Biomedical subjects

Kazuo Tanishita

Publications and source records attributed to Kazuo Tanishita.

At least 19 recordsLinked to original sources

PubMed2005

Reconstruction of 3D stacked-up structures by rat small hepatocytes on microporous membranes.

The three-dimensional (3D) culture of hepatocytes is essential for the reconstruction of functional hepatic tissues in vitro. In the present experiment, we developed a 3D-culture method in order to reconstruct hepatic cordlike structures by stacking up two-dimensional (2D) tissues composed of rat small hepatocytes (SHs), which are hepatic progenitor cells. Pairs of membranes were prepared and the cells were separately cultured on each membrane. After the SH colonies had developed, one membrane was inverted on top of the other to form an SH bilayer. Thereafter, we investigated whether the stacked cells were organized into differentiated tissues. In the 3D stacked-up structures, bile canaliculi (BC) started to form and gradually developed into anastomosing networks. Transmission electron microscopy revealed that the SHs of the upper and lower layers adhered to one another, and that BC formed between them. Bile canalicular proteins localized on the lumina of the tubular structures. Furthermore, the cells within the structures exhibited mRNA transcription of the hepatic-differentiation markers and maintained a relatively high level of albumin secretion. We conclude that highly differentiated 3D tissues, including functional BC, can be reconstructed by stacking up layers of SHs. This 3D stacked-up culture is useful for the reconstruction of tissue-engineered livers.

Albumins↗

PubMed2005

Coordinated movement of bile canalicular networks reconstructed by rat small hepatocytes.

Hepatocytes in vivo have a potential for liver regeneration, but it has been very difficult to reconstruct hepatic organoids in vitro. Recent studies have shown that small hepatocytes (SHs) can reconstruct hepatic organoids including functional bile canaliculi (BC). In the present study we analyzed the movement of BC formed in the hepatic organoids, focusing on the coordination of contraction and dilation among cells and the mechanism producing the coordination. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Time-lapse images of BC movements were taken and analyzed in cells treated with or without cytochalasin B (CB). Time-lapse images revealed that all BC, regardless of region contracted in a coordinated manner. Actin filaments were observed along the BC even after the BC networks treated with CB dilated markedly. Microinjection of dye was also carried out to investigate the flow thorough BC. Secreted fluorescein from the injected cell flowed along BC, and gap junctional protein connexin 32 was expressed along BC networks, suggesting cell-to-cell communication. Thus, groups of hepatocytes in the hepatic organoids act in a coordinated manner through intercellular communication.

Animals↗

PubMed2005

Effect of fluid force on vascular cell function.

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to fluid frictional force (shear stress) induced by blood flow, and shear stress affects the intracellular calcium ([Ca2+]i), which initiates cellular responses. Here, we studied the effect of long-term exposure of shear stress on [Ca2+]i responses in cultured ECs by using a confocal laser microscope and calcium indicator. At the initiation of shear stress of 20 dyn/cm2 (0 hr), 27% of the cells exhibited [Ca2+]i responses. This percentage gradually decreased with increasing exposure time, reaching about 4% after 24 hr of exposure. These data indicate that long-term shear-stress exposure affects [Ca2+]i responses in cultured ECs. Furthermore, we studied the effect of magnitude of shear stress on macromolecule uptake. For the low shear-stress, the uptake was enhanced, whereas the uptake was inhibited for higher shear-stress.

Analysis of Variance↗

PubMed2004

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells.

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.

Albumins↗

PubMed2004

Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model.

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.

Animals↗

PubMed2004

Localized compliance of small airways in excised rat lungs using microfocal X-ray computed tomography.

Airway compliance is a key factor in understanding lung mechanics and is used as a clinical diagnostic index. Understanding such mechanics in small airways physiologically and clinically is critical. We have determined the "morphometric change" and "localized compliance" of small airways under "near"-physiological conditions; namely, the airways were embedded in parenchyma without dehydration and fixation. Previously, we developed a two-step method to visualize small airways in detail by staining the lung tissue with a radiopaque solution and then visualizing the tissue with a cone-beam microfocal X-ray computed tomography system (Sera et al. J Biomech 36: 1587-1594, 2003). In this study, we used this technique to analyze changes in diameter and length of the same small airways ( approximately 150 microm ID) and then evaluated the localized compliance as a function of airway generation (Z). For smaller (<300-microm-diameter) airways, diameter was 36% larger at end-tidal inspiration and 89% larger at total lung capacity; length was 18% larger at end-tidal inspiration and 43% larger at total lung capacity than at functional residual capacity. Diameter, especially at smaller airways, did not behave linearly with V(1/3) (where V is volume). With increasing lung pressure, diameter changed dramatically at a particular pressure and length changed approximately linearly during inflation and deflation. Percentage of airway volume for smaller airways did not behave linearly with that of lung volume. Smaller airways were generally more compliant than larger airways with increasing Z and exhibited hysteresis in their diameter behavior. Airways at higher Z deformed at a lower pressure than those at lower Z. These results indicated that smaller airways did not behave homogeneously.

Animals↗

PubMed2004

Successive depth variations in microvascular distribution of rat somatosensory cortex.

Although hemodynamic-based functional brain imaging techniques are powerful tools to explore the brain functions noninvasively, hemodynamic-based signal is strongly affected by spatial configuration of microvessels. Understanding the quantitative relations between microvascular structure and functional activity is therefore significant to make a valid signal interpretation for the imaging techniques. In the present study, we evaluated depth profiles of microvascular distributions in rat somatosensory subfields (barrel field, forelimb region, trunk region and hindlimb region) and characterized depth variations in microvascular structures, such as locations, lengths and directions of microvessels, throughout the cortical layers (I-VI). To obtain the accurate microvascular structure, we made a customized casting method by using confocal laser scanning microscope. We observed that microvascular distribution successively varied throughout the cortical layers (I-VI) and that the maximum number density of microvessels was consistently found in middle layers (III-V). In addition, superficial layers had relatively long microvessels, almost perpendicular to the cortical surface, whereas middle layers had short microvessels propagating in all directions. These regional differences in microvascular structures were closely related to the somatosensory subfields, e.g., barrel field was the greatest number density of microvessels among the investigated subfields. Based on these observations, we compared microvascular profiles with previously reported distribution patterns of tissue partial pressure of oxygen (pO2). The results showed that tissue pO2 was correlated with microvascular distribution in some but not all of the subfields. This finding shows that detailed microvascular profiles are helpful to investigate causal relationships between microvascular structure and functional activities in cerebral cortex.

Animals↗

PubMed2004

Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells.

The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC-like structures developed between rising/piled-up cells. We investigated how BC-like structures developed with maturation of SHs and whether the structures were functionally active as BC. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Immunocytochemistry and immunoblotting for BC proteins, such as ectoATPase, 5'-nucleotidase, dipeptidylpeptidase IV, and multidrug-resistance associated protein 2, were examined and time-lapse microscopy was used for the observation of BC contractions. Secretion of bilirubin into the reconstructed BC was also observed. The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins were localized in the intercellular space that coincided with BC-like structures reconstructed between rising/piled-up cells. Tight junction-associated protein ZO-1 was also expressed along the BC-like structures. Bilirubin added to the medium were secreted into BC-like structure and accumulated without leakage. Time-lapse microscopy showed continuous contractions of reconstructed BC. In conclusion, BC-like structures reconstructed by SHs may be functional with membrane polarity, secretory ability, and motility. These results show that this culture system may suitable for investigating the mechanism of the formation of BC and their functions.

Animals↗

PubMed2004

Differences in pO2 peaks of a murine fibrosarcoma between carbon-ion and X-ray irradiation.

We measured and compared the oxygen partial pressure (pO(2)) profiles in experimental tumors after irradiation with carbon ions and with X-rays. The NFSa fibrosarcomas grown in the hind legs of C3H male mice received isoeffect single doses of carbon ions or X-rays. Coaxial oxygen microelectrodes of high spatial resolution were inserted into the tumor with 20 microm steps by a computerized micromanipulator. The number of pO(2) peaks that reached 15 mmHg were at least 0.45 per 3,000 microm in unirradiated tumors and significantly increased to 1.55 per 3,000 microm as early as day 1 of carbon-ion irradiation (p < 0.001). The tumors that received X-ray irradiation also significantly increased pO(2) peaks, but as late as day 3. The time course of pO(2) peak appearance in the present study coincides with a previous report where reoxygenation was measured by paired growth delay assay. The pO(2) peaks appeared selectively in peripheral regions of X-ray irradiated tumors, but they appeared rather homogeneously in the tumor after carbon-ion irradiation. It is concluded that carbon-ion irradiation reoxygenated the NFSa fibrosarcomas earlier in time and deeper in space than the X-ray irradiation did.

Animals↗

PubMed2004

In vivo nitric oxide measurements using a microcoaxial electrode.

Nitric oxide (NO) is a vital molecule contributing to numerous physiological phenomena in various biological systems. To investigate the physiological role of NO, a range of NO-specific electrodes allowing direct and continuous NO measurement have been developed for in vitro and in vivo NO detection. A microcoaxial electrode has also been developed for the measurement of real-time NO levels. Because the working and reference electrodes in this device are situated close together, the microcoaxial electrode is considered to be ideal for the measurement of local NO concentrations with high spatial resolution. The microcoaxial electrode has been successfully applied to the real-time measurement of NO in endothelial cells, thereby demonstrating its effectiveness as an NO-specific electrode. In this chapter, we describe our experimental protocol for performing real-time NO measurements that was developed during physiological experiments using the microcoaxial electrode.

Animals↗

PubMed2004

Intraaneurysmal flow visualization by using phase-contrast magnetic resonance imaging: feasibility study based on a geometrically realistic in vitro aneurysm model.

OBJECT: The aim of this study was to evaluate the feasibility of complex intraaneurysmal flow visualization with the currently available phase-contrast magnetic resonance (MR) imaging modality. METHODS: A geometrically realistic in vitro aneurysm model, in which detailed flow velocity analysis had already been conducted using laser Doppler velocimetry was used for this in vitro hemodynamic simulation, so that the results of phase-contrast velocity measurements could be compared with the previous reliable results. On a 1.5-tesla unit, three orthogonal components of velocity were obtained using a standard two-dimensional fast low-angle shot flow quantification sequence. Three-dimensional (3D) intraaneurysmal flow structures recorded during one cardiac cycle were depicted in one midsagittal and three axial cross-sectional planes with the aid of gray scale phase-contrast velocity maps. Isovelocity contour maps and secondary flow vectors were also created based on the phase-contrast velocity maps by using MATLAB software. The isovelocity contours in those three axial sections could demonstrate the shapes of inward and outward flow areas and their alternation over one cardiac cycle. The secondary flow vectors demonstrated twin vortices within the outward flow area adjacent to the boundary layer of inward and outward flow in all axial planes. CONCLUSIONS: The phase-contrast MR imaging method was able to depict the complex 3D intraaneurysmal flow structures in the in vitro aneurysm model. Detailed 3D intraaneurysmal flow information will be obtainable in vivo after improvements are made in spatial resolution, which is expected in the near future. The capability to visualize intraaneurysmal flow structures directly with the use of noninvasive MR imaging technology will have a positive impact on future clinical practice.

Hemodynamics↗

PubMed2003

Dual responses of tissue partial pressure of oxygen after functional stimulation in rat somatosensory cortex.

To compare the spatial heterogeneity of brain tissue partial pressure of oxygen (pO(2)) among local brain regions, we focused on functional and anatomical variations in rat somatosensory cortex. Tissue pO(2) was measured by using an oxygen microelectrode with high spatio-temporal resolution, and investigated in three somatosensory areas including hindlimb (HL), forelimb (FL), and trunk region (Tr). Their anatomical structures were determined with histological techniques (Nissl stain). In addition to the measurement of baseline tissue pO(2), we examined temporal shifts in tissue pO(2) distribution elicited by functional stimulation using the brushing stimulation to the hindlimb, forelimb, and trunk regions of the body. We observed that average tissue pO(2) in the Tr (14+/-10 Torr) was significantly lower than those in the HL (25+/-13 Torr) and FL (24+/-13 Torr). Such regional differences in tissue pO(2) were closely related to the cytoarchitectonic variations among these three areas. In addition, the functional stimulation enlarged the regional differences in the pO(2) depending on each somatosensory area; the pO(2) in the HL increased by 3.6+/-2.9% after the stimulation to hindlimb, whereas that in the Tr decreased by -2.9+/-2.5% after the stimulation to trunk region. Such dual responses of tissue pO(2) (i.e. increase or decrease) after the functional stimulation to the corresponding body regions may provide a criterion to clinically predict regions susceptible to tissue hypoxia, because considerable decrease in tissue pO(2) occurred in the Tr showing the lowest baseline pO(2).

Animals↗

PubMed2003

Three-dimensional visualization and morphometry of small airways from microfocal X-ray computed tomography.

Physiological morphometry is a critical factor in the flow dynamics in small airways. In this study, we visualized and analyzed the three-dimensional structure of the small airways without dehydration and fixation. We developed a two-step method to visualize small airways in detail by staining the lung tissue with a radiopaque solution and then visualizing the tissue with a cone-beam microfocal X-ray computed tomographic (CT) system. To verify the applicability of this staining and CT imaging (SCT) method, we used the method to visualize small airways in excised rat lungs. By using the SCT method to obtain continuous CT images, three-dimensional branching and merging bronchi ranging from 500 to 150 microm (the airway generation=8-16) were successfully reconstructed. The morphometry of the small airways (diameter, length, branching angle and gravity angle between the gravity direction and airway vector) was analyzed using the three-dimensional thinning algorithm. The diameter and length exponentially decreased with the airway generation. The asymmetry of the bifurcation decreased with generation and one branching angle decided the other pair branching angle. The SCT method is the first reported method that yields faithful high-resolution images of soft tissue geometry without fixation and the three-dimensional morphometry of small airways is useful for studying the biomechanical dynamics in small airways.

Algorithms↗

PubMed2003

Biphasic changes in tissue partial pressure of oxygen closely related to localized neural activity in guinea pig auditory cortex.

An understanding of the local changes in cerebral oxygen content accompanying functional brain activation is critical for making a valid signal interpretation of hemodynamic-based functional brain imaging. However, spatiotemporal relations between changes in tissue partial pressure of oxygen (Po2) and induced neural activity remain incompletely understood. To characterize the local Po2 response to the given neural activity, the authors simultaneously measured tissue Po2 and neural activity in the identical region of guinea pig auditory cortex with an oxygen microelectrode (tip < 10 microm) and optical recording with voltage-sensitive dye (RH 795). In addition, a laser displacement gauge and a laser-Doppler flowmeter were used to monitor the spatial displacement and regional cerebral blood flow, respectively, in the Po2 measurement region. In the activated region, tissue Po2 initially decreased during the approximately 3-seconds after the onset of acoustic stimuli, and then increased during the next approximately 5 seconds. Such biphasic changes are consistently found in cortical layers I to IV. In addition, amplitude of the biphasic change was closely related to detected peak height of the optical signal changes. The results suggest that the initial decrease in tissue Po2 is coupled to the induced neural activity and depends on response time of local increase in cerebral blood flow.

Acoustic Stimulation↗

PubMed2003

Respiratory flow in a realistic tracheostenosis model.

The possible mechanism of wheeze generation in tracheostenosis was identified by measuring inspiratory and expiratory flow in a "morphological and distensible" realistic tracheostenosis model. The shape of the model was based on CT (Computed Tomography) images of a patient that had tracheostenosis. A trachea consists of tracheal cartilage rings and smooth muscle. Spatial variation of wall distensibility was achieved in the model by varying the wall thickness based on the elastic modulus measured in pig airways. The spatial variation influenced the flow in the airway and the turbulence production rate decreased faster at smooth muscles. Using the model, we investigated the mechanism of wheeze generation by focusing on the turbulence intensity. The turbulence intensity in expiratory flow was about twice that in inspiratory flow, and larger vortices existed in post-stenosis in expiratory flow, and thus might contribute to wheeze generation.

Animals↗

PubMed2003

In vitro measurement of fluid-induced wall shear stress in unruptured cerebral aneurysms harboring blebs.

BACKGROUND AND PURPOSE: Little attention has been focused on the role of fluid-induced wall shear stress in fully developed cerebral aneurysms. The purpose of this study is to evaluate the alternation and distribution of wall shear stress over 1 cardiac cycle in patients' aneurysms. METHODS: A middle cerebral artery aneurysm and a basilar tip aneurysm with localized outpouching (blebs) in their domes were selected for this study. With the use of a stereo lithography machine, geometrically realistic aneurysm models were created on the basis of 3-dimensional CT angiograms. In vitro shearing velocity measurement was conducted with the use of laser-Doppler velocimetry at multiple points on the aneurysmal wall to calculate the value of wall shear stress. The wall shear stress was documented at multiple points in the aneurysm inflow zone, dome, and outflow zone. RESULTS: Distribution of wall shear stress was not uniform in the aneurysm walls, and particular regions were exposed to relatively high wall shear stress. The wall shear stress changed dynamically throughout 1 cardiac cycle at the point where a high value of wall shear stress was noted. The blebs of both aneurysms were exposed to high wall shear stress. Unlike previous reports in which an ideal spherical aneurysm model was used, the aneurysm inflow zone was not exposed to high shear stress. CONCLUSIONS: In vitro aneurysm models based on the patients' angiograms allowed us to conduct a more realistic evaluation of wall shear stress in the aneurysms harboring blebs. These results provide us with further indications of the correlation of wall shear stress with the natural history of cerebral aneurysms.

Angiography↗

PubMed2003

Three-dimensional blood flow analysis in a wide-necked internal carotid artery-ophthalmic artery aneurysm.

OBJECT: The aim of this study was to evaluate axial and secondary flow structures in a wide-necked internal carotid artery-ophthalmic artery aneurysm, one of the most common locations for endovascular coil placement. METHODS: A clear acrylic aneurysm model was manufactured from a three-dimensional computerized tomography angiogram. Intraaneurysm blood flow analysis was conducted using an acrylic aneurysm model together with laser Doppler velocimetry and particle imaging velocimetry. The maximal axial blood flow velocities in the inflow and outflow zones at the aneurysm orifice were noted at the peak systolic phase, measuring 46.8 and 24.9% of that in the parent artery, respectively. The mean size of the inflow zone during one cardiac cycle was 44.3 +/- 9.8% (range 35.6-58.7%) the size of the axial section at the aneurysm orifice. In the lower and upper planes of the aneurysm dome, the mean size of inward and outward flow areas were 43.3 +/- 6.7% and 43.8 +/- 6.8% the size of the axial cross-sectional plane, respectively. The axial flow velocity structures were dynamically altered throughout the cardiac cycle, particularly at the aneurysm orifice. The fastest secondary flow at the opening was also noted at the peak systolic and early diastolic phases. Axial blood flow velocity was slower in the upper axial plane of the aneurysm dome than in the lower one. Conversely, the secondary flow component was faster in the upper plane. CONCLUSIONS: The side-wall aneurysm in this study did not demonstrate a simple flow pattern as was previously seen in ideally shaped experimental aneurysms in vitro and in vivo. The flow patterns of inflow and outflow zones were very difficult to predict based on the limited flow information provided on standard digital subtraction angiography, even in an aneurysm with a relatively simple dome shape.

Aneurysm↗

PubMed2002

Intravascular inhomogeneous oxygen distribution in microvessels: theory.

Cross-sectional oxygen distribution in microvessels in most previous studies has been assumed to be homogeneous. Recent studies using phosphorescence quenching microscopy or microspectrophotometry showed a decline in oxygen profile near the arterial wall. In this study we performed theoretical analysis of intravascular P(O(2)) and S(O(2)) profiles in arterioles by using a radial diffusion model with a constant oxygen efflux from the vascular lumen, taking intravascular flow distribution into account. Theoretical calculations indicated that radial oxygen diffusion and a laminar flow pattern would create inhomogeneous intravascular oxygen profile with a decline toward the arterial wall. As mean blood flow velocity became lower, the difference between the centerline oxygen level and the inner surface level became larger. In conclusion, it is suggested that oxygen efflux from the vascular lumen and less convective supply near the vascular wall create a decline in P(O(2)) as well as S(O(2)) toward the arterial wall.

Blood Flow Velocity↗