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This paper presents the studies on the erythrocyte aggregation of density-fractionated human erythrocytes, and demonstrates the accelerated aggregation of aged cells at low shear rate. Furthermore, the functional, biochemical and cell geometrical changes (e.g., cellular deformability, content of sialic acid in cell surface, electrophoretic mobility, cell size) are studied

Red blood cells (RBCs) generally deform to adopt a parachute-like, torpedo-like, or other configuration to align and flow through a capillary that is narrower than their major axis. As described herein, even in a narrow tube (25 microm) with diameter much larger than that of a capillary, flowing RBCs at 1 mm/s align axially and deform to a paraboloid shape in a viscous Newtonian fluid (505 kDa dextran medium) with viscosity of 23.4-57.1 mPa.s. A high-speed digital camera image showed that the silhouette of the tip of RBCs fits a parabola, unlike the shape of RBCs in capillaries, because of the longer distance of the RBC-free layer between the tube wall and the RBC surface ( approximately 8.8 microm). However, when RBCs are suspended in a "non-Newtonian" viscous fluid (liposome-40 kDa dextran medium) with a shear-thinning profile, they migrate toward the tube wall to avoid the axial lining, as "near-wall-excess," which is usually observed for platelets. This migration results from the presence of flocculated liposomes at the tube center. In contrast, such near-wall excess was not observed when RBCs were suspended in a nearly Newtonian liposome-albumin medium. Such unusual flow patterns of RBCs would be explainable by the principle; a larger particle tends to flow near the centerline, and a small one tends to go to the wall to flow with least resistance. However, we visualized for the first time the complete axial aligning and near-wall excess of RBCs in the noncapillary size tube in some extreme conditions.

Reelin plays an important role in the migration of embryonic neurons, but its continuing presence suggests additional functions in the brain. We now report a novel function where reelin protects P19 embryonal cells from apoptosis during retinoic acid-induced neuronal differentiation. This increased survival is associated with reelin activation of the phosphatidyl-inositol-3-kinase (PI3 K)/Akt pathway. When PI3 K was inhibited with LY294002, reelin failed to protect against this retinoic acid-induced apoptosis. The protective effect of reelin includes activating the Src-family kinases/PI3 K/Akt pathway which then led to selective phosphorylation of Bcl-2/Bcl-XL associated death promoter (BAD) at serine-136, while the phosphorylation-incompetent mutation of BAD (S136A) suppressed this protection. These and additional studies define a novel pathway where reelin binds apoE receptors, significantly activates the PI3 K/Akt pathway causing phosphorylation of BAD which helps to protect cells from apoptosing, thus serving an important role in promoting the survival of maturing neurons in the brain.

The reactivity of endothelial cells (ECs) to proinflammatory cytokines is critically important for the pathogenesis of vascular diseases. Here, we studied functional alterations of human ECs during culture under a confluent condition; i.e., the alterations of neutrophil-activating activity, platelet-activating factor (PAF) synthesis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in cytokine-stimulated ECs. Human umbilical vein-derived ECs exhibited the increased activity in neutrophil activation, PAF synthesis, and GM-CSF production when stimulated by proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). The activity of cytokine-stimulated ECs to stimulate superoxide release in human neutrophils and to produce PAF declined markedly in parallel as ECs became growth-arrested during culture under a confluent condition. By contrast, GM-CSF production induced by cytokine stimulation was modestly increased, and up-regulation of intercellular adhesion molecule-1 (ICAM-1) and activation of mitogen-activated protein kinases were not altered. The neutrophil-activating activity of cytokine-stimulated ECs was dependent on PAF synthesis and GM-CSF production from ECs. These findings indicate that the reduced neutrophil-activating activity in growth-arrested ECs may be, at least in part, ascribed to down-regulation of PAF synthesis.

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb1 (gRb1) (C54H92O23, molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb1-induced expression of gene products responsible for neuronal death or survival, we showed that gRb1 stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-xL in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb1 treatment. Ginsenoside Rb1 appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

Electrostatic repulsive force among human red blood cells (RBCs) was observed by macromolecules-induced RBC aggregation and RBC flow behavior in narrow tube, by reducing the sialic acid content of RBCs with neuraminidase. The electrophoretic mobility of the RBCs was proportional to the sialic acid content. (1) When the sialic acid content was reduced, the RBC aggregation observed with a low shear rheoscope was enhanced with fibrinogen (MW=340,000), Ig G (MW=160,000) or Dextran T-70 (MW=70,400), but did not without the macromolecules. In coexistence of normal cells, sialic acid-reduced cells settled significantly faster, possibly due to their preferential aggregation by the macromolecules. (2) Sialic acid-reduced RBCs accumulated more to flow axis in narrow tube (20-50 μm in inner diameter), especially at acidic pH, as evaluated by the thickness of marginal cell-free layer using an image processor. The thickness was further increased with Dextran T-70 due to the RBC aggregation. Conclusively, electrostatic repulsive force among RBCs provided by sialic acid is important for understanding hemorheological behavior of the RBCs and circulatory disturbances in various diseases.

The levels of plasma HDL cholesterol and apoA-I in NFkappaB p50 subunit-deficient mice were significantly higher than those in wild-type mice under regular and high fat diets, without any significant difference in the level of total cholesterol. To examine the role of NFkappaBin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma HepG2 cells. Lipopolysaccharide-induced activation of NFkappaB reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated expression of IkappaBalpha super-repressor ameliorated the reduction. This IkappaBalpha-induced apoA-I increase was blocked by preincubation with MK886, a selective inhibitor of peroxisome proliferator-activated receptor alpha (PPARalpha), suggesting that NFkappaB inactivation induces apoA-I through activation of PPARalpha. To further support this idea, the expression of IkappaBalpha increased apoA-I promoter activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPARalpha-binding site in the apoA-I promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NFkappaB increases apoA-I and HDL cholesterol through activation of PPARalpha in vivo and in vitro. Our data suggest a new aspect of lipid metabolism and may lead to a new paradigm for prevention and treatment of atherosclerotic disease.

The effects of the oxygenation-deoxygenation process on red blood cell (RBC) aggregation were examined in relation to morphological changes in RBCs and the contribution of CO2. A low-shear rheoscope was used to measure the rate of rouleaux (one-dimensional aggregate) formation in diluted autologous plasma exposed to gas mixtures with different Po2and Pco2. RBC indexes and RBC suspension pH were measured for the oxygenated or the deoxygenated condition, and the cell shape was observed with a scanning electron microscope. In the oxygenation-deoxygenation process, the rate of rouleaux formation increased with rising pH of the RBC suspension, which was lowered in the presence of CO2. The rate increased with increasing mean corpuscular hemoglobin concentration (thus the cells shrank), which increased with rising pH and decreased in the presence of CO2. With rising pH, cell diameter increased and cell thickness decreased (thus the cell flattened). In addition, slight echinocytosis was induced in the presence of CO2, and the aggregation was reduced by the morphological change. In conclusion, RBC aggregation in the oxygenation-deoxygenation process is mainly influenced by the pH-dependent change in the surface area-to-volume ratio of the cells, and the aggregation is modified by CO2-induced acidification and the accompanying changes in mean corpuscular hemoglobin concentration and cell shape.

Aquaporin-4 (AQP4) is located on astrocyte endfeet that face blood vessels in the brain and in the pia. It is thought to play a crucial role in the development of brain edema. To confirm the notion that sex steroids and dexamethasone influence brain edema through AQP4 regulation, we investigated the effects of 17β-estradiol, testosterone, and dexamethasone on the expression of AQP4 in cultured astrocytes. Testosterone significantly up-regulated AQP4 at the level of both protein and mRNA. At a concentration of 100 nM, testosterone significantly increased AQP4 protein levels and ameliorated the osmotic fragility of astrocytes from hypoosmotic stress, suggesting that the increased levels of AQP4 facilitated the testosterone function. Moreover, this effect was attenuated by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate, which can rapidly decrease AQP4 mRNA expression, indicating that the response was specific. These results indicate that AQP4 can alter the osmotic fragility of astrocytes and that testosterone can influence brain edema through AQP4 regulation, whereas 17β-estradiol and dexamethasone cannot. © 2003 Wiley-Liss, Inc.

Microglial cells rapidly become activated in response to even minor damage of neurons, suggestive of the intimate interactions between neurons and microglial cells. Although mediators for microglia-neuron interactions have not been well identified, neurotransmitters are possible candidates transmitting signals from neurons to microglial cells. Among the neurotransmitters, we focused on the effects of norepinephrine and other adrenergic agonists on the functions of rat cultured microglial cells. Reverse transcriptase polymerase chain reaction studies revealed that microglial cells expressed mRNAs encoding alpha1A, alpha2A, beta1 and beta2 receptors. Norepinephrine and a beta2 adrenergic agonist terbutaline elevated intracellular cAMP level of microglial cells. Norepinephrine, an alpha1 agonist phenylephrine, a beta1 agonist dobutamine and terbutaline suppressed the expressions of mRNAs encoding pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor alpha. Release of tumor necrosis factor alpha and nitric oxide was suppressed by norepinephrine, phenylephrine, dobutamine and terbutaline. An alpha2 agonist clonidine and dobutamine upregulated the expression of mRNA encoding catechol-O-methyl transferase, an important enzyme to degrade norepinephrine. Norepinephrine, dobutamine and terbutaline upregulated the expressions of mRNA encoding 3-phospshoglycerate dehydrogenase, an essential enzyme for synthesis of L-serine and glycine, which are amino acids necessary for neuronal survival. Clonidine upregulated the expression of mRNA encoding an anti-apoptotic factor Bcl-xL. These results suggest that norepinephrine participates in the regulation of brain function at least partly by modulating the functions of microglia.

We investigated the effects of inhibitors of cAMP-specific phosphodiesterase type IV (PDE IV) on cultured rat microglial cells. Microglial cells expressed mRNA encoding PDE IV. Rolipram and RO-20-1724, specific inhibitors of PDE IV, elevated the intracellular cAMP level much higher than the other types of PDE inhibitors. cAMP in astrocytes but not in cerebrocortical neurons was similarly increased in response to treatment with PDE IV inhibitors examined. The PDE IV inhibitors, a beta-adrenergic agonist isoproterenol and an adenylyl cyclase stimulant forskolin suppressed the proliferation of microglial cells as revealed by PCNA-immunocytochemical staining. The PDE IV inhibitors suppressed release of TNF alpha and nitric oxide (NO) from lipopolysaccharide-activated microglial cells in pure culture, while they did not affect NO release from microglial cells in neuron-microglia coculture. The PDE IV inhibitors also suppressed superoxide anion production by phorbol ester-treated microglial cells. Isoproterenol and forskolin similarly suppressed the macrophage-like functions of activated microglial cells. However, the PDE IV inhibitors displayed novel effects distinct from those of isoproterenol, forskolin and 8Br-cAMP, regarding expression of mRNAs encoding PDE IV, metallothionein-1 and hemeoxigenase-1. The present data showed that the PDE IV inhibitors can be available to control microglial function and that their effects on glial cells should be taken into account when PDE IV inhibitors are used for treatment of brain diseases, such as multiple sclerosis.

The effects of erythrocyte aggregation on O2release were examined using O2-permeable fluorinated ethylenepropylene copolymer tubes (inner diameter, 25 μm; outer diameter, 100 μm). Measurements were performed using an apparatus built on an inverted microscope that contained a scanning-grating spectrophotometer with a photon count detector connected to two photomultipliers and an image processor through a video camera. The rate of O2release from the cells flowing in the narrow tube was determined based on the visible absorption spectrum and the flow velocity of the cells as well as the tube size. When the tube was exposed to nitrogen-saturated deoxygenated saline containing 10 mM sodium dithionite, the flowing erythrocytes were deoxygenated in proportion to the traveling distance, and the deoxygenation at a given distance increased with decreasing flow velocity and cell concentration (hematocrit). Adding Dextran T-70 to the cell suspension increased erythrocyte aggregation in the tube, which resulted in suppressed cell deoxygenation and increased marginal cell-free-layer thickness. The deoxygenation was inversely proportional to the cell-free-layer thickness. The relation was not essentially altered even when the medium viscosity was adjusted with Dextran T-40 to remain constant. The rate of O2release from erythrocytes in the tube was discussed in relation to the O2diffusion process. We conclude that the diffusion of O2from erythrocytes flowing in narrow tubes is inhibited primarily by erythrocyte aggregation itself and partly by thickening of the cell-free layer.

Microglia are well known to become activated during various kinds of neuropathological events. The factors that are responsible for the activation, however, are not fully determined. In the present study, L-Ser was shown to enhance production of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) by lipopolysaccharide (LPS)-stimulated cultured rat microglial cells. L-Ser, however, did not enhance the expression of mRNAs encoding inducible NO synthase, IL-6 and TNF alpha. On the other hand, astrocytes did not depend on L-Ser for release of IL-6 and TNF alpha. The expression of an enzyme 3-phosphoglycerate dehydrogenase (3PGDH), which is essential for L-Ser biosynthesis from a glycolytic intermediate 3-phosphoglycerate, was investigated. As revealed by Western blotting and immunocytochemical staining, 3PGDH-protein expression in vitro was the highest in astrocytes, intermediate in neurons and the lowest in microglial cells. Semiquantitative RT-PCR showed that microglial cells expressed 3PGDH-mRNA at a lower level than astrocytes. In frozen sections from rat forebrain, only astrocytes were immunoreactive for 3PGDH. The present study suggested that L-Ser is able to modulate microglial function mainly at the translation level because microglial cells cannot synthesize sufficient amount of L-Ser due to the scarce expression of 3PGDH.

To evaluate the rheological properties and the oxidative susceptibility of gamma-ray-irradiated red blood cells (RBCs).RBCs in mannitol-adenine-phosphate (MAP) medium were irradiated with 35 Gy and stored at 4 degrees 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 FeSO(4) and 5 mM ascorbate to examine the oxidative susceptibility.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.The decreased deformability of gamma-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.

Cytochrome c was detected by immunoblotting in the cytosolic fraction 3 h after 5-min ischemia in the non-ischemia-tolerant CA1 region in which about 96% of neurons had developed delayed neuronal death, while less cytosolic cytochrome c was detected in the ischemia-tolerance-induced CA1 region where many more neurons survived. In the immunohistochemical study using anti-non-native cytochrome c monoclonal antibody, immunoreactivity was observed throughout the cytoplasm in the non-ischemia-tolerant CA1 neurons, but not in the normal and ischemia-tolerant CA1 neurons. Then we determined whether Bcl-2, Bax, Bcl-xL and Bcl-xS, which regulate the release of cytochrome c from mitochondria, were altered in the ischemia-tolerant CA1 region. Bcl-2 and Bax were up-regulated in the ischemia-tolerant group, but Bcl-xL and Bcl-xS showed no apparent difference in their expression. These results suggest that cytochrome c release is prevented in CA1 neurons in gerbils in which ischemia-tolerance had been induced and that the altered ratio of Bcl-2 to Bax may play a part in this mechanism.

Adult T-cell leukemia (ATL) is characterized by infiltration of various tissues by circulating ATL cells, a finding often associated with a poor prognosis. Leukocyte migration from the circulation into tissues depends on integrin-mediated adhesion to the endothelium, and integrins are tightly regulated by several factors, such as chemokines. In this study, we focused on the interaction between chemokines and chemokine receptors on ATL cells to understand factors involved in ATL cell infiltration of lymphoid organs. We compared freshly isolated ATL cells from patients with and without lymphoid organ involvement for the expression of the chemokine receptor CCR7/EBI1, the functional receptor for secondary lymphoid-tissue chemokine (SLC), which is expressed at high levels by high endothelial venules of lymph nodes and Peyer's patches. Reverse transcriptase-polymerase chain reaction and flow cytometric analysis, using anti-CCR7 monoclonal antibody (CCR7.6B3), revealed that ATL cells from patients with lymphoid organ involvement expressed significantly more CCR7/EBI1 than control CD4+CD45RO+ T cells and ATL cells from patients without lymphoid organ involvement. Consequently, significantly more ATL cells from patients with lymphoid organ involvement than control CD4+CD45RO+ T cells and ATL cells from patients without lymphoid organ involvement adhered to surfaces coated with ICAM-1 and SLC or EBI1-ligand chemokine (ELC), another ligand for CCR7/EBI1, under static and flow conditions and migrated toward SLC or ELC at a low concentration (30 ng/ml). These findings suggest that increased CCR7/EBI1 expression plays a role in lymphoid organ infiltration of ATL cells. (Blood. 2000; 30-38)

Reactive oxygen and nitrogen species (RO/NS) such as nitric oxide (NO), hydroxyl radical (OH.), and superoxide anion (O(2)(-)) are generated in a variety of neuropathological processes and damage neurons. In the present study, we investigated the neuroprotective effects of rat astrocytes against RO/NS-induced damage using neuron-glia cocultures, and the effects were compared to those of microglial cells. Sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and FeSO(4) were used to generate NO, O(2)(-) and NO, and OH., respectively. Solely cultured neurons, which were transiently exposed to these agents, degenerated, possibly through apoptotic mechanisms as revealed by in situ detection of DNA fragmentation, whereas neurons cocultured with either astrocytes or microglial cells were viable even after exposure to RO/NS. In contrast, most neurons cocultured with meningeal fibroblasts degenerated. Astrocyte-conditioned medium partially attenuated RO/NS-induced neuronal damage. When neurons were cultured on astrocyte-derived extracellular matrix (AsECM), neuronal death induced by SNP and FeSO(4) was almost completely inhibited. AsECM contained significant amounts of laminin and fibronectin, and pure fibronectin and laminin also protected neurons against RO/NS-induced damage in the same manner as AsECM. These results suggest that astrocytes can protect neurons against RO/NS-induced damage by secreting soluble and insoluble factors.

The present study was designed to evaluate the effects of free iron on normal red blood cells (RBCs) and possible mechanisms responsible for the alteration of the RBC rheological properties. Human RBCs (Ht 5%) were incubated for 1 h at 37°C with 0–2 mM FeSO 4 in the presence of ascorbate. Thiobarbituric acid reactive substances assay was employed to estimate lipid peroxidation and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate to analyze membrane proteins. A cone-plate viscometer, and low-shear and high-shear rheoscopes were used to compare the rheological parameters in Fe 2+ /ascorbate-treated RBCs with those of diamide-treated ones. The incubation of RBCs with free iron caused a dose-dependent increase in lipid peroxidation, enhanced binding of hemoglobin to the cell membrane, diminishing of band 3 content and the concomitant increase in lower molecular weight peptides (approximately 60 and 35 kDa), while the incubation with diamide resulted in spectrin crosslinking. Both diamide and Fe 2+ /ascorbate treatment impaired RBC deformability and aggregation. The suspensions of Fe 2+ -treated RBCs manifested increased viscosity, presumably due to enhanced formation of three-dimensional aggregates. In conclusion, although in both these cases of oxidative stress the ultimate result was the deterioration of membrane structure and functions, the mechanisms of oxidative changes of the RBCs rheological properties were apparently dissimilar.

Thiol-compounds, dithiothreitol (DTT) and N -acetylcysteine (NAC), were examined for their antioxidant effects on Fe 2+ /ascorbate-treated and diamide-treated red blood cells (RBCs). Both of the compounds protected RBCs against diamide-induced spectrin crosslinking; they also prevented loss of deformability and aggregation impairment in diamide-treated cells. Neither DTT nor NAC were effective in suppressing lipid peroxidation induced by Fe 2+ /ascorbate treatment. DTT showed a limited protective effect on iron-mediated band 3 fragmentation and RBC deformability, but it was capable of preventing the decrease of rouleaux formation rate to some extent. NAC exhibited a dose-dependent band 3 protective activity, partially prevented Fe 2+ /ascorbate-induced deformability impairment and the decrease of rouleux formation rate. These findings suggest that thiol compounds are effective in modulating diamide-induced oxidative alterations of RBCs membrane constituents and rheological properties. NAC is more efficacious than DTT in protecting RBCs against iron-mediated oxidative injury, which can be attributed to its potent reductant and radical scavenging abilities.

It was previously reported that prosaposin possesses neurotrophic activity that is ascribed to an 18-mer peptide comprising the hydrophilic sequence of the rat saposin C domain. To evaluate the effect of the 18-mer peptide on ischemic neuronal damage, the peptide was infused in the left lateral ventricle immediately after occlusion of the left middle cerebral artery (MCA) in stroke-prone spontaneously hypertensive (SP-SH) rats. The treatment ameliorated the ischemia-induced space navigation disability and cortical infarction and prevented secondary thalamic degeneration in a dose-dependent manner. In culture experiments, treatment with the 18-mer peptide attenuated free radical-induced neuronal injury at low concentrations (0.002 to 2 pg/mL), and the peptide at higher concentrations (0.2 to 20 ng/mL) protected neurons against hypoxic insult. Furthermore, a saposin C fragment comprising the 18-mer peptide bound to synaptosomal fractions of the cerebral cortex, and this binding decreased at the 1st day after MCA occlusion and recovered to the preischemic level at the 7th day after ischemia. These findings suggest that the 18-mer peptide ameliorates neuronal damage in vivo and in vitro through binding to the functional receptor, although the cDNA encoding prosaposin receptor has not been determined yet.

Although microglial cells are well known to become activated in the pathological brain, mechanisms underlying the microglial activation are not fully understood. In the present study, with an aim to elucidate whether neurons are involved in the microglial activation, we compared the morphology and the superoxide anion (O2−)-generating activity of rat microglial cells in pure culture with those of cells cocultured with rat primary cortical neurons. Microglial cells in pure culture in serum-free Eagle's minimum essential medium on poly- l -lysine-coated coverslips displayed ramified morphology and suppressed activity of O2−generation. In contrast, microglial cells in neuron–microglia coculture under the same conditions as those for the pure culture displayed ameboid shape and upregulated activity of O2−generation. Electron microscopic observation revealed that microglial cells in coculture were more abundant in Golgi apparatus and secretory granules than those in pure culture and that some of microglial cells in the vicinity of neurites exhibited membrane specialization reminiscent of a junctional apparatus with high electron density between a microglial soma and a neurite. Microglial cells in coculture tended to tie neurites in bundles by extending processes. Medium conditioned by neurons significantly enhanced O2−generation by microglia, but microglial cells in contact with or in close apposition to cocultured neurons were much more intensely activated than those remote from the neurons. Furthermore, the membrane fraction of cortical neurons activated microglial cells, and this effect was abolished by treating the neuronal membrane with trypsin or neuraminidase. In conclusion, neuronal–microglial contact may be necessary to mediate microglial activation. The present findings suggest that the contact of microglia with damaged neurons in the brain is a plausible cause to activate microglia in the neuropathological pro cesses.

Cultured microglial cells usually exhibit ameboid morphology and peripheral macrophage-like properties, which are distinct from those observed in the normal mature brain. This might be caused by the inappropriate culture of microglial cells in high concentrations (approximately 200-400 microM) of Gly and Ser, although the concentrations of the amino acids in extracellular spaces of the brain parenchyma are quite low (approximately 5 microM). In the present study, we focused on the concentration-dependent effects of glycine (Gly) and serine (Ser) on microglial morphology and function. Under Gly/Ser-free and serum-free condition, the majority of rat microglial cells displayed round morphology, whereas in the presence of 5 microM Gly and 25 microM Ser, which correspond to the concentrations of Gly and Ser in the cerebrospinal fluid, they extended multiple branched processes and formed clusters of rough endoplasmic reticulum. On the other hand, Gly and Ser did not affect morphology of astrocytes. The viability of microglia was not affected by the changes in the concentrations of Gly and Ser. Metabolic activity, activities of acid phosphatase and inducible nitric oxide synthase, and superoxide anion (O2-) generation were all strongly suppressed in Gly/Ser-free medium or in medium containing physiological concentrations of both amino acids. Such activities were all enhanced in harmony with increases in the concentrations of Gly and Ser. Thus, microglial cells cultured in Gly/Ser-free medium, even though exhibiting ameboid morphology, appears to be in the functionally resting state. Furthermore, once the resting state was achieved, the microglial cells remained inactive even after the subsequent 24 h culture in serum-supplemented medium containing 400 microM of both amino acids. The medium conditioned by microglial cells that were cultured in the presence of 400 microM of Gly and Ser was toxic to cortical neurons, whereas the microglia-conditioned medium obtained in the absence of both amino acids facilitated the survival of cortical neurons. Therefore, microglial cells in the resting state, which was induced in the Gly/Ser-free condition, are likely to support neurons. Microglial cells could ramify on glass coverslips coated with astrocyte-derived extracellular matrix or on coverslips coated thinly with fibronectin and/or laminin even under the Gly/Ser-free condition. The ramified cells as induced in this way kept suppressed O2- generating activity. These findings suggest that resting ramified microglial cells with a neurotrophic activity can be induced with the combination of Gly/Ser-free medium and small amounts of extracellular matrix proteins, and that the resting state is rather stable.

We investigated lipopolysaccharide (LPS)-induced nitric oxide (NO) production by rat microglia in neuron-microglia and astrocyte-microglia cocultures to evaluate the influence of neurons and astrocytes on microglial activity. Microglial cells solely cultured in medium devoid of serine (Ser), glycine (Gly) hardly expressed inducible NO synthase (iNOS), while those cocultured with neurons and astrocytes expressed iNOS. When microglial cells and astrocytes were separately cultured by using tissue culture inserts, which allowed the microglial cells to be exposed to only diffusible factors arising from astrocytes, NO production was significantly enhanced. On the other hand, neurons, when separated from microglial cells by the inserts, could not activate microglial cells possibly due to lacking of direct contact between neurons and microglial cells. NO production in pure microglial cultures was significantly enhanced in the presence of Ser/Gly at concentrations higher than 25 microM. Conditioned media obtained from microglia culture and neuron-microglia coculture contained less than 10 microM of Ser and Gly, while media from astrocyte culture and astrocyte-microglia coculture contained 33-41 microM Ser and 20-26 microM Gly. Accordingly, astrocytes modulate the activity of microglial cells by secreting Ser and Gly. The present study proposes a novel metabolic coupling between astrocytes and microglial cells via amino acids.

In the central nervous system, interleukin (IL)-3 has been shown to exert a trophic action only on septal cholinergic neurons in vitro and in vivo, but a widespread distribution of IL-3 receptor (IL-3R) in the brain does not conform to such a selective central action of the ligand. Moreover, the mechanism(s) underlying the neurotrophic action of IL-3 has not been elucidated, although an erythroleukemic cell line is known to enter apoptosis after IL-3 starvation possibly due to a rapid decrease in Bcl-2 expression. This in vivo study focused on whether IL-3 rescued noncholinergic hippocampal neurons from lethal ischemic damage by modulating the expression of Bcl-xL, a Bcl-2 family protein produced in the mature brain. 7-d IL-3 infusion into the lateral ventricle of gerbils with transient forebrain ischemia prevented significantly hippocampal CA1 neuron death and ischemia-induced learning disability. TUNEL (terminal deoxynucleotidyltransferase–mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling) staining revealed that IL-3 infusion caused a significant reduction in the number of CA1 neurons exhibiting DNA fragmentation 7 d after ischemia. The neuroprotective action of IL-3 appeared to be mediated by a postischemic transient upregulation of the IL-3R α subunit in the hippocampal CA1 field where IL-3Rα was barely detectable under normal conditions. In situ hybridization histochemistry and immunoblot analysis demonstrated that Bcl-xL mRNA expression, even though upregulated transiently in CA1 pyramidal neurons after ischemia, did not lead to the production of Bcl-xL protein in ischemic gerbils infused with vehicle. However, IL-3 infusion prevented the decrease in Bcl-xL protein expression in the CA1 field of ischemic gerbils. Subsequent in vitro experiments showed that IL-3 induced the expression of Bcl-xL mRNA and protein in cultured neurons with IL-3Rα and attenuated neuronal damage caused by a free radical–producing agent FeSO4. These findings suggest that IL-3 prevents delayed neuronal death in the hippocampal CA1 field through a receptor-mediated expression of Bcl-xL protein, which is known to facilitate neuron survival. Since IL-3Rα in the hippocampal CA1 region, even though upregulated in response to ischemic insult, is much less intensely expressed than that in the CA3 region tolerant to ischemia, the paucity of IL-3R interacting with the ligand may account for the vulnerability of CA1 neurons to 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 microg/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.05, P.01 or P.001). The ratio of the infarcted area to the left hemispheric area in the groups treated with 0.6 microg/d of ginsenoside Rb(1) was significantly smaller than that in the saline-treated ischemic group (P.05 or P.001). The continuous infusion of ginsenoside Rb(1) (0.06 microg/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.05 or P.01). Moreover, ginsenoside Rb(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.05 or P.01). These findings suggest that ginsenoside Rb(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.

Beta-estradiol has been considered to be a neurotrophic agent, but its in vivo effect on gerbils with transient forebrain ischemia has not yet been demonstrated. In the first set of the present experiments, we infused beta-estradiol at a dose of 0.05 or 0.25 microg/day for 7 days into the lateral ventricles of normothermic gerbils starting 2 h before 3-min forebrain ischemia. Beta-estradiol infusion at a dose of 0.25 microg/day prevented significantly the ischemia-induced reduction of response latency time as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the hippocampal CA1 region as well as synapses within the strata moleculare, radiatum and oriens of the region were significantly more numerous in gerbils infused with beta-estradiol than in those receiving saline infusion. Beta-estradiol at a dose of 1.25 microg/day was ineffective and occasionally increased the mortality of experimental animals. Since the total brain content of exogenous beta-estradiol at 12 h after forebrain ischemia was estimated to be less than 145 ng, the second set of experiments focused on the neurotrophic action of beta-estradiol at concentrations around 100 ng/ml in vitro. Beta-estradiol at concentrations of 1-100 ng/ml facilitated the survival and process extension of cultured hippocampal neurons, but it did not exhibit any significant radical-scavenging effects at the concentration range. On the other hand, 100 microg/ml of beta-estradiol, even though failing to support hippocampal neurons in vitro, effectively scavenged free radicals in subsequent in vitro studies, as demonstrated elsewhere. These findings suggest that beta-estradiol at a dose of 0.25 microg/day prevents ischemia-induced learning disability and neuronal loss at early stages after transient forebrain ischemia, possibly via a receptor-mediated pathway without attenuating free radical neurotoxicity.

Our previous study showed that the oral administration of red ginseng powder before but not after transient forebrain ischemia prevented delayed neuronal death in gerbils, and that a neuroprotective molecule within red ginseng powder was ginsenoside Rb1. However, it remains to be clarified whether or not ginsenoside Rb1 acts directly on the ischemic brain, and the mechanism by which ginsenoside Rb1 protects the ischemic CA1 neurons is not determined. Without elucidation of the pharmacological property of ginsenoside Rb1, the drug would not be accepted as a neuroprotective agent. The present study demonstrated that the intracerebroventricular infusion of ginsenoside Rb1 after 3.5 min or 3 min forebrain ischemia, precluded significantly the ischemia-induced shortening of response latency in a step-down passive avoidance task and rescued a significant number of hippocampal CA1 neurons from lethal ischemic damage. The intracerebroventricular infusion of ginsenoside Rb1 did not affect hippocampal blood flow or hippocampal temperature except that it caused a slight increase in hippocampal blood flow at 5 min after transient forebrain ischemia. Furthermore, ginsenoside Rb1 at concentrations of 0.1-100 fg/ml (0.09-90 fM) rescued hippocampal neurons from lethal damage caused by the hydroxyl radical-promoting agent FeSO4 in vitro, and the Fenton reaction system containing p-nitrosodimethylaniline confirmed the hydroxyl radical-scavenging activity of ginsenoside Rb1. These findings suggest that the central infusion of ginsenoside Rb1 after forebrain ischemia protects hippocampal CA1 neurons against lethal ischemic damage possibly by scavenging free radicals which are overproduced in situ after brain ischemia and reperfusion. The present study may validate the empirical usage of ginseng root over thousands of years for the prevention of cerebrovascular diseases.

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 pi (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.

Changes in the distribution of flowing erythrocytes in a straight cylinder were studied under an inhomogeneous magnetic field. The magnetic field was applied perpendicular to a cylinder, which had a 90° side vessel at the end (oriented towards the magnetic field) to detect changes in the erythrocyte distribution within the cylinder. (1) The attraction of paramagnetic erythrocytes by the magnetic field was demonstrated by an increase in the concentration (or number) of erythrocytes drawn into the side vessel. The flow of diamagnetic, oxygenated erythrocytes was unaffected. (2) The degree of attraction of the paramagnetic erythrocytes was proportional to ``(magnetic susceptibility)'' and to ``(magnetic flux density) × (magnetic field gradient)'' up to 10 T2/m, but it saturated at high magnetic field. The onset of the saturation depended on the magnetic susceptibility of the erythrocytes. (3) The degree of attraction depended on the hematocrit of the flowing erythrocyte suspension, with a maximum value at a low hematocrit. These phenomena are explained on the basis of the balance between the paramagnetic attractive force of the magnetic field and the collision rate between erythrocytes.

It is generally accepted that process-bearing microglial cells originate from ameboid macrophage-like mesodermal cells. This transformation, often called ramification, accompanies down regulation of macrophage-like properties, but the mechanisms involved in ramification have not been clarified. We investigated factors to promote ramification in culture. Isolated ameboid microglial cells were seeded on living or paraformaldehyde-fixed astrocyte monolayers. About 80% of the cells ramified on the fixed astrocytes in astrocyte-conditioned medium as well as on the living astrocytes. In fresh culture medium, 50% of the cells on the fixed astrocytes ramified. On the other hand, ameboid cells rarely ramified on noncoated glass coverslips even in the conditioned medium. Ameboid cells cultured on extracellular matrices derived from astrocytes ramified more than on those coated with plasma fibronectin or collagen type I. A synthetic peptide containing Arg–Gly–Asp sequence or a tyrosine kinase inhibitor genistein partially reversed the ramification induced on the fixed astrocyte monolayers. These results show that some nondiffusible factors derived from astrocytes are essential for microglial ramification. A part of the nondiffusible factors are present in the extracellular matrices, and the effects might be mediated by integrins. Some diffusible factors secreted by astrocytes seem to promote ramification, if the nondiffusible factors are present. The experiments using the fixed astrocyte monolayers may be useful to identify the diffusible factors responsible for ramification.

The deformation of erythrocytes in microvessels less than 15 microns in inner diameter was analyzed using a microvascular bed isolated from rabbit mesentery. The deformation was compared with that found in glass capillaries.Human erythrocytes were perfused through two media: first, a microvascular-bed section isolated from rabbit mesentery; and second, a set of glass capillaries. Images of deformed erythrocytes were recorded on videotape under strobe light and analyzed with an image processor. The flow velocity of the erythrocytes was determined from the difference of their positions between video frames or by a dual-spot cross-correlation technique. Erythrocyte deformability was modified with diamide, diazene dicarboxylic acid bis[N,N-dimethylamide], by crosslinking spectrins.Symmetrical (parachute-like or slipper-like) deformation of erythrocytes was observed only in microvessels smaller than 13 microns in inner diameter. Erythrocytes in microvessels were less deformed than those in glass capillaries with corresponding diameters, and the marginal cell-free layer was narrower. The deformation increased by increasing the flow velocity of erythrocytes, and the cell-free layer became wider. Diamide-treated cells in microvessels were less deformed than normal cells and showed slightly narrower cell-free layers. Stronger stress in narrower microvessels induced further deformation of cells.Erythrocyte deformation in microvessels was essentially different from that in glass capillaries, and the effect of erythrocyte deformability on the flow dynamics of erythrocytes in microvessels was properly evaluated using an isolated microvascular bed.

The aggregation and sedimentation kinetics of human erythrocytes was studied by modifying the cellular properties and medium compositions simultaneously. Dextrans of average molecular weight 70400 and 494000 were used to provide suspending medium modifications, while diamide (diazene dicarboxylic acid bis(N,N-dimethylamide)) was used to alter the membrane structural properties. Laser scattering method was employed for this study, and it was compared with a kinetic method combined with a low-shear rheoscope and an image analyzer. From scattered light intensity profiles continuously obtained during aggregation of erythrocytes and sedimentation of the aggregates, characteristic kinetic parameters were computed. Kinetic parameters obtained from a phase of the one-dimensional aggregate formation and sedimentation corresponded well to the velocity of rouleaux formation obtained by the low-shear rheoscope technique. Dextrans accelerated the erythrocyte aggregation and the sedimentation, and diamide treatment suppressed the process by decreasing the erythrocyte deformability. The aggregating force by dextrans overcame the disaggregating force by the decreased deformability. However, the arrangement of erythrocytes as expressed in specific units for aggregates (i.e., rouleaux) became irregular by decreasing the erythrocyte deformability. In conclusion, the progression of erythrocyte aggregation and the structure of the aggregates were dependent on both erythrocyte properties and macromolecules in the medium.

Rheological characteristics of red cells in two patients with refractory anemia (with single chromosomal abnormality of 20q- or 13q-, respectively) were investigated with the hematological and biochemical properties. (1) Whole blood viscosity was remarkably increased, and the red cell deformability was greatly impaired (the impairments were prominent in patient with 20q-). (2) The hematocrit of both patients was about half of the normal value. Remarkable anisocytosis with elliptocytes and poikilocytes was observed in the patient with 20q-, but the anisocytosis was not so prominent in the patient with 13q-. (3) 2,3-diphosphoglycerate content in red cells was markedly increased in both patients, but adenylate content was not. (4) The red cells were slightly resistant to osmotic hemolysis, but they were not heat-labile. (5) Structural abnormality of spectrin was suggested from the impaired dimer-dimer association in red cell membrane and from the different susceptibility of spectrin to tryptic digestion. In conclusion, the rheological impairments and the abnormal shape of red cells in refractory anemia probably originated from the structural abnormality of cytoskeletal proteins in membrane, and the functional and structural abnormality may be different among patients.

The effect of an external inhomogeneous magnetic field on the flow of erythrocytes containing paramagnetic hemoglobin was studied systematically, with three experimental setups. (1) The attraction of a narrow stream of erythrocyte suspension towards stronger magnetic field, in a wide laminar flow, was found to be proportional to the magnetic susceptibility of erythrocytes χ, the product of the field strength and its spatial gradient B × d B /d z , and the reciprocal of flow velocity 1/ v , and also to the hematocrit h of the suspension. (2) A model flow of erythrocyte suspension in the vessel showed a small change in the radial distribution of erythrocytes arising from a magnetic field, which is proportional to χ, B × d d B /d z (up to 20 T 2 /m), 1/ v , and h ( B × d B /d z and h . (3) Acceleration of the sedimentation rate was detected for paramagnetic erythrocytes in an inhomogeneous magnetic field, but not with diamagnetic erythrocytes. In short, the paramagnetic attraction takes place with venous blood, and depends on the product of the field strength and its spatial gradient, the degree of deoxygenation, the flow velocity, and the hematocrit.

CPD blood, in glass containers and polyvinyl chloride (PVC) bags, was X-irradiated with 50 Gy (n = 4), and then stored for up to 4 weeks. Rheological, hematological and biochemical changes of the red cells during storage were examined. Changes in blood viscosity and various properties of red cells (deformability, shape, hematological indices and concentrations of 2,3-diphosphoglycerate and nucleotides) during storage at 4 degrees C were not altered by the irradiation. The rheological, biochemical and morphological properties of red cells were as well preserved in PVC bags as in glass containers. No abnormal and/or cross-linked proteins in red-cell membranes were induced by the irradiation. Irradiation of PVC bags did not affect the above properties of red cells through any changes in the bag constituents. In conclusion, X-ray irradiation with 50 Gy to CPD blood in PVC bags does not affect the rheological functions of red cells.

The sedimentation rate of paramagnetic erythrocytes in a vertical cylinder increased with the application of an inhomogeneous magnetic field in the horizontal direction. This phenomenon is similar to the so-called Boycott effect, which produces increased sedimentation in an inclined cylinder or channel. The detailed mechanism of this effect has not been obtained. Since the direction of force acting on the particles in a suspension can be changed continuously by changing the magnetic field strength, our method may be used to control the sedimentation rate of small paramagnetic particles in a liquid.

Relation between aggregating force (of fibrinogen and IgG) and disaggregating force (due to electrostatic repulsion among erythrocytes) in erythrocyte aggregation was investigated with a rheoscope combining a video camera, an image analyzer and a computer. (i) Erythrocyte aggregation was augmented with the increase of molecular weight of bridging macromolecules as far as examined for fibrinogen and the degradation products and IgG and the related macromolecules, and the augmentation seemed to be dependent on the molecular length of macromolecules. In accelerating the erythrocyte aggregation, fibrinogen was more effective than IgG, and some interaction between fibrinogen and IgG in their coexistence was suggested. (ii) The decrease of sialic acid content on the erythrocyte surface accelerated IgG-induced erythrocyte aggregation much greater than fibrinogen-induced one. (iii) Counteraction between aggregating force and disaggregating force in leading to erythrocyte aggregation was discussed relating to molecular length of bridging macromolecule and electrostatic repulsive force by sialic acid.

Rats were fed either a high alpha-linolenate diet or a high linoleate diet from weaning to 4 mon of age. Soybean oil was used as a control. Phospholipid compositions of erythrocytes from the three dietary groups were not significantly different. However, the difference in the alpha-linolenate (18:3n-3)/linoleate (18:2n-6) ratio of the diets was reflected in the n-3/n-6 ratios of the 20 and 22 carbon highly unsaturated fatty acids except for docosahexaenoic acid (22:6n-3) in the phospholipids. Despite the significant differences in the fatty acid compositions of phospholipids, no measurable differences were detectable in erythrocyte deformability, whole blood viscosity and hematological indices of the three dietary groups. These results indicate that the beneficial effects of the high alpha-linolenate diet, as compared with the high linoleate diet, are exerted without significant changes in these parameters.

Effect of 2,3-diphosphoglycerate (2,3-DPG) on the deformability of human erythrocytes was examined with a rheoscope under shear stress of 8-82 dyn/cm2. With increasing 2,3-DPG in erythrocytes (from 5 to 15 mM/l cells) by incubating with inosine and pyruvate in isotonic 50 mM phosphate-buffered saline, erythrocyte deformability under uniform shear stress was remarkably impaired. But reduction of 2,3-DPG (from 5 to 2.2 mM/l cells) did not affect the deformability. In 2,3-DPG-enriched erythrocytes, increased intracellular hemoglobin concentration (MCHC), decreased intracellular pH, and increased contents of ATP and IMP (and ITP) were observed. (1) When the MCHC (i.e., the internal viscosity) was normalized by suspending in hypotonic medium, the deformability of 2,3-DPG-enriched erythrocytes was greatly improved, but still decreased. (2) The change of intracellular pH between 6.5 and 7.5 (as compared adjusting to same MCHC) did not alter the deformability. (3) The changes of purine nucleotides, ATP (0.6-2.1 mM/l cells), IMP (0-0.9 mM/l cells) and ITP (0-0.5 mM/l cells) did not alter the erythrocyte deformability. In conclusion, decreased deformability of erythrocytes induced by augmentation of 2,3-DPG is due mainly to the increased internal viscosity and due partly to the increased membrane viscoelasticity.

The characteristics of red cell deformation were studied, focusing on deformation response of the cells subjected to oscillatory shear stress. Red blood cells were fractionated into subpopulations of different densities, i.e., low-density and high-density cells. The deformation response of the fractionated cells was evaluated with a rheoscope varying their intracellular viscosity and oscillation frequency of the applied shear stress, and determinants of the deformation response were compared with those of whole cell deformation under stationary shear stress. When the fractionated cells were exposed to sinusoidally oscillated shear stress, the cells underwent oscillatory deformation. The degree of deformation of the low-density cells correspond to the magnitude of the applied shear stress up to an oscillation frequency of 2.7 Hz. Meanwhile, such an oscillatory deformation as to correspond to the applied shear stress was observed up to 1.7 Hz for the high-density cells. It was suggested that intracellular viscosity was an important factor to determine the deformation response of red cells to oscillatory shear stress.

An optical spectroscopic system for determining the rate of oxygen release from flowing erythrocytes in microvessel is developed. The apparatus consists of following units attached to an inverted microscope. 1) A scanning spectrophotometer, equipped with a grating and a photon counter, was connected to an eyepiece of the microscope through a narrow light-guide, as to obtain the absorption spectrum (wave length range: 450-650 nm) of a focused spot (phi = 7 microns). 2) The velocity of erythrocyte flow was measured by dual-spots cross-correlation method, using two photomultipliers (connected to A/D converter and microcomputer) with two light-guides inserted into another eyepiece. 3) The diameter of vessel was estimated from digitized video-images, using a color image-processor. The ability of the apparatus was tested with (a) hemoglobin solution, (b) flowing erythrocyte suspension and (c) capillaries of rat mesentery. The rate of oxygen release through the vessel wall was calculated.

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.

Connective tissue growth factor (CTGF) is overexpressed in atherosclerotic blood vessels. To further investigate the role of CTGF in atherosclerosis, we examined whether CTGF is released from platelets by high shear stress, and whether the expression of CTGF along the atherosclerotic lesions depends on local hemodynamic conditions. Human platelets were subjected to 10 dyn/cm2 or 120 dyn/cm2 and analysed by Western blotting. Furthermore, longitudinal sections of 25 carotid plaques were immunohistochemically analysed for the endothelial expression of CTGF. A very low CTGF amount was secreted from platelets at low shear stress (11.4 +/- 3.9% of total CTGF in platelets). On the contrary, high shear stress caused a markedly increased CTGF release from platelets (29 +/- 13.8%, p = 0.07 vs low shear stress, n = 4). Immunohistochemical analyses showed that the mean numbers of CTGF-positive endothelial cells were significantly higher up-stream as compared with down-stream regions of the luminal surface of atherosclerotic vessels (21.3 +/- 3.6 vs 13.9 +/- 2.8 down-stream, p0.001). Moreover, in plaques undergoing intimal neovascularization, newly formed vessels accumulated particularly in up-stream parts of the lesions. In conclusion, this study demonstrated that CTGF is released from platelets by high shear stress. Furthermore, disturbed flow along atherosclerotic vessels may induce endothelial CTGF expression and contribute to the progress of atherosclerotic lesions.

The effect of triglyceride in plasma on RBC aggregation was examined, and the prospective influence on the flow of RBCs in microcirculation and the O2 release was discussed. To minimize the individual differences, blood samples were collected from one subject 2 hrs after high-fat and low fat meals. Triglyceride content in plasma was measured by an enzymatic method, and the rate of rouleaux formation was measured with a low shear rheoscope. The rate of rouleaux formation was increased with the increase of triglyceride concentration. Our previous findings suggested some functional impairment in microcirculation. (1) The enhanced RBC aggregation tends to reduce flow resistance in arterioles, but results in inhomogeneous flow of RBCs in capillaries. (2) The sclerotic change of microvessels alters flow behavior of RBCs, and thereby flow resistance is increased. (3) The enhanced RBC aggregation reduces O2 release from RBCs flowing in microvessels. In conclusion, high triglyceride level in plasma not only changes flow behavior of RBCs in microcirculation and thus increases flow resistance, but also prevents homogeneous tissue oxygenation.