Biomedical subjects

Minoru Tomita

Publications and source records attributed to Minoru Tomita.

At least 19 recordsLinked to original sources

PubMed2006

Sorbitol causes preferential selection of Muller glial precursors from late retinal progenitor cells in vitro.

PURPOSE: The replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from the brains of newborn mice. The present study was undertaken to investigate the effects of sorbitol on in vitro selection of Müller glial precursors from expanded late retinal progenitor cells (RPCs). METHODS: RPCs used in these studies were isolated from the neural retina of postnatal day one green fluorescent protein (GFP) transgenic mice. The resulting GFP positive neurospheres were dissociated into a single cell suspension and grown on poly-D-lysine/laminin coated tissue culture flasks or slides to generate adherent RPCs. These adherent cells were treated with glucose free medium containing 25 mM sorbitol for 7 days and the expression of retinal-specific cell markers was determined by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot analysis RESULTS: In vitro studies showed that sorbitol treatment of late RPCs altered cellular morphology. Immunocytochemical studies showed an increase in the proportion of cells expressing glial cell markers, most of which co-expressed CRALBP, GFAP, and vimentin. An increase in the proportion of cells expressing PKCalpha, a bipolar cell marker, was also observed. RT-PCR analysis showed down-regulation of nestin transcripts with a concomitant increase in CRALBP, GFAP, vimentin and PKCalpha. These findings were confirmed by immunoblot analysis, where down-regulation of nestin expression with simultaneous up-regulation of CRALBP, GFAP and PKCalpha was observed. CONCLUSIONS: Sorbitol treatment of multipotent late RPCs, in the absence of glucose, results in the preferential selection of Müller glial precursors and their subsequent differentiation into cells that morphologically resemble Müller cells and co-express multiple glial markers.

Animals↗

PubMed2006

Spindle-shaped constriction and propagated dilation of arterioles during cortical spreading depression.

A dual illumination technique was used to examine the behavior of parenchymal arterioles of the cortex during cortical spreading depression in six cats. K+ produced a wave-ring spread of optical density changes concentrically from the injection site. At locations where the wave was passing, arterioles first constricted at one or two spots (55+/-43% of control), forming a spindle-shape in the early phase of cortical spreading depression, and then markedly dilated (155+/-57% of control) within 1 min. The dilation started at the constricted spots, propagated bidirectionally and finally resulted in full-length dilation of the arteriole. Although it varied in magnitude and time-course, this arteriolar behavior was observed in all six cats. Despite these changes, no associated downstream tissue microvascular flow changes were discernable.

Animals↗

PubMed2006

Flow effect impacts NIRS, jeopardizing quantification of tissue hemoglobin.

It has been generally agreed that changes in light transmission detected by NIRS are not exclusively attributable to hemoglobin changes, but also include a contribution from scattering by constituents of the brain tissue, which is not transparent, but turbid. If the light scattering is constant, light transmission can be quantified by an equation similar to Lambert-Beer's law. However, upon brain activation, it is known that the blood flow invariably increases. We have shown that light scattering by blood per se increases flow-dependently, interfering with the measurement of the intrinsic signals from hemoglobin. I call this the flow effect. Here, in responses to Pourtian's comments, I present additional evidence of the flow effect, which arises from RBC aggregation/dispersion, and further point out the inappropriateness of using NIR range wavelengths for the quantification of blood hemoglobin, even though their use is unavoidable in practice, because this wavelength range offers the best transmission through the tissue. In summary, I believe that the NIRS signal is a function of both flow (flow effect, which becomes the predominant contributor when blood flow increases) and metabolism (blood oxygenation change, which becomes accordingly a minor contributor). If the contribution of the flow effect is unknown, any mathematical equation describing the hemoglobin oxygenation is a Pfaffian equation which has no solution. This does not mean that I deny the clinical usefulness of NIRS, which definitely provides an indication of some biological changes in the brain in response to stimuli. However, great care is needed in the interpretation of the signals.

Algorithms↗

PubMed2006

Contribution of the flow effect caused by shear-dependent RBC aggregation to NIR spectroscopic signals.

Near-infrared spectroscopy (NIRS) is widely used to record activation-related blood oxygenation changes in human brain tissue. However, the changes in the NIRS signal upon increased flow are influenced not only by the hemoglobin and oxyhemoglobin concentrations but also by changes in light scattering by various brain constituents. This paper points out the large contribution of flow-dependent red blood cell (RBC) aggregation as a cause of this altered light scattering, a phenomenon which has not previously been considered in the theoretical analysis of NIRS signals. Here, we show that RBCs, which constitute a major chromophore in the tissue, not only absorb light at hemoglobin molecules but also scatter it strongly at the cell membranes of aggregated RBCs, and that the blood optical density per se changes greatly with the size of the plasma gap, which varies according to flow. When local blood flow increases by 50%, the amount of the optical attenuation due to RBC dispersion/disaggregation (the flow effect) can reach 90% of the NIRS signal change for venous blood. The reasons why the optical signal due to blood oxygenation alone can be amount to less than 10% of the total are because the near-infrared lies in the most unfavorable range in the hemoglobin absorption spectrum for determining blood oxygenation, while the flow effect in the NIR range is large. We conclude that reported activation-related changes in brain blood oxygenation, at least in the peripheral region around the activation focus, based on NIRS can be mainly ascribed to the flow effect arising from RBC dispersion/disaggregation with increased flow in the venous system.

Algorithms↗

PubMed2006

Treatment and transfer of emphysema by a new bone marrow transplantation method from normal mice to Tsk mice and vice versa.

We have recently established a new bone marrow transplantation (BMT) method in which bone marrow cells are injected into the intrabone marrow (IBM). In the present study, we used an animal model for emphysema (tight-skin [Tsk] mouse) to examine whether IBM-BMT could be used to treat emphysema in Tsk mice. IBM-BMT was carried out from C3H mice into Tsk mice (8-10 weeks old) that had already shown emphysema. Six months after transplantation, the lungs of all the Tsk mice treated with IBM-BMT [C3H-->Tsk] showed similar structures to those of normal mice, whereas the [Tsk-->Tsk] mice showed emphysema, as seen in age-matched Tsk mice. Next, we attempted to transfer emphysema from Tsk mice to C3H mice by IBM-BMT. Six months after IBM-BMT, the [Tsk-->C3H] mice showed emphysema. These results strongly suggest that emphysema in Tsk mice originates from defects of stem cells in the bone marrow.

Animals↗

PubMed2006

Limitation of combination therapy of interferon and ribavirin for older patients with chronic hepatitis C.

In contrast to the United States, Japanese patients with chronic hepatitis C currently treated with interferon are generally 10 to 15 years older. Older patients, however, tend to experience more frequent adverse events. This study was conducted to clarify the effect of patient age on the efficacy and safety of combination therapy. We consecutively enrolled 208 patients with naïve chronic hepatitis C. Patients were classified into three groups according to age: younger than 50 years of age (n = 52); 50 to 59 years old (n = 83); and 60 years of age or older (n = 73). Interferon alpha-2b therapy was administered daily for 2 weeks, followed by 3 times per week for 22 weeks, while ribavirin was administered daily. Of the 208 study patients, discontinuation of therapy or dose reduction was required in 116 (56%) and was more frequent in older patient groups: 38%, 48%, and 77% for the < 50, 50-59, and > or = 60-year-old patient groups, respectively (P < .001). Multivariate analysis showed patient age to be independently associated with adherence to therapy. A sustained virological response was achieved in 77 (37%) patients, with genotype, viral load, and adherence to therapy associated with this achievement. A tendency toward a lower sustained virological response rate was seen in the older patients. In conclusion, patient age is an important factor contributing to the safety of combination therapy. Thus, treatment schedule should be modified, or other therapeutic modalities should be considered for older patients with chronic hepatitis C.

Adult↗

PubMed2006

A comparison of neural differentiation and retinal transplantation with bone marrow-derived cells and retinal progenitor cells.

Retinal progenitor cells (RPCs) are immature precursors that can differentiate into retinal neurons, including photoreceptors. Recently, it has been reported that bone marrow-derived cells may also be capable of differentiation into cells of central nervous system lineage, including retinal neurons. We compared these two cell types to evaluate their potential as a source of cells for retinal transplantation. Marrow stromal cells (MSCs) and macrophages were isolated from enhanced green fluorescence protein mice. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor to induce neuronal differentiation. RPCs were cultured under the same conditions or with 10% fetal bovine serum. Neuronal marker expression was examined and compared between MSCs and RPCs. MSCs, macrophages, and RPCs were also cultured with explanted retinas from rhodopsin knockout mice to study their potential for retinal integration. MSCs expressed neuronal and retina-specific markers by reverse transcription-polymerase chain reaction and immunocytochemistry. Both types of cells migrated into retinal explants and expressed neurofilament 200, glial fibrillary acidic protein, protein kinase C-alpha, and recoverin. RPCs expressed rhodopsin, a photoreceptor marker we never detected in MSCs. A majority of bone marrow derived-macrophages differentiated into cells that resembled microglia, rather than neural cells, in the explanted retina. This study shows that RPCs are likely to be a preferred cell type for retinal transplantation studies, compared with MSCs. However, MSCs may remain an attractive candidate for autologous transplantation.

Animals↗

PubMed2006

Capillo-venous flow in the brain: significance of intravascular RBC aggregation for venous flow regulation.

Despite numerous reports on the regulation of cerebral arterial blood flow, little work has been done on that of the capillary and venous system. We have examined capillo-venous blood flow in the rat intraparenchymal cerebral cortex, employing a high-speed video confocal fluorescence microscope and our own software (KEIOIS-2) to track individual RBCs and to document velocity changes in single capillaries and veins. We found temporal and spatial heterogeneous changes in capillary RBC density (hematocrit), RBC recruitment, oscillation of capillary flow or vasomotion, and capillary density unrelated to arteriolar diametric changes. In veins, blood flow was also quite variable in time and space, and at a high frame rate venous blood per se was observed as a moving column of amorphous RBC aggregates with irregular edges; we believe this is the first report of such an observation under physiological conditions. The formation of such intravascular RBC aggregates would enforce slowing of blood flow and vice versa: RBC aggregation was in turn entirely flow-dependent. In rapid venous flow, RBCs appeared as a straight gathering of individually separated and dispersed cells. At capillo-venous junctions, an "RBC pouring" process appeared to occur, with RBCs either being sucked up from the capillary, merging, or being held back in the capillary. Changes in venous blood viscosity due to RBC aggregation are likely to be involved in this process. These findings suggest that the capillo-venous junction somehow participates in the regulation of appropriate tissue capillary flow in toto.

Animals↗

PubMed2006

Astroglial swelling for removed rat brain enlargement incubated in deoxygenated mock cerebrospinal fluid.

The source and target of edema fluid for ischemic brain swelling clinically often observed in "malignant infarction" was examined in ex vivo. Wister rat brain hemispheres were removed and incubated air-tightly in a deoxygenated artificial cerebrospinal fluid at 37 degrees for 30 min. Ionic movement into the brain tissue was calculated from their concentration changes in the incubation fluid. We found a weight increase by 11.3+/-2.5% (p<0.01) and a decrease in Na+ from 148.0 to 139.0 +/- 8.2 mEq/l (p<0.01) and an increase in K+ from 4.3 to 11.2 +/- 1.2 mEq/l. Video tape recording revealed that the brain swelling started immediately upon the incubation, and the electronmicroscopical investigation of the swollen cortical tissue revealed that the fluid moved mainly into astroglial cells. The astroglial swelling was quite similar to that of specimen taken from clinical cases at autopsy. The driving force of the water shift can be explained by discharge of thermodynamic potential, i.e., a coupled transport of water with Na+ across the cell membrane (anomalous osmosis). The swelling was not affected by addition of aquaporin blocker, mercuric chloride. It is concluded that cerebrospinal fluid bathing the brain in situ can be the source of edema fluid for ischemic brain swelling.

Animals↗

PubMed2005

Initial oligemia with capillary flow stop followed by hyperemia during K+-induced cortical spreading depression in rats.

Local cerebral blood volume (CBV) and capillary flow changes in regions of depolarizing neurons during K(+)-induced cortical spreading depression (CSD) in the cerebral cortex of alpha-chloralose-urethane-anesthetized rats were examined employing a transillumination (550 nm) video system. Capillary flow was calculated as the reciprocal of mean transit times of blood in pixels of 40 microm x 40 microm, each of which contains a few capillaries. Potassium microinjection into the cortex evoked repetitive wave-ring spreads of oligemia at a speed of ca. 2.33 +/- 0.48 mm/min. During the spread of CSD, tracer (either saline or carbon black) was injected into the internal carotid artery. Colocated with the oligemic wave, we detected capillary flow stop as evidenced by disappearance of the hemodilution curves. At any location in the region of interest within the cerebral cortex, we observed cyclic changes of capillary flow stop/hyperperfusion in synchrony with oligemia/hyperemia fluctuations. The initial flow stop and oligemia were ascribed to capillary compression by astroglial cell swelling, presumably at the pericapillary endfeet, since the oligemia occurred before larger vessel changes. We conclude that local depolarizing neurons can decrease adjacent capillary flow directly and immediately, most likely via astroglial cell swelling, and that the flow stop triggers upstream arteriolar dilatation for capillary hyperperfusion.

Animals↗

PubMed2005

Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.

In the inflamed cornea, there is a parallel outgrowth of blood and lymphatic vessels into the normally avascular cornea. We tested whether adaptive and/or innate immune cells were actively involved in the genesis of new lymphatic vessels. Our results indicate that innate immune cells (CD11b+ macrophages, but not CD11c+ dendritic cells) physically contributed to lymphangiogenesis under pathological conditions and that bone marrow-derived CD11b+ macrophages expressed lymphatic endothelial markers such as LYVE-1 and Prox-1 under inflamed conditions in the corneal stromata of mice. Furthermore, blood vascular endothelial cells that expressed the Tie2 promoter did not contribute to newly formed lymphatic vessels under inflamed conditions. Our in vitro experiments demonstrated that CD11b+ macrophages alone were capable of forming tube-like structures that expressed markers of lymphatic endothelium such as LYVE-1 and podoplanin. The novel finding that CD11b+ macrophages are critical for the development of inflammation-dependent lymphangiogenesis in the eye suggests a new mechanism of lymphangiogenesis.

Animals↗

PubMed2005

Macrophage colony-stimulating factor (M-CSF), as well as granulocyte colony-stimulating factor (G-CSF), accelerates neovascularization.

It has been reported that bone marrow cells (BMCs) differentiate into endothelial cells of blood vessels, and that granulocyte colony-stimulating factor (G-CSF) mobilizes progenitors in the BMCs to the peripheral blood, while macrophage colony-stimulating factor (M-CSF) augments the production of monocytes. We examined whether M-CSF augments the differentiation of BMCs into endothelial cells of blood vessels using a hindlimb-ischemic model. Either G-CSF or M-CSF, or both, was administered to the hindlimb-ischemic mice for 3 days. Both M-CSF and G-CSF augmented the differentiation of BMCs into endothelial cells of blood vessels through vascular endothelial cell growth factor (VEGF), resulting in early recovery of blood flow in the ischemic limbs.

Animals↗

PubMed2004

Risk factors for hepatocellular carcinoma in Hepatitis C patients with sustained virologic response to interferon therapy.

BACKGROUND: Although a variety of papers demonstrated inhibited hepatocarcinogenesis with interferon (IFN) therapy for chronic hepatitis C, a small number of hepatocellular carcinomas (HCCs) were still observed even in sustained virologic responders. AIMS: To clarify factors affecting the development of HCC, we analyzed the frequency of HCC in sustained virologic responders over a long-term observation period. METHODS: Seven hundred and ninety-two out of the 2623 IFN-treated hepatitis C patients who had undergone liver biopsy showed sustained virologic response. Screening for development of HCC was performed periodically during an average follow-up of 5.1 years. Fibrosis of the pretreatment liver biopsy sample was graded. Risk factors for HCC were analyzed by using Cox proportional hazards regression. RESULTS: Of 792 patients, 23 developed HCC. Univariate analysis showed that stage of hepatic fibrosis, age, and alcohol consumption were significantly associated with a risk of HCC (P<0.001). There was a significant difference in the cumulative incidence between patients stratified according to these variables (P<0.001). CONCLUSIONS: Pretreatment hepatic fibrosis score, age, and alcohol consumption may affect development of HCC even in sustained virologic responders. Thus, patients with these factors should be carefully followed even after eradication of the virus.

Adult↗

PubMed2004

Choroidal neovascularization is provided by bone marrow cells.

Choroidal neovascularization (CNV) is a known cause of age-related macular degeneration (ARMD). Moreover, the most common cause of blindness in the elderly in advanced countries is ARMD with CNV. It has recently been shown that bone marrow cells (BMCs) can differentiate into various cell lineages in vitro and in vivo. Adults maintain a reservoir of hematopoietic stem cells included in BMCs that can enter the circulation to reach various organs in need of regeneration. It has recently been reported that endothelial progenitor cells (EPCs) included in BMCs are associated with neovascularization. We examine the role of BMCs in CNV using a model of CNV in adult mice. Using methods consisting of fractionated irradiation (6.0 Gy x 2) followed by bone marrow transplantation (BMT), adult mice were engrafted with whole BMCs isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP). Three months after BMT, we confirmed that the hematopoietic cells in the recipients had been completely replaced with donor cells. We then carried out laser photocoagulation to induce CNV in chimeric mice (donor cells >95%). Two weeks after the laser photocoagulation, by which time CNV had occurred, immunohistochemical examination was carried out. The vascular wall cells of the CNV expressed both EGFP and CD31. These findings indicate that newly developed blood vessels in the CNV are derived from the BMCs and suggest that the inhibition of EPC mobilization from the bone marrow to the eyes could be a new approach to the fundamental treatment of CNV in ARMD.

Animals↗

PubMed2003

Dynamic observation of oxygenation-induced contraction of and transient fiber-network formation-disassembly in cultured human brain microvascular endothelial cells.

Oxygenation-induced contraction of nonconfluent cultured human brain microvascular endothelial cells (HBECs, n = 30) was examined by video-enhanced contrast-differential interferential contrast microscopy. After administering a continuous gentle blow of pure oxygen gas to the surface of the medium just above the flattened HBEC, the plasma membrane exhibited tensioning and wrinkling, resulting in a strong contraction of the cell body by 14 +/- 7% (P < 0.001). When the cell stopped contracting, transient formation of a fiber network starting from certain spots (possibly adhesion plaques, though these were not visible in the majority of cases) and expanding to the whole cell was observed. The occurrence of fiber network formation was statistically significant (26 of 30 separate cells, P < 0.05). After cessation of oxygen delivery, the observed network of fibers broke up rapidly (in a period of 3.3 +/- 1.2 seconds) into small particles of <0.5 microm in diameter, which subsequently fused into the cellular structure. The HBEC completely recovered the control appearance. The sequential process was completed within 30 seconds and was reproduced in individual cells each time that oxygen gas was supplied. The authors conclude that the HBEC strongly contracts in response to a transient oxygenation stimulus, followed by rapid formation/disassembly of a network structure.

Brain↗

PubMed2002

Repetitive concentric wave-ring spread of oligemia/hyperemia in the sensorimotor cortex accompanying K(+)-induced spreading depression in rats and cats.

Vascular changes accompanying spreading depression (SD) remain controversial. We examined dynamic alterations of local cerebral blood volume (CBV) during SD by observing light transmission at an isosbestic point of hemoglobin (550 nm) in seven rats and five cats under alpha-chloralose/urethane anesthesia. The two species were used for comparison between the lissencephalic and gyrencephalic brains. We found that a concentrated K(+) solution microinjected into the sensorimotor cortex provoked CBV changes that appeared as a repetitive propagation of concentric wave-rings of ischemia followed by hyperemia expanding peripherally from the injection site at speeds of 1.9-3.2 mm/min. The dynamic CBV changes continued repeatedly every 1-5 min for more than 30 min in three rats, ceased within 30 min in three rats and remained at the site of K(+) injection in one rat. Similar repeated CBV changes occurred in two out of five cats.

Animals↗

PubMed2002

Dilatation of cerebral parenchymal vessels mediated by angiotensin type 1 receptor in cats.

We report the effects of angiotensin II (ANG-II), as well as angiotensin II type 1 (AT1) and type 2 receptor antagonists (CV-11974 and PD-123319, respectively) on the cerebral parenchymal microvessels in cats using the photoelectric method. ANG-II continuously and dose-dependently increased the cerebral blood volume (CBV) for 15 min. Maximum CBV increases were +0.36+or-0.11 vol% for 0.01 nmol/kg (P<0.05), +0.51+or-0.24 vol% for 0.1 nmol/kg (P<0.05), +1.87+or-0.55 vol% for 1 nmol/kg (P<0.05), and +2.14+or-0.77 vol% for 10 nmol/kg (P<0.05). Systemic arterial blood pressure increased at only 1 min following ANG-II infusion (1 and 10 nmol/kg). CV-11974 and PD-123319 per se did not change the resting CBV. CV-11974 completely inhibited the vasodilatory action of ANG-II, however, PD-123319 did not block it. We conclude that ANG-II directly dilates the parenchymal vessels through the AT1 receptor without increasing systemic blood pressure, and that intrinsic ANG-II may not be associated with maintenance of resting vascular tone.

Angiotensin II↗

PubMed2002

Immature dendritic cells (CD11c+ CD3- B220- cells) present in mouse peripheral blood.

It is well known that dendritic cells (DCs) are developed from the peripheral blood of mice when peripheral blood mononuclear cells (PBMCs) are cultured with GM-CSF. We have previously found that immature DCs are present in the blood even in humans. In the present study, we show that CD11c+ CD3- B220- cells in the mouse peripheral blood are immature DCs. The percentage of CD11c+ CD3- B220- cells in the (PBMCs) of normal mice ranges from 0.5 to 2.5%. The CD11c+ CD3- B220- cells in the PBMCs show dendrites, similar in shape to the CD11c+ CD3- B220- cells in the spleen, which are thought to be DCs definitely. However, they have practically no capacity to stimulate the proliferation of allogeneic T cells, and show a lower expression of MHC class II, B7-1 and B7-2 than CD11c+ CD3- B220- cells in the spleen. When the CD11c+ CD3- B220- cells in the PBMCs are cultured with GM-CSF, they show not only the potent ability to stimulate the proliferation of allogeneic T cells but also a higher expression of MHC class II, B7-1 and B7-2. Moreover, they migrate into the spleen when they are injected intravenously. These results suggest that CD11c+ CD3- B220- cells in the PBMCs are immature DCs, and that they migrate into the spleen, where they mature.

Animals↗