The cutaneous microvascular architecture of human diabetic toe studied by corrosion casting and scanning electron microscopy analysis.

In this morphological study, we report on the three-dimensional microvascular architecture constituting the toes of a patient affected by diabetic microangiopathy. We applied corrosion casting (CC) technique to the toes of a patient affected by Type 2 diabetes, who underwent surgery for explantation of inferior left limb due to necrotic processes of soft tissues. The toes of a foot traumatically explanted in a motorcycle accident were kept as controls. According to technical protocols, toes were injected with a low-viscosity acrylic resin (Mercox) through the major digital artery, tissues were corroded in KOH solution (8%), and resulting casts processed for SEM observations. Already at low magnification, in diabetic toes, we found an impairment of the linear track-like disposition of the vessels of plantar side, with signs of vascular disruption and obliterations, stopped resin, and leakages. Capillaries under the nail and a lot of vascular villi in eponychium and nail borders were damaged, and vascular regression phenomena acting on them were clearly visible. Resin leakages and impairment of normal vascular architecture were also observed in the root of the nail. This preliminary report represents only the first step for further investigations regarding morphological three-dimensional appearance of diabetic microangiopathy. CC and scanning electron microscopy technique well documented these morphological modifications, highlighting on both structural and ultrastructural features of diabetic toes microvessels. In conclusion, our qualitative data try to better focus on the pathophysiological mechanisms involved in diabetic dermopathy and microangiopathy, proposing CC as useful method to investigate on them.

In situ hybridization by scanning electron microscopy for painting, centromeric, and YAC localization.

The hybridization site of a DNA probe was detected using a scanning electron microscope (SEM), modifying the standard in situ hybridization (ISH) method. The experiments were performed on human metaphases obtained from lymphocyte cultures of human peripheral blood. The libraries and probes used were: 1-chromosome library for the painting of chromosome 1 (wcp 1), an alphoid centromere-specific probe of chromosome 8 (pZ8.4), and the yeast artificial chromosome (YAC) 964-C10 mapped at band p13 on chromosome 12. These probes were labeled by nick translation with biotin and displayed with a gold-conjugated anti biotin goat antibody. The gold signal was amplified by silver enhancement. The chromatides appeared as packages of thin filaments 120 nm high; some of them collapsed, probably due to ISH procedures. All the probes were clearly detected as small gold particles grouped on the surface of the target chromosomes and chromosome sites. Thus, this procedure is useful to clarify the positional relationship between the chromatin filaments and the probe.

Plexiform vascular structures in the human digital dermal layer: a SEM--corrosion casting morphological study.

This study aimed to describe the impressive diversity of vascular plexiform structures of the hypodermal layer of human skin. We chose the human body site with the highest concentration of dermal corpuscles, the human digit, and processed it with the corrosion casting technique and scanning electron microscopy analysis (SEM). This approach proved to be the best tool to study these microvascular architectures, free from any interference by surrounding tissues. We took high-definition pictures of the vascular network of sweat glands, thermoreceptorial and tactile corpuscles, the vessels constituting the glomic bodies and those feeding the hair follicles. We observed that the three-dimensional disposition of these vessels strictly depends on the shape of the corpuscles supplied. We could see the tubular vascularization of the excretory duct of sweat glands and the ovoid one feeding their bodies, sometimes made up of two lobes. In some cases, knowledge of these morphological data regarding the normal disposition in space and intrinsic vascularization structure of the dermal corpuscles can help to explain many of the physiopathological changes occurring during chronic microangiopathic diseases.

The 3D structure of the human urinary bladder mucosa: a scanning electron microscopy study.

We performed a scanning electron microscopy study on the human urinary bladder tunica mucosa. Specimens from bladder biopsies were treated with OsO4 maceration and 1N NaOH maceration methods prior to SEM observation to disclose the three-dimensional organization of the lamina propria, basal lamina and urothelium. The lamina propria housed a well developed capillary plexus just below the basal lamina; the urothelium presented a typical three-layered organization with basal, intermediate and superficial cells. The intermediate cells appeared essentially similar to basal cells in their external features and stretched from the basal lamina up to the superficial layer. The most superficial cells appeared consistently flattened and interconnected by extensive junctional complexes. They showed a peculiar specialization, their apical plasmalemma being thickened with distinctive, stiff plaques, in contrast with the underlying globular or spindle-shaped cells whose plasmalemma was only covered by short microvillosities.

Microvascularization of the human digit as studied by corrosion casting.

The aim of this study was to describe microcirculation in the human digit, focusing on the vascular patterns of its cutaneous and subcutaneous areas. We injected a functional supranumerary human thumb (Wassel type IV) with a low-viscosity acrylic resin through its digital artery. The tissues around the vessels were then digested in hot alkali and the resulting casts treated for scanning electron microscopy. We concentrated on six different areas: the palmar and dorsal side of the skin, the eponychium, the perionychium, the nail bed and the nail root. On the palmar side, many vascular villi were evident: these capillaries followed the arrangement of the fingerprint lines, whereas on the dorsal side they were scattered irregularly inside the dermal papillae. In the hypodermal layer of the palmar area, vascular supports of sweat glands and many arteriovenous anastomoses were visible, along with glomerular-shaped vessels involved in thermic regulation and tactile function. In the eponychium and perionychium, the vascular villi followed the direction of nail growth. In the face of the eponychium in contact with the nail, a wide-mesh net of capillaries was evident. In the nail bed, the vessels were arranged in many longitudinal trabeculae parallel to the major axis of the digit. In the root of the nail, we found many columnar vessels characterized by multiple angiogenic buttons on their surface.

Neurovenous superficial inferior epigastric flaps in rats.

BACKGROUND: We performed an experimental study on 46 male rats to evaluate the vitality of superficial inferior epigastric neurovenous flaps performed following different procedures. METHODS: Rats were divided into 5 groups: A (n=6), B (n=10), C (n=10), D (n=10) and E (n=10). On each rat a hemi-abdominal flap based on the superficial inferior epigastric pedicle was raised. In group A the flap was sutured into its original position, isolating it from underlying tissues by a latex glove patch. In group B an arteriectomy of the superficial inferior epigastric artery was performed. In group C the superficial inferior epigastric artery was sectioned at its origin, without dissecting it out of the pedicle. In group D and E the same procedures performed respectively in group B and C were followed, but the epigastric pedicles were immediately explanted for optic microscope observation. Flap vitality in groups A, B and C was checked after 7 days. RESULTS: Flaps in group A appeared completely vital: the glove did not interfere with flap survival. All flaps in group B looked necrotic. In group C, 9 flaps survived completely and 1 flap underwent complete necrosis. Pedicle specimens of the viable flaps (group C) were explanted for observation under the optic microscope. Each specimen from groups C, D, E revealed a rich vascular plexus in the connective tissue around the pedicle. Seven days after surgery, neo-microangiogenesis was evident. CONCLUSIONS: The results witness the importance of perivenous and perineural arterioles for neurovenous flaps survival, also considering the adaptive increase in capillary development in the days following surgery.

The extracellular matrix of the cuticle of Gordius panigettensis (Gordioiidae, Nematomorpha): observations by TEM, SEM and AFM.

The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cuticle is composed of 30-50 compact layers. The number of the layers is higher in the central part of the animal's body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately 640 nm in diameter and of indefinite length. The fibres run strictly parallel within each layer, while in adjoining layers they run at a variable angle from 45 degrees in the central body to 90 degrees in the extremities. Each fibre shows a barely detectable filamentous inner structure and is enveloped in a thin highly regular net formed by hexagonal meshes. Our results suggested that these fibres should be proteinaceous although non-collagenous. Thinner radial fibres run among the large fibres and across all the layers and span the whole thickness of the cuticle from the epithelial layer located deep underneath the large fibres up to the epicuticle on the external surface of the animal.

A new method to make vascular and bronchial casts of voluminous organs.

Vascular and bronchial endocasts represent a useful instrument to study the ramification pattern of these structures. Casts have been made from different materials, such as waxes in ancient times and, more recently, silicon-like compounds or resins (see e.g. Mercox) to study the finest details. These techniques are valuable for small specimens, whereas they are inadequate for very large organs, where technical difficulties require the development of specific instrumentation. In this study we present a new simple injection technique, based on expanded polyurethane, which allows preparing vascular and bronchial trees for macroscopic and microscopic studies. The new injection technique is very easy to carry out, since the propulsion is provided by compressed air, and it does not require special instrumentation. To this aim, endocasts of the entire tracheal-bronchial tree and casts of vascular kidney from different animals were prepared. The specimens have a very low weight, show the finest ramifications, and are very stable and resistant to mechanical stress. To examine microscopically the details of the casts, specimens from the kidney cast were also analyzed by scanning electron microscopy, revealing good preservation of microcirculatory structures, functional sphincters and endothelial cell impressions. Therefore, the technique may be useful for macroscopic studies of large specimens, retaining sufficiently fine details.

Ultrastructure and functional versatility of hirudinean botryoidal tissue.

In leeches, the botryoidal tissue is composed of two different cell types--granular botryoidal cells and flattened endothelial-like cells--localized in the loose connective tissue between the gut and the body wall sac. We have observed that the botryoidal tissue undergoes functional and structural modifications in response to the different needs arising during the life-cycle of the animal. In healthy, untreated leeches, botryoidal cells are organized in cords or clusters, sometimes surrounding few, small lacunae. Conversely, in wounded animals we have observed the transition of the botryoidal tissue from cluster/cord-like structures to a hollow/tubular architecture, typical of pre-vascular structures. We have documented in botryoidal cell cytoplasm the presence of large calcium storage. Moreover, the cytoplasm of botryoidal cells was filled with granules of different form and size, containing iron or melanin, as tested by classic histochemical methods. The presence of elements like iron and calcium was confirmed by the well-established EDS analysis. In response to a surgical wound, botryoidal tissue cells changed their shape and formed new capillary vessels. Concurrently, botryoidal cells secreted iron from cytoplasmic granules into the new cavity: this secretory activity appeared to be related to intracellular calcium fluctuations. At the end of the angiogenic process, botryoidal cells lost their contact with the basal lamina and moved freely in the circulating fluid towards the lesioned area. Interestingly, circulating botryoidal cells were found to carry melanin in the wounded area. This function is probably involved in defense processes. Thus, we have shown that stimulated botryoidal tissue displays a variety of striking structural, secretory and defensive activities.

Quantitative changes in pharmacodynamic parameters of noradrenaline in different rat aorta preparations: influence of endogenous EDRF.

1. The aim of the present study was to assess the role of endothelial cells in the modulation of vasocontractile responses to noradrenaline in rat isolated aorta when cut as standard helical strips or as ring segments. 2. Noradrenaline-potency in helical strip preparations evaluated as -logEC50 was greater than that obtained in endothelium-intact ring preparations (9.45 +/- 0.28 versus 8.69 +/- 0.09, respectively) (P < 0.05). The maximum contractile response of helical strips was significantly higher than the response of ring preparations (P < 0.05). 3. Subsequent experiments were performed on helical strips and ring preparations where the endothelium was removed by rubbing the luminal surface of the aorta with filter paper. Removal of the endothelium potentiated the noradrenaline-induced contraction in ring preparations, but not in the helical strips. 4. The nitric oxide synthase inhibitors L-NAME (3 x 10(-5)-3 x 10(-4) M) or L-NNA (1 x 10(4)-3 x 10(-4) M) which were added to the tissue bath potentiated the noradrenaline-induced contraction in the endothelium-intact ring preparations, although only L-NNA induced a statistically significant potentiation. Both L-NAME and L-NNA had no effect on the noradrenaline-contraction induced in rings without endothelium, or in helical strips with or without endothelium. 5. Vascular acetylcholine-induced relaxation is dependent on endothelium derived relaxing factor (nitric oxide). Acetylcholine (10(-9)-10(-6) M) induced a concentration-dependent relaxation in noradrenaline preconstricted intact rings. The relaxant response was strongly reduced by L-NAME (3 x 10(-5)-1 x 10(-4) M). The relaxant response to acetylcholine was very weak in ring and helical strip preparations without endothelium, but also, surprisingly, in unrubbed standard helical strips. 6. The present results suggest that the endothelium of standard helical strip preparations may be greatly damaged, a view confirmed by morphological studies. The structural and functional damage of the endothelium induced very important changes in pharmacodynamic parameters such as in the potency and the maximal responses of vascular preparations to noradrenaline. Therefore, caution must be observed when the potency and intrinsic activity of agonists evaluated on different preparations are compared, even if these come from the same vascular segment.

Synovia hyperplasia and calcification in the human TMJ disk: a clinical, surgical, and histologic study.

The aim of the study was to investigate the morphologic modifications occurring in the synovial tissue after severe derangement of the articular structures with dislocation or perforation of the disks. Light microscopic, ultrastructural, and immunocytochemical investigations were performed on 10 disks. Arthroscopic examinations had documented adhesions between the diskal surfaces and the glenoid eminence or the condylar head in all the selected cases. Histologic examination showed a remarkable hyperplasia of the synovial tissue with the formation of prominent protrusions. An evident increase of type B (fibroblast-like) cells and the presence of cells with the ultrastructural and immunocytochemical characteristics of myofibroblasts were observed. Foci of mineral precipitates and large deposits of calcified tissue were present in the synovial villi in three samples. Our observations suggest that functional failure and morphologic lesions of the synovia may be caused by arthropathy.

Endothelin and nitric oxide synthase in lymphatic endothelial cells: immunolocalization in vivo and in vitro.

BACKGROUND: Endothelin (ET) is an endothelium-derived multifunctional peptide that produces a potent, long-lasting vasoconstriction. Nitric oxide (NO), besides being the most important endothelium-derived relaxant factor in blood vessels, is supposed to be involved in regulating the interactions among endothelium, adhesive molecules, and leukocytes. METHODS: We investigated the possible occurrence and distribution of ET and constitutive nitric oxide synthase (NOs), the enzyme that generates NO from L-arginine, in bovine lymphatic vessels and primary culture of lymphatic endothelium by using immunocytochemistry. RESULTS: Specific immunostaining with both ET and endothelial constitutive NOs antisera was detectable at light and electron microscopic levels in the endothelial cell layer of lymphatic vessels, whereas no immunostaining could be observed in the muscular and adventitial layers. The immunoreaction showed a diffuse pattern throughout the cytoplasm of endothelial cells. Primary cultures of endothelial cells isolated from lymphatic vessels also displayed cytoplasmic ET- and NOs-like immunoreactivities. The endothelial nature of cell monolayers was confirmed by the positive reaction to the von Willebrand factor, a reliable marker of endothelial cells, and by ultrastructural features of cultured cells. CONCLUSIONS: These findings suggest that the endothelium is a major source of ET and NO in lymphatic vessels. Interestingly, the lymphatic endothelium maintains the capability of producing such vasoactive sustances also in vitro, thus suggesting that lymphatic endothelial cells in culture may be used in studies concerning the role of the endothelium in the generation of vasoactive molecules. According to previous functional studies, the occurrence of ET and NOs immunoreactivities in lymphatic vessel endothelium supports the view that lymphatic endothelium may play an important role in the regulation of lymphatic vascular tone and in the production of vascular contractile activity promoting lymph flow.

The articular disc surface in different functional conditions of the human temporo-mandibular joint.

The peripheral discal tissue and the surface covering layer have been studied in normal and in variously damaged human temporo-mandibular joint discs. In the normal disc the tissue consisted of dense bundles of fibers and rare fibrocytes. The surface of the disc was covered by a regular basophilic and electron-dense layer. These morphological characteristics persisted also in some pathological discs in which fibrous derangements had already occurred in the deep parts. In very deformed and damaged discs associated with serious functional anomalies, the superficial discal tissue consisted of rare fibers dispersed in a loose ground substance and of an increased number of cells. The superficial coating was formed by an irregular dense lamina and aggregates of various materials containing cellular debris, vesicles, filaments and amorphous components. These deposits are probably due to degeneration processes of discal tissue. This investigation suggests that the superficial discal tissue and the covering layer are together involved in maintaining the functional properties of the articular surfaces. Their structural modification in severe functional anomalies leads to failure in the maintenance of nonadherence conditions and to deterioration of the functional defect.

Immunohistochemical localization of lipoperoxidation products in normal human placenta.

4-Hydroxynonenal (4-HNE) is a major propagation product of lipid peroxidation that is supposed to be responsible for some of the effects associated with oxidative stress in tissues. We have investigated the possible occurrence and distribution of 4-HNE-immunoreactivity in human normal placenta using immunocytochemistry. Specific immunostaining was observed in cytotrophoblast cells, syncytiotrophoblast, some cells of the villous mesenchyme and some endothelial cells of first trimester and term placentae. The detection of 4-HNE-immunoreactivity in placenta raises the question whether lipoperoxidation products are produced locally in placental cells or represent exogenous products that derive from maternal blood flow. Since trophoblastic cells and villous macrophages are provided by a scavenger receptor, it is conceivable that these cells may play a protective role with regard to the diffusion of lipoperoxidation products from the mother to the embryo. However, since a significant degree of lipid oxidative modification does not take place in plasma, it is presumed that 4-HNE is a local product of placental metabolism. In line with this hypothesis, it is proposed that maternal low density lipoproteins, which are the major source of cholesterol for placental steroid synthesis, might be oxidized by villous cells during their traversal through the villous wall.

Presence of calcified tissue in the human temporomandibular joint disc.

Large calcified areas were observed in the articular discs of the temporomandibular joint from five patients suffering from articular dysfunctions. The calcified regions were always located inside the fibrous tissue of the discs. They had a woven bone-like morphological pattern and consisted of a compact mineralized tissue containing cells in irregular lacunae. In all the samples the calcified tissue was completely surrounded by a mineralizing border rich in cells and variously arranged collagen fibrils. Energy-dispersive spectrometry showed that mineralized regions contained large amounts of Ca and P. X-ray powder diffraction identified the crystals in these areas as hydroxyapatite.

Structure of the lymphatic microcirculation in the human urinary bladder with different intraluminal pressure and distension.

The localization, morphology and fine structure of initial lymphatic vessels in the mucosa of the empty and distended urinary bladder were studied. Endoscopic transurethral biopsies of the empty (collapsed) bladder showed under light and electron microscopy numerous intramural lymphatics with a dilated lumen and thin profile. Contacts between endothelial cells were single, overlapping, interlocking, and open while the perivascular connective tissue was filled by fascicles of collagen fibers. In the most superficial layer (subepithelial mucosa), lymphatics were not seen. Biopsies obtained under elevated intraluminal pressure and distension showed on light and electron microscopy lymphatic vessels with small lumens characteristically reduced to irregular slits. Endothelial cell contacts were simple or overlapping; open junctions were rare. The perivascular connective tissue was dense and collagen and elastic fibers often abutted one another. These findings support that with a distended or expanded urinary bladder, the effect of increased intraluminal pressure on the superficial (mucosal) layer radially pulls on the connective tissue that in turn compresses the initial lymphatics thereby restricting lymph transport.

Localization of proliferating cell nuclear antigen-immunoreactivity in human dental pulp and gingiva.

The proliferating cell nuclear antigen (PCNA) is regarded as an operational marker of proliferating cells. We have used PC10 monoclonal antibody to PCNA to reveal proliferation sites in human dental pulp and gingiva. Intense PCNA-immunoreactivity was observed in the basal layer of the gingiva lining epithelium and within some cells of the underlying connective tissues, including some endothelial and perivascular cells. PCNA-reactive cells were scattered throughout the pulp tissue, but were particularly numerous in the peripheral part. Since PCNA is an endogenous cell cycle-related molecule, we propose that PCNA-antibodies may represent useful for studying cell kinetics in human oral tissues in normal as well as pathological situations, such as tumours, wound healing and inflammation.

Occurrence and distribution of matrix metalloproteinase-2-immunoreactivity in human embryonic tissues.

Tissue development and structure is controlled by dynamic and interactive relationships between cells and the extra-cellular matrix (ECM) which they secrete. We have investigated the occurrence and distribution of metalloproteinase-2 (MMP-2), an enzyme involved in the catabolism of ECM components, in human embryonic tissues by immunocytochemistry. Cells displaying MMP-2 immunoreactivity showed a widespread distribution in human embryonic tissues and organs. Cytoplasmic staining was detected in cells deriving from all three embryonic layers. Although further studies are needed to clarify the possible substrates of MMP-2 in developing tissues, these morphological data lend support to the hypothesis that ECM remodelling and degradation may represent a physiological counterpart of ECM deposition that occur during development.