Norihiko Tateishi

Effects of GM‐CSF and ordinary supplements on the ramification of microglia in culture: A morphometrical study

Microglia transform from ameboid to ramified cells during development and display an ameboid appearance again under certain pathological conditions. Some cytokines produced by astrocytes may be responsible for the microglial transformation. In the present study, we compared the effects of cytokines, granulocyte/macrophage colony‐stimulating factor (GM‐CSF), macrophage colony‐stimulating factor (M‐CSF), and interleukin‐3 (IL‐3) on the morphology of rat cultured microglia. For quantitative evaluation, we employed “transformation index” as calculated by (perimeter of cell)2/4 π (cell area). GM‐CSF facilitated the ramification of cultured rat microglia, which was effectively induced in a serum‐free medium. However, M‐CSF and IL‐3 did not induce the ramification. A certain serum adhesion protein (possibly vitronectin) as well as other high molecular weight substances in fetal calf serum inhibited the GM‐CSF‐induced microglial ramification. Among ordinary supplements for a chemically defined medium, progesterone, insulin, and a high concentration of glucose suppressed the ramification. These findings suggest that GM‐CSF may be involved in microglial ramification and that many kinds of supplements that are added to culture media profoundly affect the morphology of microglial cells.

FLOW DYNAMICS OF ERYTHROCYTES IN MICROVESSELS OF ISOLATED RABBIT MESENTERY: CELL-FREE LAYER AND FLOW RESISTANCE

A part of microvascular bed isolated from rabbit mesentery (composed of a few branches of superior mesenteric artery) was used for the study of the flow dynamics of erythrocytes in microvessels. The flow resistance in the microvascular bed was analyzed with respect to the thickness of a cell-free layer formed along the inner wall of vessels and the suspension viscosity of erythrocytes (in terms of hematocrit and erythrocyte deformability). The thickness of the cell-free layer increased with the increase in the inner diameter of microvessels. By lowering the hematocrit, the thickness increased and the flow resistance decreased. Meanwhile, by decreasing the erythrocyte deformability with diamide, the thickness decreased and the flow resistance increased. However, the thickness was not altered in microvessels less than 10 microns by the diamide treatment. The maximum inner diameter of microvessel required to induce parachute- and/or slipper-like deformation (at flow velocity of erythrocytes less than 2 mm s-1) was 13 microns for control cells and 6 microns for diamide-treated cells. The thickness of the cell-free layer could not be closely related to the flow resistance, while a good relationship applicable to both control and diamide-treated cells was obtained between the flow resistance and the suspension viscosity. The present results conclude that the flow resistance in the isolated microvascular bed is mainly provided by both hematocrit and erythrocyte deformability.

Three dimensional motion mechanism of ultrasound probe and its application for tele-echography system

We have developed a three dimensional movable robot for an ultrasound probe for tele-echography system to apply between hospital, clinic and home. The probe movable mechanism is carefully designed not to damage the human body since miniature force sensors detect not only total contact force to the body surface but also angle of the probe to the surface. Angle and position of probe are controlled not to exceed dangerous contact force. We tried to control it via LAN and 128K ISDN by using a two-axis joystick under the Object Request Broker technique. We found that real time control was possible with an image stream. Though the delay time of the image makes the examiner stressful, we confirmed that remote diagnosis of the human abdomen is useful.

Deformation of Erythrocytes in Microvessels and Glass Capillaries: Effects of Erythrocyte Deformability

Objective: The deformation of erythrocytes in microvessels less than 15 μm in inner diameter was analyzed using a microvascular bed isolated from rabbit mesentery. The deformation was compared with that found in glass capillaries.

Gamma‐Ray‐Irradiated Red Blood Cells Stored in Mannitol‐Adenine‐Phosphate Medium: Rheological Evaluation and Susceptibility to Oxidative Stress

Background and Objectives: To evaluate the rheological properties and the oxidative susceptibility of γ-ray-irradiated red blood cells (RBCs). Materials and Methods: RBCs in mannitol-adenine-phosphate (MAP) medium were irradiated with 35 Gy and stored at 4°C for 4 weeks. The deformability of the RBCs was examined under shear flow in relation to the morphological and biochemical changes. The RBCs were further exposed to 1 mM FeSO4 and 5 mM ascorbate to examine the oxidative susceptibility. Results: The RBC deformability was decreased during storage, and the impairment was further enhanced by the irradiation, which promoted cell shrinkage and intracellular hemoglobin condensation accompanying potassium loss. Lipid peroxidation and protein aggregation of the RBC membrane as well as echinocytosis were not enhanced by the irradiation. The exposure to free iron did not stimulate the oxidation of the irradiated RBC membrane. Conclusion: The decreased deformability of γ-ray-irradiated RBCs in MAP medium was mainly induced by dehydration due to potassium loss, and the membrane lipids and proteins were stably preserved against oxidative stress.

A method for measuring the rate of oxygen release from single microvessels.

A system determining the rate of oxygen release from erythrocytes flowing in single microvessels was constructed with an inverted microscope by connecting 1) a scanning/grating spectrophotometer equipped with a photon-counting detector through a thin light guide, to obtain the visible absorption spectrum of a spot (5 microns in diameter) focused on a microvessel, 2) two photomultipliers (connected to a microcomputer via an analog-to-digital converter) through two light guides, to determine the flow velocity of erythrocytes by calculating the cross correlation between the light-intensity changes of two spots (3 microns in diameter, 5 microns apart from each other) focused on the microvessel, and 3) an image processor through a video camera, to estimate the diameter of microvessel from the digitized video images. The rate of oxygen release from single microvessels 7-25 microns in diameter in rat mesentery was measured under the superfusion of deoxygenated solution: 1) The maximal rate was obtained in capillaries, and the rate in arterial microvessels was larger than that in venous microvessels, when similar diameters were compared. 2) The rate was maximum at pH 7.0-7.2, and it decreased in more acidic and alkaline pH values. 3) The rate decreased with a decrease in temperature. The reliability of the measurement using the present apparatus was tested in detail.

Flow Behavior of Erythrocytes in Microvessels and Glass Capillaries: Effects of Erythrocyte Deformation and Erythrocyte Aggregation

Flow behavior of erythrocytes in microvessels and glass capillaries with an inner diameter of 10-50 microns was compared in relation to erythrocyte deformation and erythrocyte aggregation. This study was focused on the formation of a marginal cell-free layer, and the thickness was determined using an image processor. Human erythrocytes were perfused through a part of microvascular networks isolated from rabbit mesentery and through glass capillaries. Erythrocyte deformability was modified by treating erythrocytes with diamide, diazene-dicarboxylic acid bis[N,N-dimethylamide], and erythrocyte aggregation was accelerated by adding dextran (with a molecular weight of 70,400) to the perfusion medium. The thickness of the cell-free layer increased with an increase of the inner diameter of flow channel, with lowering the hematocrit, and with increasing the flow velocity of erythrocytes, in both microvessels and glass capillaries. Furthermore, the thickness of cell-free layer decreased with decreasing erythrocyte deformability, while it increased with accelerating erythrocyte aggregation. However, the alteration of the cell-free layer in response to the changes of these hemorheological conditions was more sensitive in microvessels than in glass capillaries. The present study concludes that flow behavior of erythrocytes in microvessels is qualitatively similar to, but quantitatively different from those in glass capillaries, as far as evaluated by the change of the thickness of the marginal cell-free layer.

Reduced oxygen release from erythrocytes by the acceleration-induced flow shift, observed in an oxygen-permeable narrow tube.

The oxygen release from flowing erythrocytes under accelerational force (0-4 g) was examined using an oxygen-permeable, fluorinated ethylenepropylene copolymer tube (25 microm in inner diameter). The narrow tube was fixed vertically on the rotating disk of a new centrifuge apparatus, and erythrocyte suspension was perfused in the direction of Earth gravity. The accelerational force was applied perpendicularly to the flow direction of cells by centrifugation. The microscopic images of the flowing cells obtained at five different wavelengths were analyzed, and marginal cell-free layer and oxygen saturation of the cells were measured. By lowering oxygen tension around the narrow tube, erythrocytes were deoxygenated in proportion to their traveling distance, and the deoxygenation was enhanced with decreasing flow velocity and hematocrit. With increase of the g-value, the shift of flowing erythrocyte column to the centrifugal side was increased, the column was compressed, and the oxygen release from the cells was suppressed. Qualitatively, similar results were obtained by inducing erythrocyte aggregation with Dextran T-70 (MW = 70,400), without accelerational force. These results conclude that both the accumulation of erythrocytes under accelerational force and the enhancement of erythrocyte aggregation by macromolecules lead to the reduction of oxygen release from the flowing cells.

Ginsenoside Rb1 prevents image navigation disability, cortical infarction, and thalamic degeneration in rats with focal cerebral ischemia

Oral administration of red ginseng powder before but not after transient forebrain ischemia prevents delayed neuronal death in gerbils. One neuroprotective molecule within red ginseng powder is ginsenoside Rb 1 . The mechanism of action(s) of ginsenoside Rb 1 remains to be determined. We performed intracerebroventricular infusion of 0.6 μg/d ginsenoside Rb 1 before or after permanent occlusion of the left middle cerebral artery in stroke-prone spontaneous hypertensive rats. Ginsenoside Rb 1 significantly decreased escape latency on repeated trials of the Morris water maze test, throughout the first to fourth trial days at 2 and 4 weeks after MCA occlusion ( P P P 1 was significantly smaller than that in the saline-treated ischemic group ( P P 1 (0.06 μg/d) was less effective and the other doses examined were ineffective in ameliorating ischemia-induced image navigation disability and reducing cortical infarct size. There were significant differences in neuron numbers in the ventroposterior thalamic nucleus and in the left-to-right ratio of the thalamic area between the saline-infused ischemic group and the ginsenoside Rb 1 -treated ischemic group ( P P 1 at concentrations of 0.1 to 100 fg/mL (0.09 to 90 fM), facilitated neurite extension and rescued cortical neurons from lethal damage caused by the free radical-promoting agent FeSO 4 , in vitro ( P P 1 protects the cerebral cortex against lethal ischemic damage possibly by acting as a neurotrophic factor-like agent and by scavenging free radicals, which are overproduced in situ during and after brain ischemia. The final link between the in vivo neuroprotective action and the in vitro neurotrophic and antioxidant activities of ginsenoside Rb 1 remains to be determined.

Electrostatic repulsion among erythrocytes in tube flow, demonstrated by the thickness of marginal cell-free layer.

Electrostatic repulsion among erythrocytes in flow was evaluated through measurement of the thickness of the marginal cell-free layer in narrow glass tubes of 20-50 microns in inner diameter. To reduce the electrostatic repulsive force, due mainly to sialic acid of the membrane glycoproteins, human erythrocytes were treated with neuraminidase. The surface negative charge of the erythrocytes, as determined from the electrophoretic mobility using free-flow electrophoresis, was found to be proportional to the sialic acid content. When erythrocytes with decreased sialic acid content flowed through narrow tubes, the thickness of cell-free layer determined using an image processor increased even in the absence of erythrocyte aggregation in the suspension. The effect was more pronounced at acidic pH. The addition of Dextran T-70 (70,400 Mol. Wt.) further increased the cell-free layer thickness due to erythrocyte aggregation. Thus, reducing the negative charge density on the erythrocyte surface by itself accelerates the axial accumulation of erythrocytes in flow due to the decreased electrostatic repulsive force between the cells, even in the absence of erythrocyte aggregation.