Enzyme activities in perikaryal and synaptic mitochondrial fractions from rat hippocampus during development.

When pharmacological or basic neurochemical systematic characterization of mitochondrial enzymatic systems correlated to energy transduction processes is attempted, studies must be based on subcellular fractions with a high degree of purity from specific brain areas and from individual animals. Distinct populations of mitochondria heterogenous with respect to biochemical enzyme characteristics from rat brain hippocampus are described. Two mitochondrial populations were derived from synaptosomes by lysis and a third consists of free non-synaptic mitochondria. The maximum rate of some cerebral enzyme activities which are part of energy transduction (citrate synthase, malate dehydrogenase; total NADH-cytochrome c reductase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase) were tested on these mitochondrial populations of 8- and 16-week-old rats. A comprehensive analysis of the data suggests that extensive but highly diversified catalytic expressions of the enzymes studied occur in the hippocampus. This is true even when a short period of the rat life span is studied. Hence the varying pattern of evolution of the differing cerebral mitochondria, probably a consequence of different metabolic functions, should be taken into account in any pharmacological study on these systems.

Morphology of epiphyseal apparatus of a ranid frog (Rana Esculenta)

Morphological, histochemical and ultrastructural investigations on epiphyseal apparatus of Rana Esculenta were made. The most important findings were the following: 1) metaphyseal cartilage is localized inside proximal diaphyseal compact bone as a plug; 2) metaphyseal cartilage do not reduce in thickness during ageing; 3) metaphyseal cartilage do not show vascular invasion and do not mineralize in degenerative zone; 4) trabecular bone was not at all evident in this animal; 5) external periosteum is well vascularized and proliferates in correspondence to marginal epiphyseal end of the diaphyseal. From these results the hypothesis that the ranid frog bone growth is not due to metaphyseal metabolism (as in avian and mammals) but to bone periosteal marginal mineralization is reached.

Ultrastructural findings of congenital dyserythropoietic sickle cell beta thal-associated anemia.

The ultrastructural findings of erythroblasts and reticulocytes in one case of congenital dyserythropoiethic anemia (CDA) associated with a haemoglobinopathy, sickle cell beta thalassemia minor (Type V CDA), is described. The observations can be summarized as follows: 1) A lot of large breaks are present in the erythroblast nuclear envelope. 2) Nuclear membrane evaginations are filled with dense loose chromatin. 3) Electron-transparent areas (moth eaten chromatin) are evident in dense chromatin. 4) Electron-dense granular material, related to altered haemoglobin chain storage, is evident in the nucleus and in the cytoplasm. 5) Iron deposits are present in mitochondrial matrix. 6) Myelinic figures are present in reticulocyte cytoplasm. For the first time the ultrastructural findings in this type of associated CDA are described and related to the double origin of clinical symptoms.

Ultrastructural aspects of human nonunion.

A histological study on the tissue of nonunion of tibias of two young patients was performed to evaluate the ability of cells to start the mineralization of the matrix. The observations can be summarized as follows: 1) Tissue vessels often appear occluded by thrombotic material; 2) Fibroblasts and chondrocytes found in the nonunion tissue seemed normal, with a good secretion apparatus; 3) The cell membranes were able to produce matrix vesicles; 4) Matrix vesicles and cell membrane looked positive to ALPase reaction, 5) Hydroxyapatite crystals could be observed in the cell matrix or inside matrix vesicles. It may be concluded that cells populating nonunion tissue are well equipped to induct the mineralization of the matrix, but the absence of a blood supply, enough to bring them a normal calcium amount, is the real reason for the nonunion.

Infantile cortical hyperostosis (Caffey disease): ultrastructural and immunohistochemical characterization of the peritrabecular cells.

The ultrastructure and the immunohistochemical pattern of the cells which are responsible for the bone resorption in the cortical infantile hyperostosis were investigated. The osteoclasts present a great positivity to MB1 antigen and a low positivity to OKM5. Mononuclear cells with primary lysosomes, looking like osteoclast ones are present in high concentration in peritrabecular spaces. These cells show a high positivity to OKM5 antigen and a low positivity to MB1 antigen. The mononuclear granulated cells are positive to tartrate-resistent acid phosphatase. The possible common origin and their co-operation in bone resorption is discussed.

The role of proteoglycans in maintaining collagen fibril morphology.

The aortic wall contains various heterogenous proteoglycan populations which interact in different ways with other components of extracellular matrix. Proteoglycans (PGs) are known to provide structural support to the vessel wall as well as to influence specific physiological functions of the tissues. The aim of the present study was to investigate the effects of Chondroitinase AC (Chase), Streptococcal Hyaluronidase (Hyase) and Heparanase on human aortic wall collagen which had been treated previously with 4M GuHCl, in order to verify the effects of selective glycanolytic treatment on type I collagen fibril ultrastructure. Following 4M GuHCl treatment, collagen fibrils are seen to have a clearly visible period. Subsequent to GuHCl and Streptococcal Hyase treatment all collagen fibrils appear to be completely swollen in thin aperiodic filaments; the typical 64 nm collagen period is completely undetectable. After GuHCl and Chase treatment a small number of collagen fibrils are seen to be swollen in thin fibrils which are mainly localized at some distance from elastic fibres. Following GuHCl and Heparanase/Heparitinase III treatment a considerable number of collagen fibrils appear to be swollen in thin fibrils; the majority of which are situated in the vicinity of elastic fibrils. The swelling of collagen fibrils underlines the fundamental role of proteoglycans in maintaining collagen fibril integrity and periodicity. It is as yet impossible to precisely map interactions between these proteoglycans and collagen fibres. The role of Hyaluronic acid requires further investigation, although the nature of this interaction is undoubtedly a matter of considerable interest.

Fine structure of the fungiform papilla in a ranid frog (Rana esculenta).

The freetop of the fungiform papilla shows a sensorial area about 100 micron in diameter, surrounded by a ring of ciliated cells. Externally to the ciliated cells, i.e., in the lateral wall, numerous large goblet cells can be seen devoid of their mucous content. The sensorial area is composed by three types of cells: mucous, supporting, and neuroepithelial cells. Mucous cells form the most superficial layer, while the cell bodies of the other two are deep, and from them basal and apical processes arise. The above mentioned cells are connected by desmosomes preferentially located between the mucous and the supporting cells, rather than between the supporting and the neuroepithelial cells. The lateral wall of the papilla is made up of a multilayered epithelium that comprises two types of cells: the first type contains electron-dense granules and an abundant rough endoplasmic reticulum, the others are ciliated cells. In the connective axis of the papilla, numerous fenestrated capillaries with endothelial vesiculated cells and nerve fibers are found.

Evidence of rat tail tendon proteoglycans at emission field scanning electron microscopy.

Alcian blue (AB) in critical electrolyte concentration (CEC) was used for scanning electron microscopy (SEM) on rat tail tendon. Segments with diameters of 4 to 9 nm are evident in the perifibrillar area with their lengths ranging from 180 to 400 nm. Frequently the segments adhere closely to collagen gap zones. Segments are not evident either in specimens fixed in glutaraldehyde or in specimens stained with AB in CEC solutions previously treated with glycanolytic enzymes. The segments suggestively correspond to electrodense filaments evident in transmission electron microscopy (TEM) if the rat tail tendon is similarly treated (Scott, 1981). It may be concluded that AB in CEC solutions can be utilized for SEM investigations and represents a good method for the detection of the three dimensional disposition of proteoglycans (PG) and their interaction with collagen.

Collagen fibril ultrastructure alters after glycanolytic digestion.

Fixed fragments of bovine nasal septum cartilage were digested for six hours either with testicular hyaluronidase or streptomyces hyaluronidase or flavobacter chondroitinase ABC, and observed with a transmission electron microscope. Collagen fibril diameters (D) were measured to evaluate the effect of enzymatic digestion on the fibril size. This resulted in an increased frequency (17% to 47%) of "thin" fibrils (80 to 32 nm), followed by a decrease (65% to 31%) of the frequency of "mid" fibrils (32 to 64 nm). The frequency of "thick" fibrils (over 64 nm) showed a moderate increase (18% to 22%). Considering the relationship between fibril diameter, fibril volume and collagen content, the apparently relevant increase in number of the "thin" fibrils corresponds to an alteration of only 4% of the total collagen. On the other hand the increase of the "thick" fibrils implies a conspicuous alteration of 20% of the total collagen. The observed fibril rearrangement after digestion may be explained in terms of the wrap of matrix proteoglycans around each fibril. The enzymatic removal of the proteoglycans could make "mid" collagen fibrils free to regress into "thin" as well as to merge together into "thick" fibrils.

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.

Recognition of cell surface modulation by elliptic Fourier analysis.

A development of elliptic Fourier analysis, consisting in an alignment of harmonics according to their clockwise or counter-clockwise rotation, resolves the discrepancy between calculated harmonic frequencies and observed morphological periodicities. The new technique is supported by consistent data of empiric and fractal contours, comparatively analyzed and visualized with and without harmonic alignment. The method is particularly suitable for the recognition of periodic modulations of the cell surface. A preliminary analysis of two different cell populations (echinocytes and chondrocytes) shows distinct patterns of surface modulation that allow an effective discrimination of the cell type, while providing relevant information about the respective cytological configurations.

The canalicular system and the osteoblast domain in human secondary osteons.

The lacunar-canalicular system in human secondary osteons was examined by two complementary techniques: light microscopy analysis of undecalcified thick sections and the SEM cortex-fractured surface technique. Unlike the earlier definitions of 'osteoblastic domain' presented as the matrix volume produced by osteoblasts in the process of osteon infilling, this study measured the domain by the length of osteoblast dendritic processes. The domain extension was defined along radial vectors advancing from the reversal line towards the central canal. According to their lengths, domains were divided into three classes: peripheral, intermediate and internal. The mean length of peripheral domains was significantly shorter than those of the intermediate and internal domains. This suggests that the infilling process is modulated by an initial preparatory phase characterised by osteoblast adhesion to the wall of the cutting cone, and a limited matrix synthesis, followed by a regular matrix volume apposition organised in concentric layers. In addition to the radial canaliculae arranged along converging vectors in planes perpendicular to the central canal, we distinguished a further class of canaliculae, the equatorial canaliculae originating from the major perimeter of the lacuna and spreading out radially in the plane of the same lacuna (therefore, perpendicularly to the radial canaliculae). The whole lacunar-canalicular network was structured as a closed system around the vascular axis of the central canal with very few canaliculae crossing the reversal line and connecting the neighbouring osteons. These anatomical observations contribute to our knowledge of lacunar-canalicular system development.

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.

Ultrastructural modifications of cartilage matrix treated with guanidinium HCl (4.0 M and 0.4 M).

Significative modifications in cartilage matrix are induced by GuHCl 4 M and 0.4 M treatment. The first treatment (4 M) induces a complete lack of interfibrillar alcianophilic particles and the swelling of collagen fibrils in 8 nm (80 A) thin aperiodic microfilaments. Among microfilaments, come alcianophilic sheets are often present ( unextractable proteoglycans?). The rectilinear pattern of filaments inside the fibrils became helicoidal. The second treatment (0.4 M) does not modify the interfibrillar morphology of alcianophilic rod like particles but may swell collagen fibril. Aldehydic fixation completely prevents such modifications. These observations show that organization of periodic collagen fibrils is probably due to proteoglycans also.

The dependency of collagen fibrillogenesis in vitro on fibroblast culture conditions. Fibroblasts in mono- and multi-layers.

The extracellular matrix produced by monolayer and tridimensional cultures of fibroblasts was investigated using histochemical and ultrastructural methods. In monolayer cultures, collagen and proteoglycans produced by fibroblasts could not be organized into morphologically recognizable structures. Tridimensional fibroblast cultures produced a well organized matrix with periodic, parallel ordered collagen fibrils of 50 nm diameter, criss-crossed by alcianophylic segments 6-10 nm thick in diameter and 100-300 nm in length, parallel to each other, perpendicular to the collagen fibrils and spaced 67 nm from each other. Some alcianophylic segments lay perpendicular to the above described ones, with maximum lengths of 65-70 nm. Alcianophylic segments are the ultrastructural evidence of structural proteoglycans. These observations suggest that the culture conditions influence the collagen and proteoglycans secretion, so that the final organization of the matrix results quite different.

Morphological aspects of rat metaphyseal cartilage pericellular matrix.

In order to verify whether it is possible to observe morphological evidence of a Ca-P amorphous phase (the first step of Ca-P crystalline deposition), the pericellular area of metaphyseal cartilage was investigated. In the pericellular zone of proliferative, maturation, hypertrophic, degeneration and calcification cartilage, many electron-opaque granules, having a very regular diameter of about 12 nm, disposed in closely-packed chains (chain granules) and increasing in number from the proliferation to the calcification zone, are evident. These chain granules, which are closely connected with proteoglycans, disappear after decalcification and are spatially related to ALPase and ATPase activities. They may be the morphological reflection of the Ca-P amorphous phase.

The foreign body reaction in total hip arthroplasties. A correlated light-microscopy, SEM, and TEM study.

An in vivo histological and ultrastructural study of the cellular reaction to particulate material currently used in orthopaedic surgery produced evidence that, on a strictly cellular level, the main damage is done by the smallest particles produced by hip prostheses, i.e. metal particles, irrespective of differences in their chemical composition. Particle size and release rate are the critical factors, although other mechanisms of cellular damage may be active once granulation tissue is formed.

Fanconi's anemia: ultrastructural observations on erythroblasts.

A case of Fanconi's anemia is reported. Particular attention was given to the ultrastructure of the erythroblasts. The main abnormalities are summarized as follows: (1) striking polymorphism of all the erythroblasts, (2) multiple nuclear cisternae showing several breaks, (3) admixture of nucleus and cytoplasm, (4) myelinic figures inside nuclear cisternae and cytoplasm, (5) moth-eaten nucleus, and (6) total absence of normal erythroblasts. These abnormalities were still visible after therapy in spite of clinical and hematologic improvement.

Electron microscopic visualization of proteoglycans with Alcian Blue.

The intercellular matrices of bovine nasal cartilage, chick embryo perichordal cartilage, and chick embryo mesenchymal cells cultured in vitro have been examined by electron microscopy after staining them with Alcian Blue in salt solutions according to Scott & Dorling (1965). Matrix granules, which are typical components of cartilage at the ultrastructural level, are not visible after Alcian Blue staining and are replaced by alcianophilic rod-like particles, varying in length and width. With tissue cultures, Alcian Blue stains 40-120 A thick filaments which display an orthogonal and longitudinal relationship to collagen fibrils. We assume that cartilage matrix granules represent linear proteoglycans that are coiled as a consequence of the usual glutaraldehyde-osmium fixation. It is thought that Alcian Blue, on the other hand, contributes to the stabilization of the proteoglycans in their original structural arrangement. This stabilizing property presumably also results in the sharp visualization of fine filaments in the tissue culture matrix.