A rapid digestive technique to expose networks of vascular elastic fibers for SEM observation

The NaOH sonication digestion technique permits rapid isolation and exposure of intact networks of elastic fibers in vascular tissue for 3-dimensional observation with the SEM. The configuration of the network of elastic fibers within the vascular wall of large elastic arteries (aorta) is generally agreed to be a flexible framework through which smooth muscle cells and collagenous fibers are interwoven. However, the configuration of elastic fiber networks in muscular arteries, medium sized veins and smaller vessels remains unknown. When the lengthy standard biochemical elastin purification techniques were applied to vessels containing lesser amounts of elastic tissue and finer elastic fibers, the vessels were completely digested. In contrast, the digestion and sonication technique isolated and exposed intact networks of delicate elastic fibers in blood vessels which do not contain large amounts of elastic tissue. Unfixed vessels were cut into short segments, placed in 0.5 N NaOH and sonicated for 20-40 min. The specimens were rinsed in deionized distilled H2O, then autoclaved for 30 min. The tissue was rinsed a second time, fixed and processed routinely for SEM. Elastic stains and enzymatic digestion with chromatographically purified elastase and collagenase confirmed that the digestion and sonication technique produced clean, isolated networks of elastic fibers. Knowledge of the configuration of the networks of elastic fibers in different vessels enhances understanding of distensibility characteristics of individual vessels and serves as a baseline for studying alterations in the elastic framework which occur during aging and disease processes such as atherosclerosis, arterial hypertension and aneurysms.     

The three-dimensional architecture of the elastic-fiber network in canine hepatic portal system

The architectural arrangement of the elastic-fiber network in the wall of canine hepatic portal veins was observed with the scanning electron microscope (SEM). Selective NaOH sonication digestion and autoclaving were used to expose and isolate the networks of elastic fibers from six selected regions of the hepatic portal vessels from seven healthy dogs. Elastic stains of adjacent segments prepared for light microscopy demonstrated that the elastic fibers were concentrated in two areas within the intact portal wall. The innermost area corresponded to the internal elastic lamina (IEL) of the tunica intima, the internal muscular layer, and the connective tissue layer of the tunica media. The second area was in the tunica adventitia. SEM specimens revealed two sleeves of elastic fiber networks which corresponded to the above regions. Small scattered bundles of radially oriented elastic fibers spanned the gap between the two sleeves. Each tunica had a different architectural arrangement of elastic fibers. The IEL had circumferentially oriented fibers which branched and anastomosed to form a continuous network on the innermost surface. The architecture of the IEL was the most variable between the different regions. The network of the IEL was the most "open" in the caudal region (splenic vein) and became "denser" toward the liver. The large elastic fibers in the tunica media were oriented at approximately right angles to the primary fibers of the IEL. These longitudinally oriented fibers anastomosed with adjacent longitudinal fibers to form a continuous network. In the tunica adventitia, thick, longitudinally oriented fibers of the continuous network fused together to form incomplete layers of fibers. The architecture of the elastic-fiber network in the canine hepatic portal vein was compared to that previously described in the systemic canine saphenous vein.     

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.     

Use of tensorial description in tissue remodeling: examples of F-actin distributions in pulmonary arteries in hypoxic hypertension

A molecular configuration tensor Pij was introduced to analyze the distribution of fibrous proteins in vascular cells for studying cells and tissues biomechanics. We have used this technique to study the biomechanics of vascular remodeling in response to the changes of blood pressure and flow. In this paper, the remodeling of the geometrical arrangement of F-actin fibers in the smooth muscle cells in rat's pulmonary arteries in hypoxic hypertension was studied. The rats were exposed to a hypoxia condition of 10% for 0, 2, 12, and 24 hr at sea level. Remodeling of blood vessels were studied at the in vivo state under normal perfusion, no-load state when small rings from blood vessels were excised, and zero-stress state after the rings were cut open radially to release the residual stress. Tissue remodeling in response to changes in blood pressure is reflected in the zero-stress state. The tensor components were determined by analyzing the configuration of phalloidin stained F-actin fibers in the media layer of pulmonary arteries. The values of P31, P32, P33 in the in-vivo state, the no-load state, and the zero-stress state are obtained. This study demonstrated the distributions of fibrous molecules in tissue remodeling can be described quantitatively using the molecular configuration tensor.     

A Modified Verhoeff-Van Gieson Elastin Histochemical Stain to Enable Pulmonary Arterial Hypertension Model Characterization

Optimal histochemical staining is critical to ensure excellent quality stained sections to enable light microscopic and histomorphometric image analysis. Verhoeff-van Gieson is the most widely used histochemical stain for the visualization of vascular elastic fibers. However, it is notoriously difficult to differentiate fine elastic fibers of small vasculature to enable histomorphometric image analysis, especially in organs such as the lung. A tissue fixation procedure of 10% neutral buffered formalin with subsequent fixation in 70% ethanol further compounds the problem of small vessel staining and identification. Therefore, a modified Verhoeff’s elastin stain was developed as a reliable method to optimally highlight the internal and external elastic laminae of small arteries (50-100 µm external diameter) and intra-acinar vessels (10-50 µm external diameter) in 3 µm thick lung tissue sections from models of pulmonary arterial hypertension. This modified Verhoeff’s elastin stain demonstrated well-defined staining of fine elastic fibers of pulmonary blood vessels enabling subsequent histomorphometric image analysis of vessel wall thickness in small arteries and intra-acinar vessels. In conclusion, modification of the standard Verhoeff-van Gieson histochemical stain is needed to visualize small caliber vessels’ elastic fibers especially in tissues fixed in 10% neutral buffered formalin followed by additional fixation in 70% ethanol.Key words: Histochemical stain, histomorphology, lung, Verhoeff-van Gieson, elastin     

Elasticity of passive blood vessels: a new concept

The mechanical properties of passive blood vessels are generally thought to depend on the parallel arrangement of elastin and collagen with linear elasticity and collagen recruitment depending on vessel strain [hook-on (HO) model]. We evaluated an alternative model [serial element (SE) model] consisting of the series arrangement of an infinite number of elements, each containing elastin with a constant elastic modulus and collagen that switches stepwise from slack (zero stress) to fully rigid (infinite stiffness) on ongoing element strain. Both models were implemented with Weibull distributions for collagen recruitment strain (HO model) and collagen tightening strain (SE model). The models were tested in experiments on rat mesenteric small arteries. Strain-tension relations were obtained before and after two rounds of digestion by collagenase. Both models fitted the data prior to digestion. However, for the HO model, this required unrealistically low estimates for collagen recruitment or elastic modulus and unrealistically high estimates for distension of collagen fibers. Furthermore, the data after digestion were far better predicted by the SE model compared with the HO model. Finally, the SE model required one parameter less (collagen elastic modulus). Therefore, the SE model provides a valuable starting point for the understanding of vascular mechanics and remodeling of vessels.     

Elasticity changes in the large arteries of human limbs in response to cycle ergometry performed with upper and lower limbs

At rest and after cycle ergometry the elastic properties of the large arteries of limbs of healthy men were examined using an original non-invasive quantitative oscillometric method. It has been shown that in response to muscle work performed with the legs there is a decrease of the effective inner radius, and an increase of the characteristic impedance modulus and bulk modulus and of the elastic resistance of the intact and relaxed wall in the large arteries in the upper limbs. All these changes testify to an increase of vascular tension in the upper limbs. In response to work performed with the hands, there is an increase of the effective inner radius of large arteries of the upper limbs, a large increase of the pulsatile blood volume increment of the intact vessels and a decrease of the characteristic impedance modulus, of the bulk modulus and of the elastic resistance of the intact arterial wall. These changes indicate a decrease of the vascular tension of these arteries. In response to work performed either with the legs or with the hands a decrease of the effective inner radius of large arteries and an increase of the elastic resistance of the relaxed arterial wall were observed in the lower limbs, all these changes indicating relatively small changes in tone of these vessels. It is concluded that the wall tension of large arteries supplying blood to the muscles of non-working limbs is increased. Vascular tension changes in the arteries in working limbs are accounted for by the superimposition of centrally originating vasoconstriction with local vasodilatation, which also affects large arteries.     

Effects of simulated weightlessness on arterial vasculature (an experimental study on vascular deconditioning)

Rats were tail-suspended to simulate microgravity and then studied for changes in arterial vasculature. Parameters investigated included vascular responsiveness at different areas of the body, morphological changes in arteries and vascular endothelium tissue, and the distribution of adrenergic nerve fibers supplying cerebral arteries and arterial vessels. The significance of the results and future research directions are discussed.     

Extracellular matrix-mediated remodeling and mechanotransduction in large vessels during development and disease

The vascular extracellular matrix (ECM) is synthesized and secreted during embryogenesis and facilitates the growth and remodeling of large vessels. Proper interactions between the ECM and vascular cells are pivotal for building the vasculature required for postnatal dynamic circulation. The ECM serves as a structural component by maintaining the integrity of the vessel wall while also regulating intercellular signaling, which involves cytokines and growth factors. The major ECM component in large vessels is elastic fibers, which include elastin and microfibrils. Elastin is predominantly synthesized by vascular smooth muscle cells (SMCs) and uses microfibrils as a scaffold to lay down and assemble cross-linked elastin. The absence of elastin causes developmental defects that result in the subendothelial proliferation of SMCs and inward remodeling of the vessel wall. Notably, elastic fiber formation is attenuated in the ductus arteriosus and umbilical arteries. These two vessels function during embryogenesis and close after birth via cellular proliferation, migration, and matrix accumulation. In dynamic postnatal mechano-environments, the elastic fibers in large vessels also serve an essential role in proper signal transduction as a component of elastin-contractile units. Disrupted mechanotransduction in SMCs leads to pathological conditions such as aortic aneurysms that exhibit outward remodeling. This review discusses the importance of the ECM—mainly the elastic fiber matrix—in large vessels during developmental remodeling and under pathological conditions. By dissecting the role of the ECM in large vessels, we aim to provide insights into the role of ECM-mediated signal transduction that can provide a basis for seeking new targets for intervention in vascular diseases.     

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC(50))) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching -5.15+/-0.79 and before stretching -6.71+/-0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.     

Zur Wandstruktur der großen Arterien der Vögel

Zusammenfassung Die Wandstruktur der großen Arterien des Schwans, der Drossel und des Stars wurde licht- und elektronenoptisch untersucht und eine Einteilung in elastische, muskuläre und Übergangsgefäße getroffen.Die Media der elastischen Gefäße besteht aus muskulo-elastischen Zylindersegmenten, die mit breiten Bindegewebslagen alternieren. Die Zylindersegmente bestehen aus plattenförmigen Lagen glatter Muskelzellen, die von elastischen Fasernetzen flankiert werden. Diese Zylindersegmente beginnen und enden in den Bindegewebslagen stark gegeneinander versetzt, so daß ein kulissenartig einander überlappendes Plattensystem entsteht. Die Bindegewebslagen bestehen neben kollagenen Fasern und Fibrozyten aus mehreren konzentrischen Lagen elastischer Fasernetze. Die elastischen Netze sind durch Verbindungsfasern zu einem dreidimensionalem, die ganze Gefäßwand durchsetzenden elastischen System verknüpft. In den Übergangsgefäßen sind die Bindegewebslagen zwischen den muskulo-elastischen Systemen weitgehend reduziert.Bindegewebige und muskuläre Wandbestandteile sind im muskulären Vogelgefäß weitgehend voneinander getrennt. Die Media besteht aus glatten Muskelzellen, die von elastischen Netzen zu Schichten zusammengefaßt werden, die Adventitia aus kollagenen Fasern, Fibrozyten und konzentrischen Lagen elastischer Fasernetze. Die glatten Gefäßmuskelzellen sind durch elastische Fasernetze zu muskulo-elastischen Einheiten zusammengefaßt. Die mechanischen Verknüpfungspunkte zwischen Muskelzellen und elastischen Fasern sind über die ganze Zelloberfläche verteilt.Die Gefäßbautypen sind durch eine Wandstärken-Lumenrelation gekennzeichnet. Sie beträgt im elastischen Gefäß 1:5 bis 1:6, im muskulären Gefäß 1:14 bis 1:16.

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The wall structure of large arteries in birds

Summary A classification of large arteries (elastic, muscular and intermediate type) in mute swan, trush and starling was undertaken with light and electron microscopy.The tunica media of elastic arteries consists of musculo-elastic cylindrical segments alternating with wide connective tissue layers. The former consists of smooth muscle cell layers, which are adjoined by a network of elastic fibers. These musculo-elastic cylinder segments overlap incompletely. The connective tissue layers consist of networks of elastic fibers concentrically arranged in addition to collagen fibers and fibrocytes. The elastic networks are joined by connecting elastic fibers, thus forming a three-dimensionalsystem. In the intermediate type of arteries the connective tissue layers between the musculo-elastic systems are greatly reduced.Connective tissue and muscular components of the wall of muscular arteries are almost completely separated. The tunica media is composed of smooth muscle cells sandwiched by networks of elastic fibers. The tunica adventitia is formed by concentric networks of elastic fibers, collagen fibers and fibrocytes.The arterial smooth muscle cells, together with networks of elastic fibers, form a musculoelastic unit. The points of mechanical attachment between smooth muscle cells and elastic fibers are scattered all over the cellular surface. The arterial types described above are characterized by a well-defined wall thickness/lumen ratio. This ratio is of the order of 1:5 to 1:6 for elastic arteries and 1:14 to 1:16 for muscular arteries.

Medizinische Dissertation unter Anleitung von Prof. Dr. Dr. H.-R. Duncker.     

Ultrastructural characteristics of elastogenesis in the major arteries of the canine hindlimb during leg lengthening

In mature dogs ultrastructural peculiarities of elastogenesis in femoral and anterior tibial arteries have been studied at various stages of the bone elongation after Ilizarov method. From the end of the 1st week of distraction, metabolic activation of intimal smooth muscle cells is revealed, from the 2d week--in the middle tunic, and on the 5th-6th week--fibroblasts of adventitia of the arteries investigated, directed to biosynthesis of intracellular predecessors of elastin and microfibrils of the elastic fibers. This results in activation of elastogenic processes, elastic structures in all three tunics of the arteries are observed to newly form and rearrange. The factor that stimulates and maintains elastogenesis is strain of extension, that occurs in the vessels during the experiment. Elastogenesis in the major arteries, when the extremity is elongated, has much in common with development of elastic components in the vascular wall in animals during the process of physiological growth.     

Early calcification patterns of the iliac arteries and their relation to the arterial structure

Summary Gross calcifications of the common iliac and internal iliac arteries represent a common finding in newborn children and infants. In both arteries, the calcific deposits regularly appear in certain areas of the arterial luminal surface only, whereas the other parts of the arterial wall remain free of gross lesions even in cases with a pronounced calcification. In the common iliac artery, the lateral wall of the vessel and the adjacent sectors of the anterior and posterior wall represent the predilection site of calcific deposits. In the internal iliac artery, the gross calcifications have been regularly demonstrated in the dorso-medial wall. The predominant localisation of the calcification in these parts of the vessels and its absence in the others depend on the definite structural features of the arterial tube and different affinity for calcium of the individual structural elements. In both iliac arteries, only the primary internal elastic membrane undergoes early calcification. However, unlike the most muscular arteries, this membrane is not developed in the whole arterial circumference of the common iliac and internal iliac arteries, but is absent in large areas of their arterial luminal layer. In these areas, the subendothelial or subintimal elastic layers are formed by the networks of longitudinally arranged elastic fibers or membraneous elastic structures which arise from the elastic networks with the further growth. These elastic elements always stay free of calcific deposits. The structural features found in both iliac arteries may be important for the development of the later pathological changes.     

Ultrastructural immunolocalization of elastic fibers in rat blood vessels using the protein A-gold technique

Identification of elastic fibers at the ultrastructural level is accomplished by a post-embedding immunohistochemical technique using the protein A-colloidal gold method. Antisera against elastins from human dermis and rat aorta have been characterized by radioimmunoassay and then applied to thin sections of rat blood vessels. Two fixative solutions and two embedding media have been tested. Both antibodies bind to elastic fibers of normal arteries and veins, indicating crossreactions among organs and species. The high sensitivity of this method is demonstrated by its application to the detection of neo-elastogenesis in the intimal thickening of aortic grafts.     

Eccrine sweat glands of rat fingertips. Scanning electron microscope observations after enzymatic digestion of dermal connective tissue

By removing epidermis with EDTA and a subsequent enzymatic digestion of dermis, eccrine sweat glands of rat fingertips were exposed and examined by scanning electron microscopy (SEM). Different protocols were tested to remove as much connective tissue as possible, while minimizing damage to other structures, and to expose the epithelial surface of secretory tubules in order to display vascular and nervous networks. SEM observations gave detailed information on the relationship between epithelial secretory cells and myoepithelial cells, as well as on the vascular and nervous networks which surround the glomeruli of glands.     

Structural and functional characteristics of the special regulatory devices in the peripheral pulmonary circulation in rabbits

The present study intended to describe in detail the several blood vessels harboring special regulatory devices in rabbit’s pulmonary tissue using light and electron microscopy and immuno-histochemistry. Numerous throttle arteries were recorded within the adventitia of the segmental and sub-segmental bronchi and within pulmonary pleura. These arteries showed characteristic narrow or obliterated lumens and some of them bear longitudinal muscular intimal bolsters. For the first time, TEM revealed some structural modifications of the vascular endothelial cells of these arteries indicating that they become more activated to perform some additional functions. Arteriovenous anastomoses (AVAs) including direct shunt vessels and glomus organs were also recognized. Direct arteriovenous shunts appeared as small connecting devices communicating between small arteries and small veins while glomus organs consisted of the tortuous glomus vessels and the related afferent and efferent vessels. Several arteries and veins showing unique unusual structural characteristics were also described. For the first time, serotonin (5-HT) was strongly expressed in the vascular endothelium and muscle fibers of throttle arteries, in glomus cells of the glomus vessels, and in vascular endothelium of some veins and venules of special structure. The exact role of 5-HT is still unknown and further investigations are required to determine the types and distribution of 5-HT receptors present in these vascular devices. We concluded that these special vascular devices can play a critical role in controlling blood flow and pressure in the peripheral pulmonary circulation; however, the exact physiological mechanisms by which they work or are controlled remain unknown providing a ripe area for further investigation.     

Decellularized omentum as novel biologic scaffold for reconstructive surgery and regenerative medicine

Homologous tissues, such as adipose tissue, may be an interesting source of acellular scaffolds, maintaining a complex physiological three-dimensional (3D) structure, to be recellularized with autologous cells. The aim of the present work is to evaluate the possibility of obtaining homologous acellular scaffolds from decellularization of the omentum, which is known to have a complex vascular network. Adult rat and human omenta were treated with an adapted decellularization protocol involving mechanical rupture (freeze-thaw cycles), enzymatic digestion (trypsin, lipase, deoxyribonuclease, ribonuclease) and lipid extraction (2-propanol). Histological staining confirmed the effectiveness of decellularization, resulting in cell-free scaffolds with no residual cells in the matrix. The complex 3D networks of collagen (azan-Mallory), elastic fibers (Van Gieson), reticular fibers and glycosaminoglycans (PAS) were maintained, whereas Oil Red and Sudan stains showed the loss of lipids in the decellularized tissue. The vascular structures in the tissue were still visible, with preservation of collagen and elastic wall components and loss of endothelial (anti-CD31 and -CD34 immunohistochemistry) and smooth muscle (anti-alpha smooth muscle actin) cells. Fat-rich and well vascularized omental tissue may be decellularized to obtain complex 3D scaffolds preserving tissue architecture potentially suitable for recellularization. Further analyses are necessary to verify the possibility of recolonization of the scaffold by adipose-derived stem cells in vitro and then in vivo after re implantation, as already known for homologus implants in regenerative processes.Key words: omentum, scaffold, decellularization, adipose tissue engineering, regenerative medicine, microvascularization     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

Heat transport by countercurrent blood vessels in the presence of an arbitrary temperature gradient

This paper presents a three-dimensional analysis of the temperature field around a pair of countercurrent arteries and veins embedded in an infinite tissue that has an arbitrary temperature gradient along the axes of the vessels. Asymptotic methods are used to show that such vessels are thermally similar to a highly conductive fiber in the same tissue. Expressions are developed for the effective radius and thermal conductivity of the fiber so that it conducts heat at the same rate that the artery and vein together convect heat and so that its local temperature equals the mean temperature of the vessels. This result allows vascular tissue to be viewed as a composite of conductive materials with highly conductive fibers replacing the convective effects of the vasculature. By characterizing the size and thermal conductivity of these fibers, well-established methods from the study of composites may be applied to determine when an effective conductive model is appropriate for the tissue and vasculature as a whole.