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To establish a highly efficient method of ex vivo expansion of hematopoietic cells (HCs), three-dimensional (3D) cocultures of HCs and stromal cell lines were performed using porous polymer scaffolds. Hematopoietic cells derived from mouse fetal livers were expanded by two successive cultures without the use of exogenous cytokines, namely, 3D cultures of stromal cells (DAS 104-8 cell line) to form stromal layers within the scaffolds, and, subsequently, by cocultures of the HCs on the stromal cell layers for 2 weeks. To expand the HCs more conveniently, in some experiments the stromal layers formed within the scaffolds were frozen (3D freezing) before the cocultures, then stored and applied to the cocultures after thawing. When the HCs were cocultured on the stromal layers of the DAS 104-8 cells, primitive HCs (c-kit + and CD34 + cells) were expanded several fold during the cocultures. In contrast, the expansion of these primitive HCs was remarkably enhanced in the cocultures using the 3D frozen-thawed DAS 104-8 stromal layers (c-kit + cells > fifteenfold and CD34 + cells > thirtyfold), and these expansions were significantly higher than those without the 3D freezing. The expansions enhanced by cocultures on the 3D frozen-thawed stromal layers were also observed in the cocultures with another stromal cell line (DAS 104-4). Because 3D frozen-thawed stromal cell lines are easy to handle, 3D coculture of HCs on frozen-thawed stromal cell lines may be an effective and convenient method for expanding primitive HCs.

The high plateaus in west China may provide suitable sites for astronomical observations with theinstitute’s middle-range telescopes and possibly with larger telescopes. Under China-Japan collaborationsfor site survey in west China, we have been conducting searches for good sites and monitoring theircharacteristics over several years. As recent results of our site survey show, sites in west Tibet arerevealed with a high possibility of good astronomical observations.Weather characteristics at Gar in Ali, Tibet, show its high clear-sky ratios, especially in winter, com-parable to Mauna Kea, Hawaii. But it has some wind problem in winter, where stronger wind speeds,over 20m/sec, occur frequently even though the sky is clear. To nd calmer sites, we have conductednumerical simulations for the Ali area using the Japan Meteorological Agency NonHydrostatic Model.We have found another site, named ZoZo Hill, near Gar. We will continue to monitor the Gar site toclarify weather characteristics over the whole year and hopefully start to negotiate for site monitoring atZoZo Hill this year.Key words: international collaboration, site test, telescope site, west Tibet1. WEST TIBET AS AN IMPORTANT LONGITUDI-NAL LOCATION FOR GLOBAL ASTRONOMICALOBSERVATION NETWORKAs west China is indicating its importance in playinga role for the global astronomical observation network(Yao 2005), we have been conducting astronomical site-monitorings at three sites in west China; Karasu (Xin-jiang Uighur), Oma (Tibet) and Gar/Ali (Tibet) undera China-Japan Astronomical Collaboration since 2007(Sasaki et al., 2008; Yao et al., 2011; Sasaki et al., 2011).2. ASTRONOMICAL SITE MONITORINGSeveral astronomical site-monitoring instruments havebeen deployed at the sites. At the Oma and Gar site,cloud monitoring cameras have revealed excellent skyconditions, especially in winter. Gar has shown betterweather conditions than Oma.On the other hand, strong winds that have been ob-served at the current monitoring site, Gar, in winter mayseriously a ect the number of observable nights (Figure1), as the imaging capabilities of telescope optics wouldbe reduced due to turbulence caused by stronger windhttp://pkas.kas.orginside the telescope dome by applying the criteria forwind speed

Fibre Multi-Object Spectrograph (FMOS) is the first near-infrared instrument with a wide field of view capable of acquiring spectra simultaneously from up to 400 objects. It has been developed as a common-use instrument for the F=2 prime-focus of the Subaru Telescope. The field coverage of 300 diameter is achieved using a new 3-element corrector optimized in the near-infrared (0.9-1.8μm) wavelength range. Due to limited space at the prime-focus, we have had to develop a novel fibre positioner, called " Echidna" , together with two OH-airglow suppressed spectrographs. FMOS consists of three subsystems: the prime focus unit for IR, the fibre positioning system/connector units, and the two spectrographs. After full systems integration, FMOS was installed on the telescope in late 2007. Many aspects of the performance were checked through various test and engineering observations. In this paper, we present the optical and mechanical components of FMOS, and show the results of our on-sky engineering observations to date. © 2010. Astronomical Society of Japan.

To develop a tissue-engineered bioartificial liver (BAL), perfusion cultures of mouse and pig fetal liver cells (FLCs) immobilized within a three-dimensional (3D) porous scaffold were performed utilizing a packed-bed reactor system. These FLCs were cultured under different medium flow rate conditions, and the effects of the flow rates on cell growth and the hepatic functions of the FLCs were investigated. In the cultures of mouse FLCs, the medium flow suppressed cell growth and the albumin secretion activity of the FLCs, and considerably lower albumin secretion than that in the 3D stationary control cultures was obtained in the perfusion cultures. In the case of pig FLCs, cell growth was also inhibited by the medium flow, however, the cells exhibited higher tolerance to the medium flow compared with mouse FLCs. The albumin secretion activity of pig FLCs was well maintained under an extremely low flow rate condition (4.8 mm/min in the reactor), and activity higher than the 3D stationary cultures was detected at a later stage (after 20 days in the perfusion cultures). These results revealed that FLCs are quite sensitive to medium flow and an extremely low shear stress is required for the perfusion cultures of FLCs.

To examine effects of local tissue cooling on contusion-induced microvascular hemodynamics and leukocytes behavior using real-time intravital microscopy.Male Wistar rats (N = 21, 130-150 g) were randomly assigned to intensive cooling group (3 degrees C, N = 7), a moderate cooling group (27 degrees C, N = 7), or control group (37 degrees C, N = 7). Contusion was induced by dropping a plastic ball on exposed cremaster muscle. After 5 min, the cremaster muscle was superfused with a saline solution for 10 min at controlled temperature of either 3 degrees C (cooling), 27 degrees C (moderate cooling), or 37 degrees C (control). Microvascular hemodynamics (vessel internal diameter, blood flow rate and erythrocyte velocity) and leukocyte behavior (rolling and adhesion) were measured from recorded videotapes in the same venules before and after contusion, and after cooling.Cooling-induced vasoconstriction was marked at 3 degrees C and moderate at 27 degrees C compared with that at 37 degrees C. Blood flow rate and erythrocyte velocity were markedly lower at 3 degrees C compared to 37 degrees C. At 27 degrees C, erythrocyte velocity was higher than that at 37 degrees C, but blood flow rate was maintained at a level similar to that at 37 degrees C. The number of rolling and adhering leukocytes at 3 degrees C and 27 degrees C were significantly less than at 37 degrees C.Our results suggest that local tissue cooling, similar to cryotherapy, improves edema and inflammatory reaction, and may be useful for reducing inflammatory response without inhibiting blood flow after contusion.

The effects of electroacupuncture stimulation (EAS) of the hind paw and the back on the mesenteric microhemodynamics in anesthetized rats were investigated using an intravital microscope system. Red blood cell (RBC) velocity in the mesenteric arterioles was measured by the dual-sensor method developed by the authors. Electrical stimulation was applied using two acupuncture needles inserted into the skin and the underlying muscles of the hind paw and the dorsal Th13-L1 level area. The hind-paw EAS evoked intensity-dependent pressor responses and increase responses in RBC velocity in mesenteric precapillary arterioles, while the back EAS evoked depressor responses and decrease responses in RBC velocity. Heart rate showed increase responses accompanying EAS either on the hind paw or the back. The pressor responses and increase responses in RBC velocity in mesenteric precapillary arterioles accompanying the hind paw EAS were abolished by an intravenous administration of alpha-adrenergic receptor antagonist (phenoxybenzamine; POB), and the tachycardiac responses were abolished by administration of beta-adrenergic receptor antagonist (propranolol). Occasional but notable reflex vasoconstrictions in the mesenteric terminal arteriole were induced by EAS either on the hind paw or the back. These vasoconstrictive responses were not affected by the administration of POB. The present study directly demonstrated that hemodynamic changes at the level of precapillary arterioles accompanying EAS either on the hind paw or the back mainly depend on the changes of systemic arterial pressure regardless of stimulus current intensities. Moreover, the results in the present study suggest some receptors other than alpha-adrenergic receptor might be involved in the mechanism of EAS-induced vasoconstriction in the mesenteric arteriole.

An overview of the current status of the 8.2 m Subaru Telescope constructed and operated at Mauna Kea, Hawaii, by the National Astronomical Observatory of Japan is presented. The basic design concept and the verified performance of the telescope system are described. Also given are the status of the instrument package offered to the astronomical community, the status of operation, and some of the future plans. The status of the telescope reported in a number of SPIE papers as of the summer of 2002 are incorporated with some updates included as of 2004 February. However, readers are encouraged to check the most updated status of the telescope through the home page, http://subarutelescope.org/index.html, and/or the direct contact with the observatory staff., Comment: 18 pages (17 pages in published version), 29 figures (GIF format), This is the version before the galley proof

White blood cells roll spontaneously in venules of intact, noninflamed rat skin. We investigated noninvasively in two experimental series which leukocyte subtypes participate in this phenomenon and the possible involvement of E-selectin. Male Lewis rats were anesthetized with sodium pentobarbital, and intravital video microscopy was performed on postcapillary venules in the nail-fold of a hind leg. In series 1 acridine yellow was infused for 15 min (50 mg per kg intravenously) to stain the leukocyte nuclei in situ. With the use of fluorescence microscopy rolling leukocytes could be classified unequivocally as polymorphonuclear (granulocytes) or monomorphonuclear (lymphocytes/monocytes) by the shape of their nucleus. Irrespective of vessel depth beneath the skin surface (25–45 µm), most identified rolling leukocytes were classified as granulocytes (72%-100%; median 89%). This percentage was independent of total rolling leukocyte flux, systemic leukocyte count, or their in vitro differentiation pattern. In series 2, rats were treated with either a synthetic, highly selective E-selectin blocking peptide or a control peptide (intravenously, 12 mg peptide per kg bolus, followed by 50 mg per kg per h). E-selectin blockade significantly reduced the leukocyte rolling level to about 50% of baseline (p

The effects of electrical stimulation of the dorsal skin area on the mesenteric arterioles were investigated in anesthetized rats by the use of an intravital microscope-television system. Changes in the diameter of the mesenteric precapillary arterioles (10-40 microm in diameter) were measured with an image processor. Blood flow velocity in the mesenteric precapillary arterioles was monitored by the dual sensor method developed by the authors. Electrical stimulation was performed through two platinum electrodes placed at the right dorsal Th5-12 level skin area by the use of an electrical stimulator (0.2 ms, 20 Hz). Continuous stimulation lasting for 30 s (1-10 mA) and intermittent stimulation lasting for 10 min (3 mA) were applied. The pressor response following the depressor response was induced by a stimulus current above 8 mA. The decrease in mesenteric blood flow velocity was induced by stimulus current above 10 mA. These responses were abolished by lidocaine injection into the subcutaneous area where the electrodes were attached. No significant change in arteriolar diameter or heart rate were induced by the stimulation for 30 s. Electrical stimulation of the skin for 10 min evoked a decrease in the diameter of arterioles (-3.4 +/- 2%, p0.01, n = 12). In the adrenalectomized group, electrical stimulation of the skin for 10 min elicited a slight increase in the diameter (1.1 +/- 0.5%, n = 6). It is therefore suggested that electrical stimulation of the skin for 30 s reflexly evoked decreases in MAP and in blood flow velocity, and that the constriction of the mesenteric precapillary arterioles induced by the stimulation for 10 min was mediated by humoral adrenaline and noradrenaline released by somato-adrenal medullary reflex.

UNLABELLED Xenon may increase cerebral blood flow and intracranial pressure (ICP). To evaluate the effects of xenon on brain circulation, we measured pial vessel diameter changes, CO(2) reactivity, and ICP during xenon inhalation in rabbits. Minimum alveolar anesthetic concentration (MAC) for xenon was established in rabbits (n = 6). By using a cranial window model, pial vessel diameters were measured at 30% and 60% xenon inhalation and in time control groups (n = 15). ICP, mean arterial blood pressure, and heart rate were recorded during 30% and 60% xenon inhalation (n = 5). Pial vessel diameters were measured during hypocapnia and hypercapnia conditions in 60% Xenon and Control groups (n = 14). MAC for xenon was 85%. Xenon (0.35 and 0.7 MAC) dilated the arterioles (10% and 18%, respectively) and venules (2% and 4%, respectively) (P < 0.05). Dilation of arterioles was more prominent than that of venules. ICP, mean arterial blood pressure, and heart rate did not change during xenon inhalation. No difference in CO(2) reactivity was observed between Xenon and Control groups (P = 0.79). Sixty percent xenon (0.7 MAC) dilated brain vessels, but venule changes were small. Xenon did not increase ICP and preserved CO(2) reactivity of the brain vessels. IMPLICATIONS Xenon might increase cerebral blood flow; however, 0.7 minimum alveolar anesthetic concentration xenon preserved both low intracranial pressure and CO(2) reactivity of the cerebral vessels in the normal rabbit.

We developed a new method for visualizing the flow behavior of erythrocytes in living microvessels by the fluorescent labeling technique. Intravital microscopic observation of the microcirculation in the rat mesentery using this technique enabled quantitative analyses of a few hydrodynamic aspects of the two-phase blood flow; e.g. diameter-flow relationships in a consecutively branched vascular network and the distribution of erythrocytes in an arteriolar bifurcation.

To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2 x 2 x 2 mm, mean pore size: 100, 250 or 500 microns) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6-11.3 x 10(7) hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2 x 10(7) cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

The role of hemodynamics on platelet thrombus formation was studied in venules and arterioles of the rat mesentery. Thrombus formation was induced by the fluorescent dye/light method for examination of the following factors: 1) the effect of wall shear rate on thrombus initiation, 2) the effect of wall shear rate on the growth of thrombi, and 3) the relation between platelet thrombus initiation and intraluminal velocity profile. The range of wall shear rate was up to approximately 1,000 1/sec in venules and from 640 to 2,900 1/sec in arterioles. Platelet thrombus initiation occurred more rapidly at higher wall shear rate in venules and at lower wall shear rate in arterioles. Thrombus initiation time was shortest around a wall shear rate of 900 1/sec in venules and around 700 1/sec in arterioles. Thrombus growth rate in venules was greatest at a wall shear rate of 1,500-2,000 1/sec. Thrombus initiation and its relation to blood flow was also examined in branched and curved microvessels. In these vessels platelet thrombi were also first initiated at the sites of higher wall shear rate in venules and of lower wall shear rate in arterioles.

Recent studies indicate a possible role of nitric oxide (NO) in regulating leukocyte-endothelial cell interactions, which plays a key role in the tumor immunity. The purpose of the present study is aimed to observe the tumor hemodynamics intravitally and to clarify the effect of NO on tumor microcirculation by means of a real-time confocal laser-scanning microscope using NO-reactive indicators. Visualization of localization of NO and the leukocyte behavior was made in the mesenteric microvessels of an experimental tumor model rat. Production of NO was clearly visualized along the endothelium of the tumor-free rats, but scarcely found in the newly formed tumor microvessels. A higher level of NO production was observed in a solid tumor region, where a more marked decrease in the leukocyte-endothelial cell interactions was observed. Local administration of a NO synthase (NOS) inhibitor increased leukocyte adhesion. This indicates that tumor-derived NOS creates the tumor specific microenvironment of the immature angiogenic tumor vessels, thereby modulating leukocyte behavior.

UNLABELLED The adhesion of polymorphonuclear leukocytes to the capillary endothelium is one of the key events in the pathophysiology of hemorrhagic shock. We studied sodium nitroprusside (SNP) for its ability to modulate leukocyte-endothelial cell interactions induced by hemorrhagic shock and reinfusion of blood by using intravital microscopy of the rat mesentery. Administration of SNP at a dose of 0.1 microg x kg(-1) x min(-1) infusion neither significantly decreased mean arterial blood pressure nor significantly altered bleedout volumes in hemorrhagic rats, indicating that SNP at this dose did not modify the severity of the shock protocol. Resuscitation from 1 h of hemorrhagic shock (mean arterial blood pressure approximately 45 mm Hg) significantly increased the number of adherent and emigrated leukocytes in the rat mesenteric microcirculation. However, infusion of SNP, started 15 min before hemorrhage, and continued over the entire experimental period, markedly reduced the leukocyte adhesion after reinfusion and emigration during hemorrhagic shock and after reinfusion. We concluded that the nitric oxide donor SNP is effective at reducing the leukocyte-endothelial interaction after blood reinfusion after hemorrhagic shock in rats. IMPLICATIONS The i.v. infusion of 0.1 microg x kg(-1) x min(-1) of sodium nitroprusside, a dose that does not exert a significant vasodilator effect, reduces leukocyte adhesion and emigration after hemorrhagic shock.

An intravital fluorescence videomicroscope system was used to investigate the pharmacological effects of endothelin-1 (ET-1) on the coronary microcirculation in isolated beating hearts of rats. The heart was perfused by retrograde aortic steady flow with an oxygenated Krebs-Ringer solution containing FITC-dextran. Cumulative injections of ET-1 (1-30 pmol) elicited a dose-dependent increase in perfusion pressure from 52 +/- 15 mm Hg (mean +/- SEM; n = 6) before the ET-1 injection to 104 +/- 23 mm Hg at the ET-1 dose of 30 pmol. A dose-dependent narrowing of microvessels was also observed on a monitor screen. This diffuse vasoconstriction was especially prominent in small arterioles; the maximum vasoconstriction of the smaller arterioles was significantly higher than that of the larger arterioles. These findings suggest that ET-1 may have an important role in governing the coronary resistance and regulating the capillary flow in myocardium.

The aim of the present study was to visualize the superficial glomeruli of the Munich-Wistar (MW) rat and to characterize the responses of the renal microvasculature to endothelin-1 (ET-1). We first examined the distribution of superficial glomeruli of the MW rat compared to that in a control strain (Wistar rat). Secondly, we examined the effects of ET-1 on the renal microcirculation of the MW rat. The right kidney was perfused with a Krebs-Ringer solution containing fluorescein isothiocyanate dextran (FITC-dextran) and was visualized under an epi-illuminated fluorescence microscope system. Changes in perfusion pressure and diameter of the microvessels accompanying the administration of ET-1 (10 fmole-300 pmole) were measured. The number of superficial glomeruli was greater in the MW rat than in the Wistar rat. ET-1 had long-lasting and dose-dependent pressor effects. Perfusion pressure showed a 3.5-fold increase compared with the control, and the afferent arterioles showed greater dose-dependent vasoconstriction than the efferent arterioles. These findings suggest that the MW rat is a useful animal model for the study of renal microcirculation and that the renal microcirculation is extremely sensitive to ET-1.

We compared the thrombolytic activity of a novel modified tissue-type plasminogen activator (t-PA; del 92-173, 275Arg-->Glu), YM866, with that of t-PA in a platelet-rich thrombosis model. Thrombus was induced in guinea pig mesenteric artery by irradiation with filtered light in combination with intravenous (i.v.) administration of fluorescent dye. When occlusion by the thrombus extended to 99% of the luminal area of the vessel, test drug (YM866, t-PA, or saline) was administered by i.v. bolus injection under heparinization. Both YM866 and t-PA exhibited dose-dependent thrombolytic activity; however, the improvement in occlusion rate and the incidence of successful thrombolysis induced by YM866 were three times higher than those induced by t-PA. With YM866 1 mg/kg, alpha 2-plasmin inhibitor levels decreased significantly to 58% of saline group values, but no change was noted in fibrinogen levels. YM866 antigen levels at this dose were seven times higher than those of t-PA. These results suggest that YM866 in single bolus injection is a thrombolytic agent superior to t-PA in platelet-rich thrombi without systemic fibrinolytic activation and that this efficacy is due to the prolonged half-life (t1/2) of the drug.

As part of our attempt to develop a hybrid artificial liver support system using cultured hepatocytes, we investigated the long-term metabolic function of hepatocytes incubated in a packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20 mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250 mum, 2 x 2 x 2 mm), together with conventional monolayer culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism and urea synthesis activities were evaluated quantitatively based on reaction kinetic analyses. Initial rates of ammonium metabolism and urea-N synthesis, as well as GPT enzyme activities, were adopted as indexes of the metabolic performance of the reactor and activities of the cultured hepatocytes.When serum-free medium was used in the perfusion cultures, ammonium metabolic and urea-N synthetic rates showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayer cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayer culture experiments.We concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain the metabolic function of immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming the difficulties involved in the development of a hybridtype artificial liver support system. (c) 1994 John Wiley & Sons, Inc.

The intravascular platelet aggregation and the subsequent thrombus formation were found to be induced in the microvasculature by the irradiation of filtered light in combination with the intravascular administration of fluorescein sodium. Effects of several factors such as light intensity, dye concentration and vessel diameter were examined in venules of rat mesenteries. It was confirmed that the platelet thrombus was induced with excellent reproducibility depending on the combined condition of the light intensity and the dye concentration. In venules within the range of 20 to 80μm in diameter, almost the same values of the initiation time of thrombus formation were obtaind. On the other hand, the time to totally occlude the vessel lumen by the thrombus prolonged significantly with the increase of vessel diameter, which phenomenon seemed to be induced by almost the same growth rate of thrombus in the radial direction in all venules with different diameters. Inhibitory effects of prostaglandins (PGD2, PGE1) for the thrombus formation were also examined by the proposed model. The platelet aggregation in venules was significantly inhibited by higher concentration of these doses. This result well corresponded to that of in vitro experiments reported by other researchers. The present model seems useful for various purposes in studying the kinetics of intravascular thrombus formation as well as evaluating the effects of anti-platelet aggregating drugs.

By use of a newly developed automatic erythrocyte sedimentation curve recorder (sedimentimeter), ESR curves of normal subjects and several patients with augmented ESR were recorded, and the hemorheological characteristics of them were analyzed based on the semi-empirical model of Puccini.The automatic sedimentimeter differs in principle from the existing ones in that 1) the optical image of the whole sedimentation tube is focussed on a small photoelectronic image sensor (Reticon®) and (2) the output signals from this image sensor are electronically processed with the resulting curve of the time course of blood sedimentation. Performance of the apparatus was found excellent.Characteristics of erythrocyte sedimentation process were estimated quantitatively in terms of two parameters of the Puccini's equation, i. e., t50 and 1/b. The accuracy of the approximation was found over 0.96 of correlation coefficient, which seemed satisfactory for the practical purposes.It was confirmed in normal subjects that t50's increased almost linearly with the values of hematocrit, while 1/b's were nearly constant. Blood samples obtained from the diseased group which contains one case of diabetes mellitus, rheumatoid arthritis, Hodgkin's disease, thromboangitis obliterans, malignant thymoma (post op.), aneurysm (chronic DIC) and atherosclerotic occlusive disease (post amputation) showed mostly augmented ESR. Parameters of t50 and 1/b in these patients were found in the range of ±175% of normal values, which shows the possible use of these parameters as a diagnostic measure of clinical conditions in some of these diseases.