Archives of histology and cytology最新文献

We have recently demonstrated that indentation-type atomic force microscopy (IT-AFM) is capable of detecting early onset osteoarthritis (OA) (Stolz, 2009). This study was based on biopsies, using a desk-top commercial atomic force microscope (AFM). However, cartilage analysis in the knee joints needs to be non-destructive to avoid new seeding points for OA by the taking of biopsies. This requires bringing the probe tip in contact with the articular cartilage (AC) surface inside the joint. Here we present our recent progress towards a medical instrument for performing such IT-AFM measurements for in-vivo knee diagnostics. The scanning force arthroscope (SFA) integrates a miniaturized AFM into a standard arthroscopic sleeve, and is used for direct, quantitative, in situ inspection of AC (Imer et al., 2006). The stabilization and the positioning of the instrument relative to the surface under investigation were performed by means of eight inflatable balloons. An integrated three-dimensional, piezoelectric scanner allowed raster scanning and probing of a small area of cartilage around the point of insertion. An AFM probe with an integrated deflection sensor was mounted at the distal end of the instrument. Using this instrument, several measurements were performed on agarose gel and on porcine cartilage samples. The load-displacement curves obtained were analyzed and the dynamic elastic moduli | E(*) | were calculated. A good correlation between these values and those published in the scientific literature was found. Therefore, we concluded that the SFA can provide quantitative measurements to detect early pathological changes in OA.

To develop an experimental model and evaluate the effects of the magnitude and duration of pressure, the rat abdominal wall (25x20 mm) was subjected to compression either by a weight or by two magnets. In the weight compression tests, a steel plate was inserted under anesthesia into the rat peritoneal cavity, and the abdominal wall was compressed in situ between the underlying steel plate and a weight placed on the abdominal wall. This method resulted in moderate changes in the subcutaneous connective tissue and muscle at 100 mmHg (13.3 kPa) for 4 h, while some muscle damage was observed at 50 mmHg (6.7 kPa) for 4 h and at 100 mmHg for 2 or 3 h. In the magnet pinching tests, a magnet was inserted into the peritoneal cavity, and another magnet overlaid on the skin. Then the abdominal wall was compressed by the two magnets with or without anesthesia. The compression without anesthesia produced significant edema and injuries of the abdominal wall at 50 mmHg for 4 h and at 100 mmHg for 3 or 4 h, while the injuries incurred at 100 mmHg for 2 h were mild. Susceptibility to pressure was high in the muscle, moderate in the subcutaneous connective tissue, and low in the skin. The compression with anesthesia produced significantly milder injuries than those under anesthesia. These findings indicate that the difference in the extent of injuries between the weight compression and magnet compression models are clearly attributable to the pentobarbital anesthesia induced during the compression. Results therefore show that experimental pressure ulcers should be examined in a waking condition and that magnet compression is a useful model for studying the pathogenesis of pressure ulcers.

Iron in the brain is utilized for cellular respiration, neurotransmitter synthesis/degradation, and myelin formation. Iron, especially its ferrous form, also has the potential for catalyzing the Fenton reaction to generate highly cytotoxic hydroxyl radicals. The amount of iron in the brain must therefore be strictly controlled. In this study, we focused on the cellular and subcellular localizations of nonheme ferric (Fe(III)) and ferrous (Fe(II)) iron in the adult female rat brain using light and electron microscopic histochemistry. Although Fe(II) deposition was much less dominant than Fe(III), the brain contained iron in both forms. Among the cellular elements of the brain, oligodendrocytes were numerically the most prominent and heavily iron-storing cells. Pericapillary astrocytes and sporadic microglial cells also showed dense iron accumulation. Large neurons involved in the motor system were relatively strongly iron-positive. Subcellularly, Fe(III) and Fe(II) were mainly localized in lysosomes, and occasionally in the cytosol and mitochondria. Furthermore, capillary endothelial cells had Fe(III)-positive reactions in lysosomes and the cytosol, with Fe(II)-positive reactions on the luminal membrane. With advancing age, both Fe(III) and Fe(II) became more extensively distributed and accumulated more numerously in oligodendrocytes and astrocytes. These findings suggest that age-related increases in Fe(II) accumulation may raise the risk of tissue damage in the normal brain.

The tracheal epithelium can be induced to move as a cellular sheet by heterotopic transplantation, which offers the opportunity to observe migrating cells as a group in an in vivo environment. We therefor investigated the ultrastructural characteristics of migrating tracheal epithelial cells with special reference to the moving front using this transplantation. The migrating epithelial cells underwent squamous metaplasia and lost their differentiated characteristics such as cilia or secretory granules. Several unique observations were made concerning the mechanism of mobility: one is that epithelial cells in the front were elongated in a direction perpendicular to the course of movement, different from previous reports in vitro. The second is that lamellipodia, which are regarded as the major locomotive machinery in the adult wound epithelium, did not make up the major part of the front; the major portion of the anterior fringe of the moving front was usually smooth and gently curved, and actin cables parallel to the elongated cells were observed by confocal laser microscopy, indicating that the purse-string mechanism of epithelial wound healing takes place. The third finding is that the cells in the front had irregular bleb-like structures on their antero-basal surface, which were formed even in the portion where the cells did not attach to the matrix. Few organelles were recognized in these structures. From their location, one might propose that these bleb-like structures play a role in the recognition of the substrate and thus the movement of the cell sheet.

Dipyridamole, an inhibitor of adenosine uptake as well as a cGMP phosphodiesterase inhibitor, is commonly used in prophylactic therapy for patients with angina pectoris. However, the effects of dipyridamole on systemic blood vessels, especially on the peripheral vascular system, are not well understood. Therefore, the effect of dipyridamole on ATP-induced arteriole contraction was examined with special reference to intracellular Ca(2+) concentration ([Ca(2+)](i)) using real-time confocal microscopy. In cases of 0.1-10microM range, dipyridamole induced only slight [Ca(2+)](i) decreases in smooth muscle cells of both testicular and cerebral arterioles. However, 100microM dipyridamole induced substantial [Ca(2+)](i) decreases in the cells. In the presence of 10microM dipyridamole, changes in ATP-induced [Ca(2+)](i) were found to be inhibited in smooth muscle cells of testicular arterioles but not in those of cerebral arterioles. In addition, alpha, beta-methylene ATP-induced [Ca(2+)](i) increases in testicular arteriole smooth muscle cells were also partially inhibited in the presence of dipyridamole. When testicular arterioles were perfused with dipyridamole, no increases in nitric oxide levels were detected. High levels of K(+) induced a [Ca(2+)](i) increase in testicular arterioles that was also partially inhibited by dipyridamole. In the presence of substances that affect protein kinase A or G, ATP-induced [Ca(2+)](i) was not completely inhibited. These findings suggest that dipyridamole may act not only as an inhibitor of adenosine uptake and as a cGMP phosphodiesterase inhibitor, but also as a calcium channel blocker in arteriole smooth muscle cells.

5-hydroxytriptamine (5-HT) is an important transmitter for vessel constriction. The present study was performed to clarify the effect of 5-HT on smooth muscles in large- and small-sized cerebral and testicular arterioles by confocal microscopy, with special reference to intracellular Ca2+ concentration ([Ca2+]i) dynamics. In cerebral vessels, 5-HT induced a [Ca2+]i increase and the contraction of smooth muscle cells in large- and midsized arterioles (external diameters>50 microm) but not in small-sized arterioles. Conspicuous [Ca2+]i changes by 5-HT were especially observed in the portions close to the cerebral arterial circle, and the 5-HT-induced responses were caused by both Ca2+ influx and mobilization. Experiments using agonists and antagonists also revealed that cerebral arteriole smooth muscles possess 5-HT1a, 1b, 2 (G-protein-coupled type), and 3 (ion channel type) receptors; specifically, 5-HT2 plays a major role in these responses. On the other hand, in testicular vessels, there were few regional differences among responses to 5-HT, and both large- and small-sized arterioles responded to 5-HT. The responses were caused by only Ca2+ mobilization mediated 5-HT1a and 2. These results indicate that arterioles in different tissues may respond to 5-HT in different manners. Regional differences and the size-dependent manner of responses to 5-HT in cerebral blood vessels also indicate that the regulatory mechanism of blood circulation is highly differentiated in each region of the central nervous system.

Continuously growing rodent incisors have a special epithelial structure for maintaining adult stem cells that shows a bulbous epithelial protrusion at the apical end and is referred to as an "apical bud". Guinea pig cheek teeth (premolars and molars), also continuously growing teeth, have a complex crown shape consisting of plural cusps. The present study clarifies the existence of apical buds in guinea pig premolars/molars as it examines the relationship between the crown shape and the orientation of the apical buds by micro-computed tomography (micro-CT) and immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU). One premolar and three molar teeth in each side of the maxillae and mandibles assumed characteristic features: each horizontally-sectioned tooth showing a complex zigzag shape was composed of a core of dentin covered by a layer of enamel on all axial surfaces except the buccal of the uppers and the lingual of the lowers. Furthermore, four bulbous epithelial protrusions--including the stellate reticulum--were recognized in the apical end of each tooth, where slow-cycling long-term label-retaining cells resided 20 days after a peritoneal injection of BrdU. These data indicate that guinea pig premolars/molars have four apical buds where the epithelial adult stem cells reside. In contrast, rodent incisors, which show a single cone appearance, are covered by enamel on the labial side and possess only one apical bud. The results of this study suggest that plural apical buds, being arranged bucco-lingually and mesio-distally, produce the crown mold in a zigzag fashion.

Regulation of the intracellular calcium ion concentration ([Ca(2+)](i)) is critical, because calcium signaling controls diverse and vital cellular processes such as secretion, proliferation, division, gene transcription, and apoptosis. Store-operated calcium entry (SOCE) is the main mechanism through which non-excitable cells replenish and thus maintain this delicate balance. There is limited evidence which indicates that SOCE may be inhibited during mitosis, and the mechanisms leading to the presumed inhibition has not been elucidated. In the present study, we examined and compared the [Ca(2+)](i) dynamics of COS-7 cells in mitotic and non-mitotic phases with special reference paid to SOCE. Laser scanning confocal microscopy to monitor [Ca(2+)](i) dynamics revealed that SOCE was progressively inhibited in mitosis and became virtually absent during the metaphase. We used various cytoskeletal modifying drugs and immunofluorescence to assess the contribution of microtubule and actin filaments in SOCE signaling. Nocodazole treatment caused microtubule reorganization and retraction from the cell periphery that mimicked the natural mitotic microtubule remodeling that was also accompanied by SOCE inhibition. Short exposure to paclitaxel, a microtubule-stabilizing drug, bolstered SOCE, whereas long exposure resulted in microtubule disruption and SOCE inhibition. Actin-modifying drugs did not affect SOCE. These findings indicate that mitotic microtubule remodeling plays a significant role in the inhibition of SOCE during mitosis.

The morphofunctional organization of the exocrine cells in the foregut of the red-eared slider turtle, Trachemys scripta, was investigated by histochemistry (PAS, AB pH1.0 and pH 2.5, HID-AB, Bowie), lectin-histochemistry (WGA, SBA, UEA, ConA, PNA, DBA, sialidase-SBA, sialidase-PNA, Paradoxical ConA), and immunohistochemistry (antipepsin, anti-alpha-H+,K+ ATPase) to detect regional differences and verify the existence of an oro-aboral gradient in gastric juice secretion. Observations showed that pharyngeal goblet cells have mucins with terminal residuals of GalNAc and sialic acid. In the oesophagus, sulphomucins in the goblet cells are progressively substituted by sialomucins and no glands are found. Gastric surface cells secrete mostly sialomucins with residuals of GlcNAc and GalNAc. The cardias presents glands with cells secreting mostly sialomucins, differing from the surface ones in having GalNAcalpha1,3GalNAc sequences. The fundus presents complex glands with main and lesser tubules. Cells in the main tubules secrete stable, class-III mucins with sialylated residuals of glucose and/or mannose, GalNAcalpha1, 3GalNAc, and Galbeta1,3GalNAc sequences. In the lesser tubules, the oxynticopeptic cells are found, presenting pepsinogen granules and an affinity to the anti-alpha-H+,K+ ATPase, without any oro-aboral variation. The pyloric glands have a secretion similar to that of the neck cells of the fundic glands, consisting of stable, class-III mucins mostly sialylated with a high heterogeneity of residuals such as glucose and/or mannose, fucose, GlcNAc, and GalNAc. Mucins in the foregut are probably involved in several functions such as lubrication, protection against gastric juice, osmotic regulation to increase intestinal absorption, and protection against microbial injuries.

The expression of stress-responsive proteins, such as nestin and a 27-kDa heat-shock protein (HSP27), was immunohistochemically examined in order to demonstrate glial responses in the rat olfactory bulb following sensory deprivation. At 3 days to 1 week after sensory deprivation, numerous nestin-expressing cells appeared within the glomerulus of the olfactory bulb. These cells were regarded as reactive astrocytes since they were immunoreactive for glial fibrillary acidic protein and showed hypertrophic features. The glomeruli, in which nestin-immunoreactive astrocytes were localized, were filled with degenerating terminals of olfactory receptor neurons and migrated microglia. A small population of nestin-immunoreactive cells was positive for a proliferating cell marker, Ki67 (8.0-9.7% at 3 days; 3.1 - 5.0% at 1 week). At 3 weeks, nestin-immunoreactive astrocytes were occasionally detected. At 6 weeks, when the olfactory receptor neurons had completely recovered, no nestin-immunoreactive astrocytes were detected. HSP 27 was also expressed within the glomerular astrocytes and showed a similar spatiotemporal expression pattern to nestin. The present study suggests that reactive astrocytes may be involved in axonal regeneration and synaptic remodeling in the olfactory system, through the recapitulation of developmentally regulated proteins, such as nestin and HSP27.