Liposome-encapsulated hemoglobin improves energy metabolism in skeletal muscle ischemia and reperfusion in the rat.

The effect of liposome-encapsulated hemoglobin (LEH) was tested in a rodent model of limb ischemia and reperfusion--causing local reperfusion injury and a cascade of systemic responses. Intracellular pH (pHi) and phosphocreatine (PCr)/inorganic phosphate (Pi) ratio were serially monitored using ³¹P-nuclear magnetic resonance spectroscopy with a 2-cm solenoid coil on a rodent hind limb. After baseline measurements, the right hind limb underwent ischemia for 70 min, followed 10 min later by intravenous administration of LEH (10 mL/kg, n = 6), homologous red blood cells (RBCs, n = 6), saline (n = 6), or no treatment (n = 6). Reperfusion was then observed for an additional 60 min. While pHi decreased precipitously after the onset of ischemia and even following reperfusion, LEH-treated rats had significantly milder intracellular acidosis compared with all other groups during ischemia, and after reperfusion as well throughout the observation with the saline-treated rats. In contrast, the PCr/Pi ratio decreased regardless of treatment after ischemia until reperfusion, when the ratio returned toward normal or the energy status improved only in the LEH-treated rats, while the ratio remained depressed in the control animals receiving RBC, saline, or no treatment. Morphological studies 7 days later revealed a tendency toward suppressed mononuclear cell infiltration with preservation of muscular mass and structure in the LEH-treated rats. LEH treatment after early limb ischemia appeared to improve intracellular energy metabolism and eventually preserve skeletal muscle in a rodent model of limb ischemia and reperfusion.

Direct activation of glomerular endothelial cells by anti-moesin activity of anti-myeloperoxidase antibody.

BACKGROUND: Glomerular neutrophil infiltration has been thought to be a key pathological event in the development of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis involving glomerulonephritis. Accordingly, we sought to explore the molecules responsible for glomerular neutrophil accumulation. METHODS: Glomerular neutrophil infiltration and renal chemokine expression in mice treated with anti-MPO IgG were evaluated. Chemokine expression in vitro induced by anti-MPO IgG was measured in the primary mouse glomerular endothelial cells (mGEC). The target molecule reacted with anti-MPO IgG on the mGEC was determined by peptide mass fingerprint analysis. RESULTS: A significant glomerular neutrophil infiltration was observed in the mice administered with anti-MPO IgG. The expressions of CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), were significantly increased in the renal cortex, indicating that these chemokines contribute to the neutrophil infiltration. Based on the previous findings of upregulation of adhesion molecule expression in mGEC treated with anti-MPO IgG, we examined whether mGEC secrete these chemokines in response to anti-MPO IgG. Indeed, anti-MPO IgG induced secretion of KC and MIP-2, leading to neutrophil chemotaxis in vitro. Furthermore, complete depletion of MPO in mGEC and serum using MPO-deficient mice showed an upregulation of intercellular adhesion molecule-1, indicating cross-reactive molecule(s) were existing on mGEC. We identified the molecule as moesin by a proteomic approach. CONCLUSIONS: The endothelial CXC chemokines, KC and MIP-2, contribute to infiltration of neutrophils in MPO-ANCA-associated vasculitis involving glomerulonephritis. The activation of glomerular endothelial cells by anti-MPO IgG appeared to directly involve a signaling through moesin.

Sensory stimulation triggers spindles during sleep stage 2.

STUDY OBJECTIVES: Toward understanding the function of sleep spindle, we examined whether sensory stimulation triggers sleep spindles. PARTICIPANTS: Eleven normal subjects participated in the experiments. INTERVENTION: The subjects had a nap in the afternoon, and sensory stimulation was applied during sleep stage 2. MEASUREMENTS: 21-channel EEG was recorded during the 2-3 hour experiment carried out between 13:00 and 16:00. Somatosensory, auditory, or visual stimulation was performed over a 5-minute period during stage 2. The frequency and duration of spindles were compared in 2 different segments of 5 minutes, with and without sensory stimulation. The latency from the onset of a sensory stimulus to the succeeding spindle was also analyzed. To estimate the active brain regions during a spindle, the EEG recordings were modeled with a single equivalent moving dipole (SEMD) model. RESULTS: In the period with stimulation, spindle frequency and duration increased compared with the period without stimulation. Statistical tests revealed that with stimulation, the interval between 2 consecutive spindles was significantly shorter (p < 0.05, regardless of the modality) and that the duration of the spindles was significantly longer with stimulation (p < 0.05, regardless of the modality). The latency was approximately 2 s. During a spindle after somatosensory stimulation brain activities were observed near the somatosensory area, while with auditory stimulation active regions were observed near the auditory cortex. CONCLUSIONS: A sensory stimulus appeared to trigger a sleep spindle during sleep stage 2. SEMD trajectories suggest that active brain regions during spindle are different according to the modality of the preceding stimulus.

Re-evaluation of tropicamide in the pupillary response test for Alzheimer's disease.

In 1994, a pupillary response test using very dilute (0.01%) tropicamide, a cholinergic antagonist, evoked remarkable pupil dilation in subjects with Alzheimer's disease (AD) and has since been considered a diagnostic tool for AD. However, since this test was first reported, there have been studies suggesting it cannot provide a differential diagnosis of AD. Thus, the present study re-evaluated the pupillary dilation of AD (n=17) and non-AD (n=20) subjects at a 0.01% concentration of tropicamide and found that non-AD subjects, including young subjects, showed the same extent of pupil dilation as AD subjects. Furthermore, there was no significant difference between the average dilation rate of the two groups (P>0.05). When the tropicamide concentration was diluted to half of the initial concentration and performed for AD (n=14), vascular dementia (VD) (n=14), and young (n=16) subjects, the AD subjects showed a large dilation (mean pupil dilation rate: 133.8+/-15%) while the non-AD subjects did not show hypersensitivity to the new concentration of eye drops (105.4+/-9%). These differences in pupillary dilation between AD and non-AD patients were statistically significant (P<0.001). Based on these results, the most effective cutoff point of 0.005% tropicamide for differential diagnosis was 114.5% of the average pupil dilation rate for 60 min.

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.

Development of catheter-type optical oxygen sensor and applications to bioinstrumentation.

A catheter-type optical oxygen sensor based on phosphorescence lifetime was developed for medical and animal experimental use. Since the sensor probe should have biocompatibility and high oxygen permeability in vivo, we focused attention on acceptable polymer materials for contact lenses as the substrates of probes. Pd-porphyrin was doped in silicone-based polymer, and was fixed at the edge of an optical fiber inserted in a catheter tube. The shape of the probe was 600 microm in diameter and 100 microm in thickness, and the probe had high oxygen permeability of Dk value 455. In accuracy evaluation, there found an excellent correlation between the pO2 values measured through phosphorescence lifetime using the oxygen sensors and those measured as the calibrating data using oxygen electrodes. The response time required to achieve 90% from reversible default value to be from 150 to 0 mmHg, and from 0 to 150 mmHg was 15.43 and 7.52 s, respectively. In addition, other properties such as temperature and pH dependency, response, and durability of our optical oxygen sensor were investigated. In animal experiments, the catheter-type oxygen sensor was inserted via the femoral artery of a rat, and arterial oxygen pressure was monitored under asphyxiation. The sensor was valid in the range of oxygen concentration sufficient for biometry, and expected to be integrated with an indwelling needle.

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.

A new method for measuring the oxygen diffusion constant and oxygen consumption rate of arteriolar walls.

Oxygen transport is believed to primarily occur via capillaries and depends on the oxygen tension gradient between the vessels and tissues. As blood flows along branching arterioles, the O(2) saturation drops, indicating either consumption or diffusion. The blood flow rate, the O(2) concentration gradient, and Krogh's O(2) diffusion constant (K) of the vessel wall are parameters affecting O(2)delivery. We devised a method for evaluating K of arteriolar wall in vivo using phosphorescence quenching microscopy to measure the partial pressure of oxygen in two areas almost simultaneously. The K value of arteriolar wall (inner diameter, 63.5 ± 11.9 µm; wall thickness, 18.0 ± 1.2 µm) was found to be 6.0 ± 1.2 × 10(-11) (cm(2)/s)(ml O(2)·cm(-3) tissue·mmHg(-1)). The arteriolar wall O(2) consumption rate (M) was 1.5 ± 0.1 (ml O(2)·100 cm(-3) tissue·min(-1)), as calculated using Krogh's diffusion equation. These results suggest that the arteriolar wall consumes a considerable proportion of the O(2) that diffuses through it.

Combinations of hydrostatic pressure and shear stress influence morphology and adhesion molecules in cultured endothelial cells.

Endothelial cells are exposed to mechanical stimuli from blood flow and blood pressure. However, it is not yet fully understood how their simultaneous exposure affects endothelial function. Firstly, in this study we investigated the effect of combined stress on morphology of cultured human aortic endothelial cells (HAECs). In the results, HAECs exposed to steady flow (a pressure of 100 mmHg, and a shear stress of 1.5 Pa) were more elongated than those exposed to a hydrostatic pressure of 100 mmHg and HAECs exposed to pulsatile flow (a pressure of 80/120 mmHg, and a shear stress of 1.2/1.8 Pa) were more elongated than those exposed to steady flow. Similarly, HAECs exposed to pulsatile flow were most oriented to the flow direction among these three stresses. Secondly, we investigated the effect of combined stress on gene expression of cell adhesion molecules in HAECs. After stress exposure to HAECs the mRNA of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin were measured by real time RT-PCR. In the results, the exposure of steady flow increased the mRNA levels of ICAM-1 compared to the exposure of hydrostatic pressure; however, the exposure of pulsatile flow decreased the mRNA level of ICAM-1 compared to the exposure of steady flow. These findings suggest that gene expression of cell adhesion molecules induced by combined stress were different to the superposition of individual stress and that not only difference in the components of combined stress but also difference in the magnitude of the components of combined stress are important.

Platelet and leukocyte adhesion in the microvasculature at the cerebral surface immediately after subarachnoid hemorrhage.

OBJECTIVE: Pathophysiology after subarachnoid hemorrhage (SAH) caused by aneurysmal rupture has not been well examined. The purpose of this study was to observe platelet-leukocyte-endothelial cell interactions as indexes of inflammatory and prothrombogenic responses in the acute phase of SAH, using an in vivo cranial window method. METHODS: Subarachnoid hemorrhage was induced in C57Bl/6J mice by using the endovascular perforation method. Intravital microscopy was used to monitor the rolling and adhesion of platelets and leukocytes that were labeled with different fluorochromes. Regional cerebral blood flow was measured with laser Doppler flowmetry. The platelet-leukocyte-endothelial cell interactions were observed 30 minutes, 2 hours, and 8 hours after SAH. The effect of P-selectin antibody and apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, on these responses was examined at 2 hours after SAH, and compared with a different SAH model in which autologous blood was injected into the foramen magna. RESULTS: SAH was accompanied by a 60% decrease in regional cerebral blood flow, whereas no changes in regional cerebral blood flow were observed on the contralateral side. SAH elicited time- and size-dependent increases in rolling and adherent platelets and leukocytes in cerebral venules. All of these interactions were attenuated by treatment with a P-selectin antibody or apocynin. There was no significant blood cell recruitment observed in the blood-injected SAH model. CONCLUSION: SAH at the skull base induced P-selectin- and oxygen radical-mediated platelet-leukocyte-endothelial cell interactions in venules at the cerebral surface. These early inflammatory and prothrombogenic responses may cause a whole-brain injury immediately after SAH.

Angiotensin II type 1 receptor signaling contributes to platelet-leukocyte-endothelial cell interactions in the cerebral microvasculature.

Angiotensin II type 1 (AT(1)) receptor signaling has been implicated in cerebral microvascular alterations associated with ischemia, diabetes mellitus, hypercholesterolemia, and atherosclerosis. Platelets, which express AT(1) receptors, also appear to contribute to the thrombogenic and inflammatory responses that are elicited by these pathological conditions. This study assesses the role of AT(1) receptor activation on platelet-leukocyte-endothelial cell interactions elicited in cerebral microvasculature by ischemia and reperfusion. Intravital microscopy was used to monitor the adhesion of platelets and leukocytes that were labeled with different fluorochromes, whereas dihydrorhodamine-123 was used to quantify oxygen radical production in cerebral surface of mice that were either treated with the AT(1) receptor agonist Val-angiotensin II (ANG II) or subjected to bilateral common carotid artery occlusion (BCCAO) followed by reperfusion. ANG II elicited a dose- and time- dependent increase in platelet-leukocyte-endothelial cell interactions in cerebral venules that included rolling platelets, adherent platelets on the leukocytes and the endothelial cells, rolling leukocytes, and adherent leukocytes. All of these interactions were attenuated by treatment with either P-selectin or P-selectin glycoprotein ligand 1 (PSGL-1) antibody. The AT(1) receptor antagonist candesartan and losartan as well as diphenyleneiodonium, an inhibitor of flavoproteins including NAD(P)H oxidase, significantly reduced the platelet-leukocyte-endothelial cell interactions elicited by either ANG II administration or BCCAO/reperfusion. The increased oxygen radical generation elicited by BCCAO/reperfusion was also attenuated by candesartan. These findings are consistent with an AT(1) receptor signaling mechanism, which involves oxygen radical production and ultimately results in P-selectin- and PSGL-1-mediated platelet-leukocyte-endothelial cell interactions in the cerebral microcirculation.

Up-regulation of adhesion molecule expression in glomerular endothelial cells by anti-myeloperoxidase antibody.

BACKGROUND: Anti-neutrophil cytoplasmic antibody directed against myeloperoxidase (MPO-ANCA) has been implicated in pauci-immune crescentic glomerulonephritis. It stimulates primed neutrophils to adhere to glomerular endothelial cells (GECs), thereby releasing reactive oxygen and other toxic substances and ultimately damaging the GECs. Though, a pathogenic role for MPO-ANCA is not fully understood, we hypothesized that MPO-ANCA modulates GEC functions by the increases in expression of adhesion molecules. METHODS: A polyclonal rabbit anti-recombinant mouse MPO antibody (anti-rmMPO IgG) was evaluated in mouse GEC (mGEC) for its effect on adhesion molecule expression. The primary culture of mGEC was incubated with anti-rmMPO IgG or isotype control and the expression of intercellular adhesion molecules-1 (ICAM-1) was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis and ICAM-1 cell ELISA. RESULTS: The real-time RT-PCR analysis showed that a treatment with 100 microg/ml anti-rmMPO IgG increased the expression of mRNAs for ICAM-1, vascular cell adhesion molecule-1 and E-selectin by approximately 12.5, 7.5 and 10.5-fold, respectively. ICAM-1 cell ELISA also substantiated increased expression of ICAM-1. This enhancement of ICAM-1 expression was mediated by the antigen specificity of anti-rmMPO IgG. In addition, there were several proteins in mGEC specifically immunoprecipitated with anti-rmMPO IgG. CONCLUSIONS: These results showed that anti-MPO antibody activates not only neutrophils, but also GEC, indicating that anti-rmMPO IgG-induced direct activation of GEC contributes to neutrophil adhesion to GEC, thereby increasing glomerular neutrophil infiltration in initiation and progression of pauci-immune glomerulonephritis.

A modified back-propagation method to avoid false local minima.

The back-propagation method encounters two problems in practice, i.e., slow learning progress and convergence to a false local minimum. The present study addresses the latter problem and proposes a modified back-propagation method. The basic idea of the method is to keep the sigmoid derivative relatively large while some of the error signals are large. For this purpose, each connecting weight in a network is multiplied by a factor in the range of (0,1], at a constant interval during a learning process. Results of numerical experiments substantiate the validity of the method.

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.

Roles of reactive oxygen species in monocyte activation induced by photochemical reactions during photodynamic therapy.

This study attempts to investigate the mechanism of the vascular shut down (VSD) effect during photodynamic therapy (PDT) with zinc coproporphyrin III tetrasodium salt as a photosensitizer. PDT is a treatment based on photochemical reactions and the resultant cytotoxic reactive oxygen species (ROS). Platelet thrombus formation leading to stasis observed in vivo during PDT is called the VSD effect. Leukocytes play an important role in the VSD effect in vivo, but the mechanism how activated monocytes generate ROS is not known in detail. To evaluate ROS generation by activated monocytes is especially important to clarify leukocyte-endothelium interactions in the VSD mechanism. The dichlorofluorescein fluorescence intensity of monocytes with four types of free radical scavenger was investigated by confocal laser scanning microscopy. The fluorescence intensity of monocytes that had been incubated with superoxide dismutase and incubated and added with L-histidine was decreased by about 20 and 30%, respectively. The result affirms the predominant role of singlet oxygen and superoxide anion radicals in monocyte activation in the VSD effect during PDT.

Chronic stress evaluation using neuro-fuzzy.

The purpose of this research was to evaluate chronic stress using physiological parameters. Wistar rats were exposed to sound stress for 14 days. Biosignals were acquired hourly. To develop a fuzzy inference system that can integrate physiological parameters, the parameters of the system were adjusted by the adaptive neuro-fuzzy inference system. Of the training dataset, the input dataset was the physiological parameters from the biosignals and the output dataset was the target values from the cortisol production. Physiological parameters were integrated using the fuzzy inference system, then 24-hour results were analyzed by the Cosinor method. Chronic stress was evaluated from the degree of circadian rhythm disturbance. Suppose that the degree of stress for initial rest period was 1. Then, the degree of stress after 14-day sound stress increased to 131, and increased to 1.47 after the 7-day recovery period. That is, the rat was exposed to 37%increased amount of stress by the 14-day sound and did not recover after the 7-day recovery period.