Ultrastructural distribution of 36-kD microfibril-associated glycoprotein (MAGP-36) in human and bovine tissues.

We observed the ultrastructural distribution of MAGP-36 by immunoelectron microscopy in human and bovine tissues. MAGP-36 was present in microfibrils associated with tropoelastin in skin, aorta, and spleen. It was not detected in microfibrils from the ocular zonule and kidney mesangium that were not associated with tropoelastin. In skin, MAGP-36 was present in both early immature elastic fibers and mature elastic fibers. In mature elastic fibers, MAGP-36 was localized around amorphous elastic cores at the elastin-microfibril interface and in electron-dense bundles. Localization of MAGP-36 in elastic fibers coincided with the distribution of lysyl oxidase, an enzyme that plays a pivotal role in the deposition of tropoelastin. These findings suggest that MAGP-36 may be involved in elastogenesis.     

Distribution of elastic system fibers in the peripheral nerves of mammals

Various nerves of 6 representative species of mammals (including the human) were studied by the comparative association of the selective staining methods of light microscopy with the ultrastructural observation after tannic acid-glutaraldehyde fixation, which provided a reliable means of characterizing the different elastic system fibers. Although mature elastic fibers are not present in nerves, elastic-related fibers are frequently observed: oxytalan fibers are found mainly in the endoneurium, whereas elaunin fibers predominate in the epineurium. These fibers are longitudinally disposed, in a parallel orientation to the axons. The fact that these findings were consistently observed in the nerves of all species studied argues strongly in favor of the existence of a uniform structural pattern of distribution of elastic system fibers in nerves as a general phenomenon in mammals.     

Simultaneous observation of collagen and elastin in normal and pathological tissues: analysis of Sirius-red-stained sections by fluorescence microscopy

In order to observe collagen and elastic fibers simultaneously, sections of human aorta, skin, lung, liver, and bladder were stained by Sirius red and analyzed by fluorescence microscopy. In all cases, the fibers of collagen presented the characteristic fluorescent red-orange color that results from the interaction of this extracellular protein with the dye, whereas elastic fibers showed strong green fluorescence (intrinsic fluorescence). This method efficiently detects collagen and elastic fibers when these two structures are present and could have valuable applications in processes that involves both fibers.L.F.B. received a doctoral fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.     

Resorption of elastic fibers in monkey gingival connective tissue: ultrastructural and immunocytochemical evidence

Little is known about the remodeling of elastic fibers in gingival connective tissue. In this study, elastic fibers in the lamina propria of monkey gingiva were examined by transmission electron microscopy and immunocytochemistry. Some elastic fibers were localized at invagination on the surface of the narrow processes of fibroblasts distributed among dense assemblies of collagen fibrils, and also within coated pits, which were pinching off as coated vesicles. At a higher magnification, the coated vesicles contained filamentous structures, as well as pentagonal structures similar those previously reported in elastic fibers. Immunoelectron microscopy demonstrated positive staining for fibrillin, one of the main components of microfibril, localized either in the coated pits or vesicles. These observations indicate that at least some elastic fibers were resorbed by fibroblasts, and that, in spite of the general belief that little remodeling of elastic fibers occurs under normal conditions, resorption of elastic fibers does occur in monkey gingival connective tissue. The functional significance of this is not yet clear, but it may be involved in facilitating the delicate and efficient adaptation of tissue to physical requirements during mastication.     

Elastica-positive material in the atrial endocardium. Light and electron microscopic identification

The elastic layer of the endocardium is studied in various laboratory animals (mouse, rat, rabbit, cat, and dog) and in man. Coarse elastica-positive fibers form a tightly woven layer in the endocardium of the left atrium; the elastic layer consists of loosely arranged delicate fibers in the endocardium of the right atrium. Electron microscopy shows the elastic material to consist of homogeneous elastin (E) and of elastic fiber microfibrils (EFM). Elastic material in the endocardium of the left atrium is mainly formed of E with few EFM present. By contrast, the portion of EFM predominated that of E in elastic fibers from the right atrium, where some elastica-positive fibers even appear as pure bundles of microfibrils. This was also observed in human material obtained from aged individuals (8th decennium). It is concluded that EFM are not only progenitors of E but represent an independent fibrous component of the connective tissue.     

Ultrastructure of the pulmonary trunk wall during the prenatal period of ontogenesis

By means of transmissive electron microscopy stages of morphogenesis have been studied in the pulmonary trunk wall of 12 healthy human embryos and fetuses of 5-24-week-old. There is a close dependence in development of the smooth muscle component of the middle tunic, elastic and collagenous fibers of the pulmonary trunk wall. In the early prenatal morphogenesis of the pulmonary trunk fibrillogenesis develops intensively, overtakes the differentiation time of myocytes from mesenchymal cells, the collagenous fibers appearing earlier than the elastic ones. The structures, that ensure solidity of the vascular wall (collagenous fibers), appear in embryogenesis earlier that those, ensuring its elasticity (ability to reversible deformity).     

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.     

Subepithelial elastic system fibers of oblique mucosal folds in the rat proximal colon.

The proximal colon of the rat is characterized by a 'herring bone' pattern of oblique mucosal folds (OMF) which are arranged in a parallel array. By light and electron microscopy the OMF exhibited rich subepithelial elastic system fibers which bound the epithelial basement membrane and the smooth muscle cells of the lamina propria together. The elastic system fibers usually consist of elastic, elaunin and oxytalan fibers. However, the subepithelial elastic system fibers of the OMF were composed of relatively thin elastic fibers with a few microfibrils, and elaunin and oxytalan fibers which were almost indiscernible. Areas other than the OMF were quite poor in subepithelial elastic system fibers. The interpositions between each of the OMF were composed of typical components: elastic, elaunin and oxytalan fibers. The composition of the subepithelial elastic system fibers of the OMF does not correspond to that of any other organs previously reported. The present study suggests that the OMF of the rat proximal colon might be equipped in such a way to resist to distension or compression.     

Fluorescent staining of elastic tissue with Rhodamine B and related xanthene dyes

Summary Frozen sections, cut at 8 from various fresh tissues, were dried for 5 minutes over sulfuric acid, and then stained for 4 minutes in a 0.1% aqueous solution of Rhodamine B (Chroma) at pH 8.O. After soaking twice in butanol for 2 minutes each, the sections were kept on a warmer at 60° C for 2 minutes and then mounted in oil of cedar. Under ultraviolet microscopy, elastic fibers selectively stained yellow-orange against a pale blue background. No autofluorescence of elastic fibers was observed in guinea pig, rat, hamster or rabbit tissue in contrast to the autofluorescence of elastic fibers typically seen in human tissue.Similar fluorescent staining of the elastic tissue could be achieved by using a number of related xanthene dyes including pyronine, eosin B, acridine orange, pholoxin, phloxin B. More distantly related auranine O and thiazol yellow G were likewise used with success as fluorochromes for elastic fibers.Supported by a John A. Hartford Foundation Grant.     

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections

 We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin auto-fluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results. Accepted: 28 August 1996     

A tissue-engineered human dermal construct utilizing fibroblasts and transforming growth factor β1 to promote elastogenesis

Numerous studies have shown that extracellular matrix (ECM)-based scaffolds are suitable for dermal constructs for the differentiation of various cell types in vitro and for constructive tissue remodeling after implantation in vivo. However, a shortcoming of these ECM materials is its limited elastogenesis. Elastic fibers constitute an essential component of mammalian connective tissue and the presence of elastic fibers is crucial for the proper function of the cardiovascular, pulmonary, and intestinal systems. Since it is still largely unknown how cells coordinate the molecular events of elastic-fiber assembly, understanding the ability to regenerate elastic fibers in tissues remains a significant challenge. For this reason, human neonatal dermal fibroblasts (HDFneo) were analyzed for their potential to serve as a cell culture model for elastic fiber assembly. Using optical technologies such as multiphoton laser-scanning microscopy (MPSLM) we demonstrate that HDFneo stimulated with transforming growth factor β1 (TGF-β1) are able to produce a distinct and complex elastic fiber system in vitro. As shown by the desmosine and isodesmosine content, crosslinked elastic fibers were formed within the 3D ECM-based scaffold. This tissue-engineered dermal construct may prove to be an effective template for the development of medicinal approaches in regenerative soft skin tissue reconstruction through TGF-β1 induction.     

Elastic fibers in the tunica propria of the seminiferous tubules. Light and electron microscopic investigations.

Histological examination of numerous biopsies from mature testes, repeatedly showed irregularities of the elastin staining in the tunica propria of the seminiferous tubules. Even when abundant elastic fibers were visible by light microscopy, no elastic fibers were demonstrable in the electron microscope in ultrathin sections of testicular tubules embedded in Epon and contrasted with phosphotungstic acid (PTA). In 43 biopsies from 22 men aged 17-39 years (19 investigations of sterility and three patients with hypogonadotropic hypogonadism) we therefore checked for the occurrence of elastic fibers in the wall of the seminiferous tubules. Matrix loci of elastic fibers could indeed by demonstrated by electron microscopy using PTA and potassium permanganate (KMnO4), but only after embedding in araldite. Under these conditions, light and electron microscopic findings agreed with each other. The appearances of moderate and severe testicular tubular atrophy differed slightly from one another with regard to the amount of elastin. In the "Sertoli cells only syndrome", elastic fibers were demonstrable only outside the hyalinized inner layer. In the Klinefelter syndrome, only "uncertain" elastin loci were present, but greatly increased microfibrils were to be seen using the electron microscope. No elastic elements and only very sparse microfibrils were present in the tunica propria of the tubules of young men with hypogonadotropic hypogonadism.     

SEM observations of the elastic networks in canine femoral artery

Scanning electron microscopy was used to study the normal architectural arrangement of elastic tissue in a medium-sized muscular artery. Selective NaOH sonication digestion or formic acid digestion was used to expose and isolate the elastic networks in the femoral arteries of four healthy dogs. The digested segments were neutralized and freeze-dried before mounting for scanning electron microscopy (SEM) observation. The fenestrated internal elastic lamina (IEL) had a smooth surface with scattered regions of the fine elastic fibers that made up lacy networks protruding from the luminal surface. Prominent ellipsoid fenestrae, randomly scattered across the surface, were grouped into small and large sizes based on their mean diameter. The openings of most fenestrae were bridged by elastic fibers to give the fenestrae a sieve-like appearance. Large, transversely oriented, fusiform gaps were randomly scattered along the length of the IEL. These gaps, filled in by an elastic fiber network, sometimes spanned as much as a quarter of the vessel circumference. It is suggested that these gaps represent splits in the IEL that have been repaired. The tunica media contained a complex network of anastomosing elastic fibers and lamellae that were primarily circumferential in orientation. A well-defined external elastic lamina formed a solid sheet at the junction of the tunica media and the tunica adventitia. The tunica adventitia contained 8-10 incomplete lamellae of large, interconnecting, longitudinally oriented fibers. The architecture of the elastic network in canine femoral artery was compared with that previously described in medium-sized canine veins and in the rat femoral artery.     

Elastic fiber formation in monolayer and organ cultures of chondrocytes isolated from auricular cartilage.

Chondrocytes were isolated from auricular cartilage of immature rabbits and maintained in monolayer or organ culture for 14 days. In both types of culture the chondrocytes formed conspicuous elastic fibers. In monolayer culture the fibers could be identified by orcein staining in the culture dish. Electron microscopy of organ cultures revealed the presence of two basic components of elastic fibers, i.e. microfibrils and elastin.     

Arrangement and structure of sinus hair muscles in the big-clawed shrew,Sorex unguiculatus

The arrangement and structure of sinus hair muscles in the snout of the shrew, Sorex unguiculatus, were studied by electron microscopy and serial section light microscopy. Both striated and smooth muscles are directly associated with sinus hair follicles. The striated muscle fibers originate from the base of a follicle and insert onto the superficial portion of adjoining caudally positioned follicles. Some fibers insert into the corium instead of inserting into a follicle. The fibers show a fine structure typical of red fibers. Smooth muscle cells form a network with elastic fibers beneath the corium. Some cells are directly attached to the capsule of the sinus, thus forming a type of M. arrector pili. Striated muscle fibers that appear to end in the corium are connected with the smooth muscle network through the elastic fibers which appear to function as the tendon of these two types of muscle cell.     

A selective histochemical method for the quantitative estimation of elastic fibers by computerized morphometric analysis. Effect of colchicin treatment

A morphometric technique is reported that uses a new selective staining of the elastic system fibers in skin biopsy specimens to facilitate the quantitative evaluation of the volume fraction occupied by these elastic fibers in the tissue. The study of elastic fibers in the dermis of 30 patients, before and after six months of treatment with Colchicin, was carried out with a Quantimet 720 system. Preelastic (oxytalan and elaunin) fibers and mature elastic fibers were quantitated separately. Compared to the average volume fraction (surface occupied by the elastic fibers) before treatment with Colchicin (1.449 +/- 0.64%), the mean values after treatment were significantly increased (2.076 +/- 0.61%). The same results were found for the preelastic fibers: 0.807 +/- 0.51% before treatment and 1.025 +/- 0.54% after treatment. These results demonstrate the advantages of our monochromatic staining method for automatic quantitation of elastic fibers as well as the possibilities of the quantitative study of the elastic fibers in human dermis. This methodology should be applicable to other inherited or acquired diseases affecting skin elastic fibers as well as to other tissues containing elastic fibers.     

Videostroboscopic and morphological aspects of voice disturbances in patients with larynx atrophy and coexisting hypopharynx cancer

Vocal folds play a crucial role in voice production. The physiological vibrations of vocal folds depend on the unchanged multilayered structure of the vocal folds mucosa. Morphological changes of mucosa are the cause of voice quality disorders - dysphonia. The aim of this study was to determine the morphological base of dysphonia in patients with vocal folds atrophy. A group of 24 patients with larynx atrophy confirmed by endoscopic (VLS) and stroboscopic (VLSS) examination of the larynx was included in the study. The morphological assessment of the larynx mucosa was carried out with the use of the transmission electron microscopy (TEM). Ultramorphological examinations revealed changes in the epithelium, basal membrane and lamina propria of the vocal folds mucosa. An increased number of collagenous fibers, fibroblasts with signs of vacuolar degeneration inflammatory cells and a decreased number of blood vessels and pericytes were observed. Morphological changes found in the epithelium, basal membrane and lamina propria of the vocal folds mucosa were the cause of disorders of vocal folds vibrations registered in the stroboscopic examination of the larynx (VLSS).     

Development of elastic fibers of nuchal ligament, aorta, and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study

The morphogenesis of elastic fibers of the nuchal ligament, aorta, and lung of sheep was studied by light microscopy, transmission electron microscopy, and immunohistochemical methods for the detection of elastin. The degree of maturation of the amorphous materials of elastic fibers was assessed morphologically in preparations stained by the tannic acid and periodic acid methenamine-silver methods. With both of these methods, the amorphous components of mature fibers stained less intensely than did those of immature fibers. Elastic fibers in early stages of development consisted of many microfibrils and few, small, branching masses of immature amorphous material. Thicker fibers were formed by the coalescence of growing masses of amorphous materials. In late stages of formation of elastic fibers, the mature amorphous materials were associated with few microfibrils; and they were partially surrounded by immature amorphous materials associated with many microfibrils. Antielastin antibody reacted evenly with amorphous materials in very early stages of elastic-fiber development, but reacted only with the other zones of amorphous materials in later stages; it also reacted with the microfibrils in all stages. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin on their surfaces. This conclusion is in agreement with ultrastructural observations showing 1) that development of microfibrils precedes that of the amorphous material and 2) that the microfibrils adjacent to the immature amorphous materials are covered with small amounts of tannic acid-positive amorphous materials. These observations suggest that microfibrils serve as sites for elastin deposition, both in early elastogenesis and in subsequent growth of elastic fibers. However, the nature of the interaction between elastin and microfibrils remains unknown.     

3‐photon fluorescence imaging of sulforhodamine B‐labeled elastic fibers in the mouse skin in vivo

Elastic fibers are key constituents of the skin. The commonly adopted optical technique for visualizing elastic fibers in the animal skin in vivo is 2‐photon microscopy (2 PM) of autofluorescence, which typically suffers from low signal level. Here we demonstrate a new optical methodology to image elastic fibers in animal models in vivo: 3‐photon microscopy (3 PM) excited at the 1700‐nm window combining with preferential labeling of elastic fibers using sulforhodamine B (SRB). First, we demonstrate that intravenous injection of SRB can circumvent the skin barrier (encountered in topical application) and preferentially label elastic fibers, as verified by simultaneous 2 PM of both autofluorescence and SRB fluorescence from skin structures. Then through 3‐photon excitation property characterization, we show that 3‐photon fluorescence can be excited from SRB at the 1700‐nm window, and 1600‐nm excitation is most efficient according to our 3‐photon action cross section measurement. Based on these results and using our developed 1600‐nm femtosecond laser source, we finally demonstrate 3 PM of SRB‐labeled elastic fibers through the whole dermis in the mouse skin in vivo, with only 3.7‐mW optical power deposited on the skin surface. We expect our methodology will provide novel optical solution to elastic fiber research.     

Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin

Muscle needs an elastic framework to maintain its mechanical stability. Removal of thin filaments in rabbit skeletal muscle with plasma gelsolin has revealed the essential features of elastic filaments. The selective removal of thin filaments was confirmed by staining with phalloidin-rhodamine for fluorescence microscopy, examination of arrowhead formation with myosin subfragment 1 by electron microscopy, and analysis by SDS-PAGE. Thin section electron microscopy revealed the elastic fine filaments (approximately 4 nm in diameter) connecting thick filaments and the Z line. After removal of thin filaments, both rigor stiffness and active tension generation were lost, but the resting tension remained. These observations indicate that the thin filament-free fibers maintain a framework composed of the serial connections of thick filaments, the elastic filaments, and the Z line, which gives passive elasticity to the contractile system of skeletal muscle. The resting tension that remained in the thin filament-free fibers was decreased by mild trypsin treatment. The only protein component that was digested in parallel with the decrease in the resting tension and the disappearance of the elastic filaments was alpha-connectin (also called titin 1), which was transformed from the alpha to the beta form (from titin 1 to 2, respectively). Thus, we conclude that the main protein component of the elastic filaments is alpha-connectin (titin 1).