Possible Mechanisms of Axonal Transport Disturbances in Mouse Spinal Motoneurons Induced by Hypogravity.

The data obtained by transcriptome analysis of lumbar spinal cord segments, sciatic nerve, and the respiratory diaphragm of the mice performed after a space flight on board Bion-M1 biosatellite were processed by bioinformatic methods aimed at elucidation of the regularities in hypogravity-induced transcriptome changes in various compartments of motor neurons. The study revealed abnormalities of axonal transport in spinal motor neurons provoked by weightlessness. These data agree with the results of electron microscopy examination of the spinal cord in experimental animals. In space group mice sacrificed on the landing day, the content of perinuclear ribosomes in lumbar motoneurons surpassed that in control mice or in the recovery group examined 1 week after the flight. The data corroborate our hypothesis on contribution of axonal transport disturbances into pathogenesis of hypogravity motor syndrome. They can be employed as a launching pad for further study of hypogravity-triggered motor disorder mechanisms in order to elaborate the preventive therapy against the development of hypogravity motor syndrome in space flights.

Backscattered electron image of osmium-impregnated/macerated tissues as a novel technique for identifying the cis-face of the Golgi apparatus by high-resolution scanning electron microscopy.

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three-dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis-trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis- or trans-faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis-Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis-elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron-mode SEM. Prolonged osmication by osmium impregnation (2% OsO4 solution at 40°C for 40 h) and osmium maceration (0.1% OsO4 solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.

Differences in the pharmacokinetics of Cyp3a substrates in TSOD and streptozotocin-induced diabetic mice.

The pharmacokinetics of drugs can change in diabetes mellitus and even among diabetics. They may differ between type I diabetes (T1DM) and type 2 diabetes (T2DM). As triazolam was administered orally to Tsumura, Suzuki, obese, diabetes (TSOD) mice and streptozotocin (STZ) mice, clearance per body (CL/F) in TSOD mice did not differ compared with Tsumura, Suzuki, non-obesity (TSNO) mice. In STZ mice, CL/F was greater than in control mice. Small intestinal cytochrome P450 (Cyp) 3a expression in TSOD mice was significantly lower than in TSNO mice. No significant difference existed in small intestinal Cyp3a expression between STZ mice and control mice. In insulin-treated mice, small intestinal Cyp3a expression was significantly lower than in control mice. These results suggested that the differences in changes in small intestinal Cyp3a expression between T1DM and T2DM may be due to differences in plasma insulin concentrations. This may be a factor in the difference in the drug pharmacokinetics between T2DM and T1DM patients.

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.

Imaging of human metaphase chromosomes by atomic force microscopy in liquid.

Human metaphase chromosomes were observed using an intermittent contact mode of atomic force microscopy (AFM) in a phosphate-buffered saline solution to clarify their conformation close to that in the physiological state. In the AFM images in liquid, symmetric alternating ridges and grooves were evident on their surface of the paired sister chromatids. The number of the ridges and grooves were rather specific to the type of the chromosome. The structural changes of chromosomes caused by trypsin treatment were also directly observable using AFM in liquid. These results suggest that the intermittent contact mode AFM is useful not only for analyzing the structure of chromosomes in a liquid condition but also for studying the effect of chemical treatments on chromosomes in relation to their structural changes.

Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy.

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells.

The three-dimensional organization of collagen fibrils in the human cornea and sclera.

The organization of collagen fibrils in the human cornea and sclera was studied by scanning electron microscopy, after digestion of cellular elements by sodium hydroxide, and by conventional transmission electron microscopy. The collagen fibrils in the cornea had a uniform diameter of about 25 nm. In Bowman's layer, individual collagen fibrils were interwoven densely to form a felt-like sheet. In the stroma, most of the collagen fibrils ran abreast in lamellae, with varying widths and thickness. These lamellae were arranged basically parallel to the corneal surface but often communicated with those of adjacent layers by interchanging their fibrils. In the innermost stromal region adjacent to Descemet's membrane, collagen fibrils were oriented in various directions and interlaced, forming loose fibrillar networks. The sclera, however, was composed of collagen fibrils with various diameters ranging from 25-230 nm. Although these collagen fibrils formed bundles, they were not parallel but were entangled in individual bundles. These collagen bundles varied in width and thickness, often gave off branches, and intertwined with each other.

Distribution and ultrastructure of the autonomic nerves in the mouse pancreas.

Peripheral innervation of the mouse pancreas was studied by scanning and transmission electron microscopy, as well as by light microscopy (cholinesterase technique). Major nerve bundles usually ran with arteries in the connective tissue septa. They gave off delicate branches that formed plexuses around arteries and arterioles. When reaching the capillaries, nerve fibers left the arterioles and formed very loose networks in the interacinar spaces. The nerves accompanying the arteries also sent off branches toward the islets of Langerhans and formed a dense plexus around the islets. A few delicate nerve fibers were also present around the pancreatic ducts. Thus, the intrapancreatic nerves formed four plexuses: perivascular, periductal, periacinar and peri-insular. The plexuses were networks of unmyelinated nerve fibers consisting of axons with varicosities and Schwann cells. Intrapancreatic ganglia were found in the interlobular connective tissue; ganglia were often closely associated to islets of Langerhans. Our findings indicate that the "interstitial cells" described by light microscopists correspond to Schwann cells. Axons in the nerve plexuses contain transmitter vesicles and therefore represent an autonomic terminal apparatus. The rich innervation of arterioles and islets suggests that neural regulation of secretory function is mediated by control of pancreatic blood flow.

Cytoarchitecture of the smooth muscles and pericytes of rat cerebral blood vessels. A scanning electron microscopic study.

The three-dimensional cytoarchitecture of the smooth muscles and pericytes of rat cerebral blood vessels was studied by scanning electron microscopy after removing extracellular connective tissue matrices with the KOH-collagenase digestion method. The tunica media of major intracranial arteries such as the internal carotid, vertebral, basilar, and other cerebral arteries measuring more than 100 microns in outer diameter consisted of spindle-shaped smooth-muscle cells arranged circularly to the long axis of the vessel. Muscle cells at the branching points, however, showed a variety of shapes, sizes, and arrangements. As the vessel size decreased, smooth-muscle cells showed bi- or trifurcations at the cell poles. In the precapillary arterioles, smooth-muscle cells which had helically surrounded the endothelial tubes had bulging cell bodies with various cytoplasmic processes extending from the cell poles. Distinct specializations presumed to be sphincters were not found on the arteries or arterioles. Pericytes of the capillary had become extended along the vessel axis, having fusiform cell bodies with longitudinally oriented long cytoplasmic processes. Cells located periendothelially in the venules and veins were stellate in shape with many cytoplasmic processes which were interwoven to form complicated cellular networks around the endothelial tube.

Three-dimensional cytoarchitecture of rat pulmonary venous walls: a light and scanning electron microscopic study.

The three-dimensional architecture of the rat pulmonary veins was studied by light microscopy (LM) and scanning electron microscopy (SEM). For LM, the left lungs were fixed with formalin, sectioned and immunostained with an anti-alpha-smooth muscle actin (alpha-SMA) antibody in addition to conventional staining. For SEM, the specimens were fixed with glutaraldehyde and immersed in 30% KOH solution for 8 min followed by treatment of collagenase solution for more than 5 h. By LM, the smooth muscle cells stained with anti-alpha-SMA showed discontinuous, periodical thickenings of circular bundles in the wall of the venules, but they became thin and continuous in the larger vessels (or veins) that had a cardiac muscle layer on the outside. Under SEM, the smooth muscle cells formed circular-oriented bundles at constant intervals along the venules less than 100 microm in diameter. These bundles had circumferential constrictions in the lumen. The cardiac muscle cells, which appeared in large pulmonary veins of more than 100 microm, ran in a circular or oblique direction and completely surrounded the vessel wall outside of the thin continuous layer of smooth muscle cells. The muscle arrangements were considered to play a significant role in the return blood flow in rat pulmonary veins.

Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton.

Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments--actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity.

Different enzyme activities in coronary capillary endothelial cells.

Differential distributions of alkaline phosphatase (AP) and dipeptydylpeptidase IV (DPPIV) were studied in coronary microvascular endothelial cells. Endothelial cells were obtained by the perfusion of coronary vessels with 0.1% trypsin PBS solution and cultured in uncoated culture dishes. Staining of cultured endothelial cells with AP- and DPPIV-sensitive reagents revealed blue or red staining, respectively. Most colonies showed cells of only one color, blue or red, even at the fifth passage. AP-sensitive cells, which were originally elongated, shortened and widened, proliferating to form monolayer colonies of cobble stone-like cells. AP-stainability became weak with repeated passages. DPPIV-sensitive endothelial cells remained elongated even after repeated passages. The cell shape and stainability seemed to be coupled and maintained through the five passages studied.

Immunohistochemical demonstration of bovine viral diarrhoea virus antigen in the pancreatic islet cells of cattle with insulin-dependent diabetes mellitus.

The pancreatic islets were studied in seven cattle with insulin-dependent diabetes mellitus (IDDM) associated with persistent bovine viral diarrhoea virus (BVDV) infection. BVDV antigen was detected immunohistochemically in the pancreatic islet cells. There was a decrease in the size and number of islets, vacuolar degeneration of residual islet cells, and lymphocytic insulitis. The atrophied islets were composed of small uniform cells with limited amounts of cytoplasm, containing a small number of insulin- and chromogranin-positive granules. Enlarged islets consisting of islet cells with vacuolated cytoplasm were also frequently observed. Many of the vacuolated islet cells differed from the cells of normal islets in containing fewer cytoplasmic insulin- and chromogranin-positive granules. Mild lymphocytic insulitis was observed frequently in enlarged islets but rarely in atrophied islets. Immunoreactivity with BVDV antibody was found in the acinar cells of the pars exocrina in all seven cattle and in the residual cells of the islets of Langerhans of four cattle. BVDV antigen-positive cells were seen more frequently in the enlarged islets than in the atrophied islets. Some islets with lymphocytic infiltrates showed a small number of antigen-positive cells. These findings suggest that autoimmune IDDM was induced by persistent BVDV infection, resulting in gradual destruction of the islet beta cells.

[Observation of human corneal and scleral collagen fibrils by atomic force microscopy].

PURPOSE: We attempted to analyze the three-dimensional ultrastructure of human corneal and scleral collagen fibrils with an atomic force microscope (AFM). METHODS: A normal eye removed from a 66-year-old male was used in the study. Suspended corneal and scleral collagen fibrils were individually attached to glass slides by centrifugation. These collagen fibrils were air-dried and observed with a non-contact mode AFM in air. RESULTS: AFM imaging provided information on the surface topography of both corneal and scleral collagen fibrils. The corneal collagen fibrils had a height of 11.9 +/- 1.0 (mean +/- standard deviation) nm and the scleral fibrils of 82.5 +/- 35.6 nm. A periodic banding pattern of grooves and ridges was clearly found in both types of fibrils; the D-periodicity and the groove depth were 65.7 +/- 0.8 nm and 1.46 +/- 0.50 nm in the corneal fibrils, and 67.3 +/- 1.1 nm and 6.16 +/- 1.23 nm in the scleral fibrils. CONCLUSIONS: Surface topographic images of human corneal and scleral collagen fibrils were clearly obtained with the AFM. This technique provides quantitative information on the surface morphology of the collagen fibrils at high resolution.

[The three-dimensional ultrastructure of the collagen fibers, reticular fibers and elastic fibers: a review].

Fibrous components of the connective tissue are light-microscopically classified into three types: collagen fibers, reticular fibers and elastic fibers. The present paper reviews the three-dimensional ultrastructure of these fibrous components, mainly based on our studies by scanning electron microscopy. The collagen fibers are shaped like tapes or cords about 1 to 20 microns in diameter. Each fiber is a bundle of fibrils running roughly parallel to each other. These collagen fibrils vary in diameter from 30 to 300 nm depending on their locating area of the body, and show a repeating pattern of depressed and protruding segments on the surface. The reticular fibers consist of collagen fibrils about 20-40 nm in diameter, which run singly or in small bundles. They are usually interwoven elaborately to form thin lace-like sheets or sheaths attaching to basal laminae of such cells as epithelial, endothelial and muscular cells. These fibers are considered to play an important role not only in adhering the cells to the collagen fibers, but also in constituting the skeletal framework suitable for individual cells and tissues. The elastic fibers consist of two different components: elastin and fibrillin. Elastin forms unit fibrils of 0.1-0.2 micron thickness which are arranged in bundles or laminae specific to individual organs and tissues. Fibrillin, on the other hand, forms microfibrils about 10 nm in diameter running in or along elastin bundles. These microfibrils also form delicate networks separate from elastin components. For a comprehensive understanding of the fibrous components in the connective tissue, the author proposed categorizing them into two systems: the collagen fibrillar system as a supporting framework of tissues and cells, and the fibrillin-elastin fibrillar system for distributing stressing forces uniformly in tissues.

Microhardness evaluations of resin-dentin bonding areas by nano-indentation.

The purpose of this experiment was to determine the hardness values of the hybrid layer and its surroundings through the continuous use of a microhardness measuring device. Black's Class V cavities were prepared in nine dog teeth. The cavities were divided into four groups according to the dentin adhesive system applied. The adhesive systems were: "Bond One System", "Liner Bond II sigma System", "One Step System", and "Single Bond System". The treated teeth were observed at seven days post-application. Specimens were cross-sectioned perpendicularly or horizontally to the resin-dentin interface and embedded in epoxy resin. Their surfaces were polished. The microhardness of the resin-dentin bonding area was measured with a nano-indentation tester. The hardness values at a point of 10 microns distant from the interface in the direction of the dentin differed between systems. It appeared that this was influenced by the presence of the decalcified dentin not impregnated by resin, differences in the chemistry forming the hybrid layer, and the composition of the bonding resin. The hardness of the dentin-bonding interface and its surroundings was determined, and these areas were observed using SEM. Three layers were confirmed the healthy dentin layer, the composite resin layer, and the hybrid layer, (in which decalcified dentin impregnated by resin and that not impregnated by resin are considered to be mix). In the hybrid layer, no impression was found by SEM although the hardness in the bonding interface was significantly different. These layers appear to be more elastic and softer than the healthy dentin.

Development of nanomanipulator using a high-speed atomic force microscope coupled with a haptic device.

The atomic force microscope (AFM) has been widely used for surface fabrication and manipulation. However, nanomanipulation using a conventional AFM is inefficient because of the sequential nature of the scan-manipulation scan cycle, which makes it difficult for the operator to observe the region of interest and perform the manipulation simultaneously. In this paper, a nanomanipulation technique using a high-speed atomic force microscope (HS-AFM) is described. During manipulation using the AFM probe, the operation is periodically interrupted for a fraction of a second for high-speed imaging that allows the topographical image of the manipulated surface to be periodically updated. With the use of high-speed imaging, the interrupting time for imaging can be greatly reduced, and as a result, the operator almost does not notice the blink time of the interruption for imaging during the manipulation. This creates a more intuitive interface with greater feedback and finesse to the operator. Nanofabrication under real-time monitoring was performed to demonstrate the utility of this arrangement for real-time nanomanipulation of sample surfaces under ambient conditions. Furthermore, the HS-AFM is coupled with a haptic device for the human interface, enabling the operator to move the HS-AFM probe to any position on the surface while feeling the response from the surface during the manipulation.

High-speed AFM of human chromosomes in liquid.

Further developments of the previously reported high-speed contact-mode AFM are described. The technique is applied to the imaging of human chromosomes at video rate both in air and in water. These are the largest structures to have been imaged with high-speed AFM and the first imaging in liquid to be reported. A possible mechanism that allows such high-speed contact-mode imaging without significant damage to the sample is discussed in the context of the velocity dependence of the measured lateral force on the AFM tip.

The olfactory route for cerebrospinal fluid drainage into the peripheral lymphatic system.

Drainage of the cerebrospinal fluid through the olfactory nerves into the nasal lymphatics has been suggested repeatedly. To investigate precisely the morphology of this pathway, India ink was injected into the subarachnoidal space of the rat brain, and samples including the olfactory bulbs, olfactory tracts and the nasal mucosa were observed by light and electron microscopy. Under the dissecting microscope, ink particles were found within the subarachnoid space and along the olfactory nerves. At the nasal mucosa, a lymphatic network stained in black was identified near the olfactory nerves, which finally emptied into the superficial and deep cervical lymph nodes. Light microscopically, ink particles were found in the subarachnoid space, partially distributed around the olfactory nerves and within the lymphatic vessels. By electron microscopy, the subarachnoid space often formed a pocket-like space in the entrance of the fila olfactoria. The olfactory nerves were partially surrounded by ink particles within the space between perineurial cells and epineurial fibroblasts. At the nasal mucosa, the lymphatics were frequently located close to the nerves. These results indicate that the cerebrospinal fluid drains from the subarachnoid space along the olfactory nerves to the nasal lymphatics, which in turn, empties into the cervical lymph nodes. This anatomical communication, thus, allows the central nervous system to connect with the lymphatic system. The presence of this route may play an important role in the movement of antigens from the subarachnoidal space to the extracranial lymphatic vessels, resulting in inducement of an immune response of the central nervous system.

A scanning electron-microscopic study of the rat thymus with special reference to cell types and migration of lymphocytes into the general circulation.

The three-dimensional structure of the rat thymus was studied by combined scanning- and transmission electron microscopy. The thymus consists mainly of four types of cells: epithelial cells, lymphocytes, macrophages, and interdigitating cells (IDCs). The epithelial cells form a meshwork in the thymus parenchyma. Cortical epithelial cells are stellate in shape, while the medullary cells comprise two types: stellate and large vacuolated elements. A continuous single layer of epithelial cells separates the parenchyma from connective tissue formations of the capsule, septa and vessels. Surrounding the blood vessels, this epithelial sheath is continuous in the cortex, while it is partly interrupted in the medulla, suggesting that the blood-thymus barrier might function more completely in the cortex. Cortical lymphocytes are round and vary in size, whereas medullary lymphocytes are mainly small, although they vary considerably in surface morphology. Two types of large wandering cells, macrophages and IDCs, could be distinguished, as well as intermediate forms. IDCs sometimes embraced or contacted lymphocytes, suggesting their role in the differentiation of the latter cells. Perivascular channels were present around venules and some arterioles in the cortico-medullary region and in the medulla. A few lymphatic vessels were present in extended perivascular spaces. The present study suggests the possible existence of two routes of passage of lymphocytes into the general circulation. One is via the lymphatics, while the other is through the postcapillary venules into the blood circulation. Our SEM images give evidence that lymphocytes use an intracellular route, i.e., the endothelium of venules.