuronal damage.

Background. The immunosuppressants cyclosporine and FK506 have been used successfully in clinical transplantation, but both agents have various side effects. We have previously found that cyclosporine is prothrombotic and that FK506 is antithrombotic in an in vivo system. The aim of the present study was to assess the effects of these agents on platelet reactivity and coagulation using an in vitro shear-induced hemostatic platelet plug-forming instrument, the hemostatometer. Methods. A purpose-built hemostatometer was constructed in our laboratory. The effects of cyclosporine and FK506 on platelet reactivity and coagulation were assessed under high shear stress using non-anticoagulated rat and human blood. Results. FK506 significantly inhibited both platelet reactivity and coagulation. Cyclosporine also significantly inhibited coagulation, but a proaggregatory effect was observed at a final blood concentration of 0.05 mg/ml. Conclusions. The present in vitro results support our previous in vivo findings regarding the prothrombotic and antithrombotic effects of cyclosporine and FK506, respectively.

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.

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.

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.

Biological effects of magnetic field and their safety criteria, especially effects of gradient magnetic field on the cerebral and pulmonary circulation during functional brain mapping are still unclear. Here we estimated that magnetically induced artifacts for the blood oxygenation level- and flow- based functional magnetic resonance imaging are less than 0.1%, and disturbance in the pulmonary circulation is less than 1.3% even if the field strength of magnetic resonance system is risen up to 10 tesla. These paramagnetic effects are considered to be small and harmless during human brain mapping.

A technique called optical coherence tomography (OCT) was applied to in vivo observation of microcirculation in the rat cerebral cortex. The OCT system used in this study provided cross-sectional images of the cerebral cortical tissue up to about 1 mm depth with longitudinal resolution up to 8 microm. It could visualize cross-sectional structure of the dura, arachnoid membrane, cortical tissue, and pial microvessels through the cranial window. Pial microvessels with diameter larger than several 10 microm could be detected to observe their cross-sectional shape, while the microvessels within the cortical tissue with smaller diameter were not discernible. The OCT observation revealed that the pial microvessels showed different spatial configurations depending on the cerebral preparations with intact dura and without dura. Stimulus responses of the somatosensory cortices were also different among the preparation methods; Delayed swelling of the cortical surface appeared in the somatosensory cortex following the electrical stimulation of the hind paw in the case of dura removal, which was restricted to a thin surface layer with less than several 10 microm. It is considered to reflect the reactive hyperemia accompanying the neuronal activation. Doppler frequency shift due to the blood flow was detected in pial arterioles. This phenomenon is promising to provide the velocity profile within microvessels and may be applicable to the functional imaging of the brain.

BACKGROUND: The correlation between regional changes in neuronal activity and changes in hemodynamics is a major issue for noninvasive neuroimaging techniques such as functional magnetic resonance imaging (fMRI) and near-infrared optical imaging (NIOI). A tight coupling of these changes has been assumed to elucidate brain function from data obtained with those techniques. In the present study, we investigated the relationship between neuronal activity and hemodynamic responses in the occipital cortex of humans during visual stimulation and in the somatosensory cortex of rats during peripheral nerve stimulation. METHODS: The temporal frequency dependence of macroscopic hemodynamic responses on visual stimuli was investigated in the occipital cortex of humans by simultaneous measurements made using fMRI and NIOI. The stimulus-intensity dependence of both microscopic hemodynamic changes and changes in neuronal activity in response to peripheral nerve stimulation was investigated in animal models by analyzing membrane potential (fluorescence), hemodynamic parameters (visible spectra and laser-Doppler flowmetry), and vessel diameter (image analyzer). RESULTS: Above a certain level of stimulus-intensity, increases in regional cerebral blood flow (rCBF) were accompanied by a decrease in regional cerebral blood volume (rCBV), i.e., dissociation of rCBF and rCBV responses occurred in both the human and animal experiments. Furthermore, the animal experiments revealed that the distribution of increased rCBF and O2 spread well beyond the area of neuronal activation, and that the increases showed saturation in the activated area. CONCLUSIONS: These results suggest that above a certain level of neuronal activity, a regulatory mechanism between regional cerebral blood flow (rCBF) and rCBV acts to prevent excess O2 inflow into the focally activated area.

Previous investigations using an He-Ne laser-induced thrombosis method have shown that stroke-prone spontaneously hypertensive rats (SHRSP) have an enhanced thrombotic tendency in vivo compared to normotensive, Wistar Kyoto rats (WKY). In addition, studies in the presence of acetylcholine have suggested the presence of endothelial dysfunction in SHRSP. In contrast, shear-induced platelet reactivity in vitro appeared to be depressed in SHRSP. The aim of the present study was to investigate endothelial function in SHRSP using a new physiological in vivo model, and to determine the response of platelets to nitric oxide (NO) in non-anticoagulated blood using a shear-induced platelet function in vitro method (haemostatometry). Endothelial function was estimated by measuring flow-mediated vasodilation (FMV) of the femoral artery. Vessels were exposed and blood flow was arrested using a silicone-coated arterial clamp. Vasodilation was measured by computer-assisted image analysis 3 min after release of stasis. Arterial vasodilation was observed in the femoral artery of WKY, but not in SHRSP. Vasodilation was seen in both WKY and SHRSP; however, in response to the NO donor, 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC 5). In contrast, 100 µM NOC 5 did not affect platelet reactivity in SHRSP. The NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, sodium salt (carboxy-PTIO) and the NO synthase inhibitor, NG-nitro-L-arginine methyl ester, hydrochloride (L-NAME), did not affect shear-induced platelet reactivity. NOC 5 at 10 µM (final concentration) inhibited shear-induced platelet reactivity in WKY. These results confirm the presence of endothelial dysfunction in SHRSP and indicate that platelets are non-responsive to NO in this hypertensive model. The data suggest that defective endothelial reactions or disturbed thrombogenic mechanisms outweigh the platelet hyporeactivity and contribute to the prothrombotic status in SHRSP.

Platelet reactivity in stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were compared. In vivo platelet reactivity was tested by the He-Ne laser-induced thrombosis model. The number of laser pulses needed to reach thrombotic occlusion of the targeted vessel was used as an index of thrombogenicity. SHRSP rats needed significantly less number of irradiation to reach occlusion than WKY rats (SHRSP vs. WKY, 5.1+/-0.3 vs. 8.1+/-0.6), indicating enhanced thrombotic response in SHRSP rats. Further, acetylcholine administration significantly increased the number of laser pulses until occlusion in WKY but not in SHRSP rats. This suggests an impaired thrombotic reaction in acetylcholine-treated WKY but not in SHRSP rats. Platelet reactivity in vitro was measured in native blood by a shear-induced haemostasis test (haemostatometry). Indexes of this test (H1/H2), which inversely correlated with platelet reactivity, were significantly greater in SHRSP than in WKY rats (SHRSP vs. WKY, H1: 1815+/-192 vs. 763+/-75; H2: 7547+/-723 vs. 3536+/-264). This suggests reduced platelet reactivity in SHRSP compared with WKY rats. Thus, the present findings show increased thrombotic tendency in SHRSP rats in vivo despite reduced platelet reactivity in vitro. To explain this contradiction, we suggest that an increased in vivo thrombotic tendency may be due to impaired nitric oxide (NO) release from endothelial cells in SHRSP rats, and that a reduced platelet reactivity in vitro may be due to an adaptation of SHRSP rats to survive at extremely high blood pressure.

SummaryInhibitors of angiotensin converting enzyme (ACE) have been developed recently for therapeutic purposes in hypertension and ischemic cardiovascular diseases. Ogiku et al. (1) reported that one such inhibitor, imidapril, significantly prolonged survival in stroke-prone spontaneously hypertensive rats (SHRSP). The present study was designed to investigate the effect of imidapril on cerebral blood vessels in SHRSP to clarify role of the ACE inhibitor in mechanisms of cerebral thrombosis and stroke. Imidapril was administered orally at 1.0 and 5.0 mg/kg/day for 3 weeks from the age of 7 weeks, and was shown to prevent the usual increase in blood pressure seen in these animals. It also delayed He-Ne laser-induced cerebral thrombosis and increased significantly the plasma concentration of nitric oxide metabolites (NO2/NO3). To confirm the association between nitric oxide (NO) and these effects of imidapril, an inhibitor of nitric oxide synthase, NG - nitro-L-arginine methyl ester hydrochloride (L-NAME) was dissolved in drinking water and administered to the animals for 3 weeks. Four of six rats died from stroke when L-NAME was given alone. When imidapril (5.0 mg/kg/day) was administered with L-NAME, however, the animals showed no signs or symptoms of stroke. In these instances, therefore, the concurrent administration of L-NAME with imidapril reversed significantly the effects of imidapril. Intravenous injection of imidaprilat (100 µg/kg), an active metabolite of imidapril, also decreased blood pressure significantly and increased the plasma levels of NO2/NO3 after 5 min. Moreover, imidaprilat enlarged arteriolar diameters and caused an increase in red cell velocity and mean blood flow in pial arterioles after 15 min.The results strongly suggested that imidapril protects cerebral vessels in SHRSP by elevating the release of NO, thereby improving the cerebral circulation and reducing the tendency to thrombosis and stroke.

The effects of exercise and catecholamines on platelet reactivity or coagulation and fibrinolysis appear to be inconsistent. This may be partly due to the methods employed in previous studies. In the present study, we investigated the effects of acute aerobic exercise and catecholamines on the thrombotic status by a novel in vitro method, shear-induced hemostatic plug formation (hemostatometry), using nonanticoagulated (native) blood. Aerobic exercise (60% maximal O2consumption) was performed by healthy male volunteers for 20 min, and the effect on platelet reactivity and coagulation was assessed by performing hemostatometry before and immediately after exercise. Exercise significantly increased shear-induced platelet reactivity, coagulation, and catecholamine levels. The effect of catecholamines on platelet reactivity and coagulation was assessed in vitro by adding catecholamines to blood collected in the resting state. The main findings of the present study are that elevation of circulating norepinephrine at levels that are attained during exercise causes platelet hyperreactivity and a platelet-mediated enhanced coagulation. This may be a mechanism of an association of aerobic exercise with thrombotic risk.

Thrombotic potential and hemodynamic changes were assessed in the cerebral microcirculation in normal rats (WKY), non-stroke-prone spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP) at the age of 4, 16 and 32 weeks. Whole blood platelet aggregation revealed that the platelet aggregability in vitro was significantly depressed in SHRSP compared to WKY at 16 and 32 weeks using 2 μg/ml collagen but was similar or higher than WKY at 32 weeks using 20 or 40 μg/ml collagen. Platelet-rich thrombi were induced using a helium-neon (He-Ne) laser technique in vivo. The numbers of laser pulses required to induce an occlu-sive thrombus in arterioles were similar in the three strains at the age of 4 weeks. In contrast, the numbers of laser pulses needed to induce vascular occlusion in SHR (5.5 ± 0.7; n = 4) and SHRSP (4.9 ± 0.3; n = 4) were lower than in WKY (7.4 ± 0.3; n = 5) at the age of 16 weeks. Similar differences were observed at 32 weeks (SHR 5.8 ± 0.2; n = 6; SHRSP 4.3 ± 0.1; n = 4; WKY 7.0 ± 0.2; n = 7). Red blood cell velocities were measured in pial arterioles using a fiber-optic laser Doppler anemometer microscope. Red cell velocities and wall shear rates in SHR and SHRSP were significantly lower than those in WKY (p < 0.05) at the age of 16 weeks and were markedly lower in SHRSP than in either WKY or SHR at the age of 32 weeks. The plasma concentration of nitrite/nitrate determined by the Griess reaction was significantly reduced in SHRSP at 32 weeks compared with 4-week-old rats. These changes could contribute to the enhanced tendency to cerebro-vascular stroke in SHRSP.

Red cell velocity was measured by a fiber-optic laser-Doppler anemometer microscope in microvessels of the mesentery of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto control rats (WKY) with high temporal resolution. Based on the velocity data, the propagation velocity of flow pulse along single microvessels as well as the arteriovenous distributions of the mean volumetric flow, the amplitude of flow pulsation, and the phase lag of the flow relative to the systemic pressure were analyzed in relationship to the vessel wall rheology and the network topology. The propagation velocity was 3.5–134 cm/s in arterioles of 12–43 microns in diameter, and it increased with increasing diameter. The mean volumetric flow exhibited a power law relationship to the vessel diameter, in which the exponents were 3.16 and 3.30 for arterioles and 3.03 and 2.85 for venules in SHR and WKY, respectively. The exponents for arterioles were > 3 (P < 0.1) and different between SHR and WKY (P < 0.12). The amplitude of the flow pulsation also varied in a power law with the vessel diameter, whose exponents for arterioles were 3.41 and 3.52 for SHR and WKY, respectively. The flow phase lag increased gradually with decreasing vessel diameter in arterioles and increased comparatively rapidly with increasing vessel diameter in venules, which suggests more compliant vessel walls for venules than arterioles. The slope of the increase in the flow phase lag with the vessel diameter in arterioles of SHR was larger than that for WKY (P < 0.05). This difference in the flow phase lag distribution and the difference in the exponent of the mean flow rate vs. diameter relationship may reflect that the number of small arterioles relative to large ones is reduced in SHR microvasculature compared with that in WKY.