Elastogenesis in the wall of the human thoracic aorta

The results of this study dealing with the human thoracic foetal aorta testify that even in the middle of the fifth month of development the internal elastic membrane is not yet completely continuous. Furthermore they show that elastogenesis in the tunica media of the human thoracic aorta does not begin directly below the internal elastic membrane, as it does in the foetal aorta of the laboratory rat, but, as it can be seen in our material, somewhat deeper in the developing tunica media. A thin layer of less differentiated tunica media cells persists for a long time in the vicinity of the internal elastic membrane. In the middle of the fifth month, the fusing elastic membrane segments in the tunica media still consist of very immature elastic tissue with a large proportion of the microfibrillar component. The collagen fibrils in the intercellular spaces in the whole depth of the wall of the developing aorta do not become a part of the elastic membranes. Their bundles merely accompany all the elastic membranes in the wall of the thoracic aorta, including the internal elastic membrane.     

Observations on the fine structure of the baroreceptors and adrenergic innervation of the guinea-pig carotid sinus

We examined the fine structure of the baroreceptors and the adrenergic innervation of the guinea-pig carotid sinus. The tunica adventitia contained many nerve bundles whose perineuria enclosed unmyelinated nerve fibers, alone or together with myelinated nerve fibers. Baroreceptors, which lay close to elastic and collagen fibers in the adventitia and media, were surrounded by “terminal” cells with ultrastructural features characteristic of Schwann cells and contained inclusions of various types. Morphologic features of the baroreceptors included densely packed mitochondria, osmiophilic lamellated and homogeneous bodies, clear and granular vesicles, lamellar systems, glycogen granules, neurofilaments, neurotubuli, and vacuolated mitochondria. In animals that had been treated with 6-hydroxydopamine, occasional electrondense endings (or fibers) were observed in the adventitial layer. The baroreceptors in the guinea-pig carotid sinus appear to have most of the morphologic features reported for other species.     

The elastic cartilage in the normal rat epiglottis

Summary Chondrocytes of the rat epiglottis contain large amounts of glycogen and lipids, which often make the cells resemble fat cells. The content of lipids is interpreted as being related to the function of the cells. The membranes of some of the large vacuoles are stained with ruthenium red. The cells give rise to long cytoplasmic processes. As in hyaline cartilage the intercellular substance consists of a fine network containing proteoglycan granules together with thicker cross striated fibers. Furthermore elastic fibers are found, consisting of amorphous and microfibrillar parts. In the matrix, both lysosome-like granules and more or less empty vesicles are observed. Accumulations of a finely particulate electron dense material and of a translucent amorphous material containing membrane bound granules are found in some lacunae situated in the outer part of the cartilage. These accumulations are possibly related to the development of collagenous and elastic fibers.     

Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey.

In primates the membrane separating the seminiferous epithelium from the interstitial space is composed of one to three (monkey) or two to six layers (man) of myoid cells associated with one to two layers of fibrocyte-like adventitial cells. All these cells are separated from each other by irregular spaces filled with various connective tissue intercellular components. Subjacent to the elements of the seminiferous epithelium is a continuous, often redundant, basement membrane. A similar basement membrane-like material forms a layer next to and over small areas of the plasma membrane of myoid cells. Collagen fibrils grouped in bundles of various sizes are seen in all connective tissue layers but are particularly abundant in the space between the seminiferous epithelium and the innermost layer of myoid cells. Elastic fibrils demonstrated by the Verhoeff iron hematoxylin technique are also present. Composed of a homogeneous material, the elastic fibrils are short, irregular, branching entities with a diameter comparable to or smaller than that of collagen fibrils. In addition, an abundance of microfibrils with a diameter of 12-15 nm is present in the various connective tissue layers. These microfibrils have a densely stained cortex and a lightly stained core. When seen close to the myoid cells, bundles of micro fibrils appear to insert on well defined areas next to the plasma membrane. These areas commonly face the patches of electron-dense material observed on the inner aspect of the plasma membrane of the myoid cells and in which the actin filaments are inserted. Bundles of microfibrils often span the gap between myoid cells of the same layer as well as those of adjacent layers. Microfibrils are also closely related to the surface of elastic fibrils and are seen intertwining with collagen fibrils. Thus microfibrils appear to bridge and bind together adjacent myoid cells and anchor the surface of these cells to the bundles of elastic and collagen fibrils present in the intercellular spaces of the limiting membrane.     

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus

Summary The wall structure of arteriovenous anastomoses in the rabbit ear was investigated. (1) Clusters of epithelioid smooth muscle cells form 3–4 longitudinally oriented plicae. The channel shows a single, irregularly outlined lumen, and its wall is very thin between adjacent plicae. (2) Endothelial cells covering the plicae protrude into the lumen, thus suggesting active contraction or shortening of the plicae. (3) The tunica adventitia is composed of 4–6 sheaths of flat fibroblasts, which may serve as a barrier to prevent loss of neurotransmitters. Processes of some of the fibroblasts also extend into the tunica media. (4) The tunica media is composed of an outer circular layer of typical smooth muscle cells, and an inner longitudinally running plica of ramified smooth muscle cells. Wide intercellular spaces between these ramified cells are filled with collagen fibrils, microfibrils, amorphous intercellular substances, and fibroblasts. Fibroblasts form close membrane contacts with each other, and with the smooth muscle cells. (5) Fibroblasts and other connective tissue components may function as an elastic support during active motility of the anastomotic channel.     

Investigation of relationships between collagens, elastin and proteoglycans in bovine thoracic aorta by immunofluorescence techniques

Types I, III and V collagens and proteoglycan were localized in the aorta by indirect immunofluorescence techniques. Type I collagen was more prominent in media and adventitia than in intima while type III collagen predominated in intima and media but appeared less significant in adventitia. Type V collagen was observed in intima and media only and was seen surrounding smooth muscle cells. Type I collagen was located between elastic fibres but type III collagen appeared to envelop the fibres, suggesting an interaction between elastic fibres and type III collagen. Pretreatment of sections with testicular hyaluronidase caused no changes in staining for type I collagen, but adventitial areas showed increased staining for type III collagen. After digestion with chondroitinase ABC, intimal and medial areas showed increased staining for type III collagen. Therefore, type III collagen forms stronger interactions with proteoglycans and hyaluronic acid than does type I collagen and type III collagen in adventitia is largely masked by hyaluronic acid, while type III collagen in intima and media is associated with proteoglycan. Thus, type III collagen is a more significant component of adventitia than previously recognized. Proteoglycan was also partly localized along elastic fibres. It is, therefore, suggested that elastic fibres are coated with type III collagen, which itself is coated with proteoglycan.     

A histological study on the coronary artery of the indigenous black Bengal goat in Bangladesh.

The coronary artery of the black Bengal goat was studied by light microscopy. The wall of the coronary artery consisted of the tunica intima, tunica media and tunica externa. The tunica intima consisted of a single layer of flattened endothelium. The tunica media was well-developed and composed of mainly of smooth muscle cells together with some fine elastic fibers. The tunica externa consisted of predominant collagen fibers, and some elastic fibers and smooth muscle cells. Elastic fibers in the tunica externa formed a circular arrangement around the tunica media. Sex differences were not observed. The media with well-developed smooth muscle cells may be responsible for changes in functional physiological conditions of the heart.     

The effect of ascorbate on the synthesis of minor (non-interstitial) collagens by cultured bovine aortic smooth muscle cells

Ultrastructural and biochemical studies were carried out on bovine aortic smooth muscle cells cultured in the presence or absence of ascorbate. In its absence, electron microscopic examination of cultures revealed that the extracellular components consisted primarily of microfibrils. Morphologically identifiable collagen fibrils were only observed in the matrix upon ascorbate supplementation. Smooth muscle cells grown in ascorbate-free media synthesized large amounts of type VI collagen. The identity of the latter was confirmed by ion exchange chromatography, slab gel electrophoresis, and amino acid analysis. Addition of ascorbate resulted in a stimulation of type I collagen production, levels of the type III remained constant, and types V and VI were decreased. Since, in the absence of ascorbate, smooth muscle cells are known to synthesize predominantly elastin, the present data support the contention that the type VI collagen and the microfibrillar component of elastic tissue are either identical or similar.     

The autonomous innervation of the buffalo (Bubalus bubalis) testis. An immunohistochemical study

The innervation pattern in the buffalo testis was determined by using histochemical and immunohistochemical methods. Nerves were concentrated in the tunica albuginea and septula testis, and did not show an uniform distribution. The tunica albuginea at the lateral and medial sides and at the free border of the testis is most densely innervated than at the epididymal border. At the cranial pole thick nerve bundles were observed between albugineal vessels and muscle bundles. Rare parenchymal nerves were found in perivascular position between seminiferous tubules and their occurrence is confined to lobules at the cranial and caudal testicular poles. An intense NPY immunoreactivity occurred in nerve bundles and in solitary varicose fibres. Nerves were concentrated in the tunica albuginea at the lateral and medial side and at the free border of the testis, and in the lobules at the cranial and caudal testicular poles. Sub P immunoreactivity was occasionally detected in some thicker nerve bundles and solitary fibers, in the tunica albuginea and in the wall of blood vessels, showing a similar distribution but less intensity and density than NPY immunoreactivity. TH immunoreactivity stained nerve fibers in the buffalo testis with a distribution pattern similar to that obtained with general neuronal markers. The histochemical reaction for AchE was negative, so cholinergic fibers cannot be detected in the buffalo testis. The histochemical NADPHd reaction stained rare nitrergic nerve bundles and solitary fibers. The majority of NADPHd activity was confined to the vascular endothelium, and rarely to the interstitial Leydig cells, whereas the Sertoli and germ cells did not show any reaction.     

A morphological comparison of aortic elastin from five species as seen with the scanning electron microscope

The elastic laminae were extracted from thoracic aortas of adult animals including sheep, dogs, rabbits, cats and rats by treating them in hot alkaline solution (0.1 N NaOH at 75 degrees C) and observed with a scanning electron microscope. The elastic laminae are comprised of sheet-like internal elastic lamina, fibrous and membraneous elastin in tunica media, interlamellar fibers and hollow spaces which we presume were formerly filled with smooth muscle cells in the tunica media. These structures are the same in all five species except that the number of layers and the total thickness of the wall differs.     

Ultrastructural Study of Changes in Cell Morphology and Extracellular Matrix in Prospective Endodermal Cells of Newt Embryos (Cynops pyrrhogaster) before and during Gastrulation

The cell morphology, cell-to-cell contact behavior and extracellular matrix (ECM) of inner cells (prospective endodermal cells) of newt ( Cynops pyrrhogaster ) embryos were examined from the morula to gastrula stage by light and electron microscopy. The inner cells showed increased cell-to-cell contact from the early blastula to early gastrula stage. The cells formed blebs (5–15 μm in diameter) during the blastula stage, and started to form filopodia and lamellipodia before gastrulation. Alcian blue and lanthanum nitrate treatment revealed ECM components on the cell surface in the early blastula stage and these components increased in amount from the late blastula to early gastrula stage. It is suggested that the increase in ECM components on the cell surface may have some relation with changes in cell-to-cell contact and formation of processes on the cell surface. Besides the cell surface ECM components, glycogen-like granules were observed in intercellular spaces. From the distribution of granules in gastrulae, it is suggested that these may be important in maintaining intercellular spaces for migration of invaginating cells.     

Evidence for fluid-phase pinocytosis of extrahepatic bile duct cells isolated from normal rats in culture.

In order to elucidate the physiological function of extrahepatic bile duct cells, we isolated epithelial cells from the rat extrahepatic bile duct by digesting resected segments of the extrahepatic bile duct with 0.15% trypsin in ice-cold Ca(2+)-free Hanks' balanced salt solution supplemented with 0.25 mM EDTA overnight. As a result, the epithelial cells were collected as aggregates and attached to culture dishes coated with type I collagen. Approximately 95% of the cells cultured for 24 hrs were found to be positive for gamma-glutamyl transpeptidase and cytokeratin-19, but negative for vimentin. These characteristics were identical to the features of rat extrahepatic biliary epithelial cells in situ. Ultrastructurally, the cells were long and columnar in configuration on the 2nd day in culture, and possessed numerous microvilli at the apical surface and well-developed junctional complexes at the lateral surface. These findings also indicate that the cells maintain an epithelial nature and are morphologically polarized. When the cells were exposed to a low dose of horseradish peroxidase (HRP) on the 2nd day in culture, which was followed by fixation and treatment with 3-3'-diaminobenzidine, HRP was found preferentially in the cytoplasmic vesicles near the apical surface. HRP was then observed in the intercellular spaces; however, the electron-dense tracer, ruthenium red, did not permeate into the intercellular spaces, and HRP was found in neither cytoplasms nor intercellular spaces when the cells were incubated in HRP-containing medium at 4 degrees C for 30 min. These results suggest that the extrahepatic bile duct epithelial cells are involved in the reabsorption of bile constituents.     

Intercellular accumulation of type V collagen fibrils in accordance with cell aggregation

We reported previously that human fibroblasts form clumps when cultured on a dish coated with reconstituted type V collagen fibrils. Essentially all the type V collagen fibrils, initially coated on the dish, were recovered in the cell clumps that had eventually formed during the culture. We interpreted that type V collagen fibrils adhere to cells more strongly than to the dish and are detached by cell movements. In this study, type V collagen was suspended with fibroblasts to examine the fate of the type V collagen fibrils and to determine whether the fibrils affect the behaviour of the cells directly adherent to the dish. The added type V collagen accumulated in the intercellular space concomitantly with the local aggregation of fibroblasts. scanning electron microscope examination indicated that type V collagen fibrils were found in the vicinity of cells in cultures without ascorbic acid where essentially no collagen secretion takes place. These results indicate that type V collagen forms fibrils and the fibrils are accumulated in the intercellular spaces. The accumulated type V collagen fibrils work as a cementing material for cell clump formation. This phenomenon is discussed in relation to the possible involvement of type V collagen fibrils in tissue organization.     

The infundibular process of rodents of the genus Meriones

Summary The infundibular process of the desert-living rodents Meriones differs from those of other mammals particularly in having extremely large perivascular spaces. These may extend between nerve fibres which are also much more loosely packed than in other species. Typical neurosecretory fibres are present and may project into the perivascular spaces. Both microvesicles and neurosecretory granules may occur in clumps bound by a membrane, forming multivesicular or multigranular bodies.     

The innervation and fine structure of paraneuronic cells in an amphibian pulmonary artery

Summary The pulmonary artery of Bufo marinus contains large numbers of bipolar cells situated in the tunica adventitia and in the outer layers of the media. These cells show a bright green-yellow fluorescence (emission spectra 485 nm) after formaldehyde pre-treatment suggesting that they contain a primary monoamine. The most characteristic fine-structural feature of these cells is the presence of numerous dense-cored vesicles (80—300 nm diameter) in their cytoplasm. The cells are in close contact (20 nm gap) with both agranular and granular nerve fibres. Both EM-cytochemical and formaldehyde-induced fluorescence tests indicate that the granule-containing nerve fibres are adrenergic. The agranular nerve fibres form discrete synaptic contacts with pre-and post-synaptic membrane thickenings on the cells. This was never observed with respect to the adrenergic fibres. Each process of the cells is about 45 m long. The processes do not bear any special relationship to either vessels of the arterial vasa vasorum or medial smooth muscle cells. Their location in the wall of the artery suggests that they are functionally significant with respect to activity of the arterial media.     

Ultrastructural Studies of Carboxyl-Terminal Truncation Mutants of the Neuronal Intermediate Filament Protein Peripherin

Abstract: We have prepared carboxyl-terminal truncation mutants of the neuronal intermediate filament (IF) protein peripherin and examined the assembly characteristics of these mutant proteins in SW13 cells in the presence and absence of vimentin. In the absence of vimentin, tailless peripherin protein (Per-C424) self-assembles into bundles and clumps as observed by immunofluorescence, whereas a peripherin mutant that is missing the tail as well as a small portion of the rod (Per-C356) appears as spherical aggregates. Similar phenotypes are observed when vimentin-positive cells are transfected with Per-C424 or Per-C356. In these cells, the entire IF network is disrupted, and vimentin colocalizes with the mutant peripherin proteins. To examine the morphology of the bundles and clumps formed by Per-C424 at the electron microscopic level, we prepared stable cell lines expressing different levels of this mutant protein. By immunofluorescence, Per-C424 appears as either clumps or bundles of filaments depending on the expression level of the mutant protein. However, under electron microscopy, it is apparent that both clumps and bundles are composed of tightly packed IFs. We were unable to obtain stable cell lines expressing Per-C356, indicating that this mutant may prevent cell proliferation. Using a vector containing an internal ribosomal entry site, we prepared a construct that expresses Per-C356 and green fluorescent protein as a single mRNA, and we were able to isolate cells that expressed Per-C356 by fluorescence-activated cell sorting. Electron microscopic analysis of these cells showed that these aggregates are solid and contain no obvious filamentous structures.     

Ultrastructural observations on rapid formation of neuro-muscular junctions in vitro

Summary The development of neuro-muscular junctions (mouse, rat) from the time of first contact between neurons and myotubes in culture and the changes which lead to the formation of functional synaptic contacts have been investigated using light microscopy and ultrastructural techniques.An extensive basal lamina was present when the neuronal cell population was added to the developing myotubes in culture. The nerve cells were initially strongly attracted to each other and nerve cell aggregates formed rapidly. It was only when nerve fibres began to grow out of these aggregates to contact developing myotubes that changes within the cytoplasm of the two adjacent cells were observed. These developments included accumulations of filaments, membrane densities, mitochondria and large clear vesicles within both cells in the region of contact. In addition, collections of glycogen granules and an extensive membrane reticular complex were found within myotubes, and an extensive granular material filled many of the nerve processes. The basal lamina within the intercellular space appeared more electron-dense than elsewhere and was traversed by strands linking the two cell membranes. These features all appeared to be stages in the initial formation of neuro-muscular junctions. It was only after these events had occurred that presynaptic vesicles gradually appeared within the future nerve terminal. The results of this paper therefore support the view that synaptic transmission at developing mammalian neuromuscular junctions is not necessarily dependent on the presence of presynaptic vesicles.     

Heterotopic allotransplantation of isolated aortic cells

Summary Smooth muscle cells were enzymatically isolated from the tunica media of the aorta of 5-day-old rats. Following in vitro exposure to colloidal thorium dioxide particles the cells displayed numerous lysosomes filled with these particles. Intramuscular injection of isolated cells into the tongues of young rats of the same strain, resulted in the formation by both freshly isolated and thorium dioxide-labeled cells of a characteristic tissue similar to that found in the intact aortic wall. Thus, the transplants consisted of typical smooth muscle cells surrounded by an extracellular matrix consisting of a microfibrillar network, patches and fibers of elastin, collagen fibrils and small polygonal granules believed to represent proteoglycans. This system can be used for experimental studies on the production of extracellular matrix components and on other functions of arterial smooth muscle cells growing outside the vascular wall.The technical assistance of Ms. Karin Blomgren and the secretarial assistance of Ms. Inger Åhrén are gratefully acknowledgedFinancial support was obtained from the Swedish Medical Research Council (proj. no. 12X-03355), the King Gustaf V 80th Birthday Fund, the M. Bergvall Foundation, the A.O. Swärd Foundation, and from the Funds of the Karolinska Institutet     

Filament arrangements in negatively stained cultured cells: the organization of actin

Treatment of spread, cultured cells with Triton X-100 followed by negative staining reveals the organization of the unextracted intracellular filamentous elements: actin, microtubules and the 100 angstrom filaments. The present report describes the organization of the actin-like filaments in human skin fibroblasts and mouse 3 T 3 cells. As shown in earlier studies, the cytoplasmic stress fibres were seen to be composed of bundles of colinear actin-like filaments. In addition to these large stress fibres much smaller bundles of thin filaments as well as randomly oriented thin filaments were also observed. A thick bundle of thin filaments, 0.2 microm to 0.5 microm in diameter, was found to delimit the concave cell edges most prominent in well-spread stationary cells. The leading edge and ruffled border of human skin fibroblasts appeared as a broad web, of meshwork of diagonally oriented thin filaments interconnecting radiating, linear bundles of thin filaments about 0.1 microm in diameter. These bundles corresponding to the microspikes described earlier ranged from about 1.5 microm in length and were separated by 1 microm to 3 microm laterally. The leading edge of 3 T 3 cells showed a similar organization but with fewer radiating thin filament bundles. Both the filaments in the bundles and in the meshwork formed arrowhead complexes with smooth muscle myosin subfragment - 1 which were unipolar and directed towards the main body of the cell. The findings are discussed in relation to the mechanisms of non-muscle cell motility.     

The effect of agarose and dexamethasone on the nature and production of extracellular matrix components by elastic cartilage chondrocytes

Chondrocytes isolated enzymatically from rabbit ear cartilage, were cultivated in vitro in the presence of 2% agarose or 0.1 mumol/l dexamethasone. Freshly-isolated chondrocytes suspended in either Eagle's medium or 2% agarose were auto-transplanted intramuscularly. Samples were then examined by light microscopy and transmission electron microscopy. The cells cultivated in vitro rapidly formed confluent multiple overlapping layers filled with a loose matrix consisting of single collagen fibres, proteoglycans and scarce elastic fibres. The number and maturity of the elastic fibres increased substantially after dexamethasone was added. The chondrocytes in intramuscular transplants produced a larger amount of intercellular matrix with many elastic fibres than those cultured in vitro. Addition of agarose to in vitro and in vivo systems selectively suppressed the elastin production but did not diminish the production of elastic fibre microfibrils and other matrix components. This made cultures and transplants of elastic chondrocytes resemble rather hyaline cartilage than the original tissue. It seems that the lack of elastin in the matrix does not result simply from inhibition of elastin secretion or increased elastolysis. It may be related to a reversible change of genetic expression of elastic cartilage chondrocytes under the influence of agarose.