Regulation mechanisms of endocrine disruptors on vasodilation and vasoconstriction: Insights from <i>ex vivo</i> models

Cardiovascular diseases (CVD) are one of the leading causes of death worldwide. The knowledge and understanding of CVD are based on the study of vascular physiology and how the smooth muscle cells and tissues perform their different functions. Exposure to endocrine disruptors (EDCs), such as phytoestrogens, polycyclic aromatic hydrocarbons, flame retardants, plasticizers, pesticides, and cosmetics, is an integral and fundamental part of human exposure. Humans are exposed to EDCs by multiple pathways including air, food, water, and consumer products. However, this exposure can lead to several adverse effects on human health, including on the cardiovascular (CV) system. The negative impact that EDC toxicity has on human CV health is a serious problem that must not be overlooked. In this point of view, we proposed the use of the human umbilical artery as a human model to study the direct effects of EDCs on the vascular level. Several works where these cells were directly exposed to EDC’s were presented to highlight this well-established model as a great strategy to be used. In the future, we emphasize the need to continue to carry out different investigations using HUA to unveil and understand the vascular toxicity of EDCs and improve human CV health.     

Angiogenesis of buffalo choroid plexuses: structural and immunocytochemical study

Mammalian choroid plexuses (CPs) are vascularized structures involved in numerous exchange processes that supply nutrients and hormones to the brain, and that remove deleterious compounds and metabolites from the brain. Studies in the adult Mediterranean buffalo have investigated the morphology of CPs using histochemical and immunohistochemical techniques. To date, however, there have been no studies conducted on ruminants regarding this removal process which serves to repair functional vascular damage in the CPs. Each of these vascular repair processes is a very complex and none of these has not yet been completely understood. Then, the aim of the present study is to investigate the morphological processes during angiogenesis in the CPs of healthy adult buffaloes, utilizing transmission electron microscopy (TEM), scanning electron microscopy (SEM), and immunogold-labeling SEM analysis (biomarkers: angiopoietin-2 [Ang-2], vascular endothelial growth factor receptor-3 [VEGFR-3], and CD133). At TEM, the inner surface of the blood capillaries sometimes showed pillar-like cells, which in contact with endothelial cells formed prominences, which in turn formed neo-blood capillaries. With immunogold-labeling SEM analysis, the CP blood capillaries showed Ang-2 and VEGF-3, respectively, in positive particles and spheroid formations. In addition, the external surface of the blood capillaries showed spheroid formations that originated from the neo-vascular capillaries whose terminals formed a capillary network, positive to CD133. On the basis of these results, the following hypothesis can be made, namely, that these CPs are vascular structures which play a fundamental role in maintaining brain homeostasis and self-repairing of functional vascular damage, independently of the presence of rete mirabile in this species.     

Comparison of Two Digestive Techniques for Preparation of Vascular Elastic Networks for SEM Observation

Two digestion procedures are now available to expose and isolate networks of vascular elastic fibers for three-dimensional SEM observation. This study was designed to observe and elucidate the differences between the two types of digestion (sodium hydroxide vs. formic acid) and the differences between the two types of dehydration (ethanol-critical-point drying vs. freeze drying) used in each procedure. Canine venous valve segments, containing delicate networks of elastic fibers, and femoral arteries, containing large elastic lamellae, were used to compare the effects of the digestion and dehydration procedures on two types of vessels with different content and organization of elastic tissue. Results indicated there was no significant difference in the architecture of the elastic networks of either vessel based on the method of digestion. The major architectural changes in the elastic networks occurred as a result of the dehydration procedure used following digestion. Freeze drying is probably the best for arterial specimens due to their prominent lamellae, which give added support to maintain their normal architecture. It is suggested that both methods of dehydration be used on corresponding venous specimens containing delicate elastic networks. In this way, the investigator can benefit from the advantages of each method and overcome their respective disadvantages to get a more accurate picture of the three-dimensional architecture of these delicate networks.     

Angioarchitecture of the ventral surface of the tongue from Wistar rats

The goal of this study was to describe the angioarchitecture of the ventral surface of the tongue from Wistar rats using a vascular corrosion casting technique associated with scanning electron microscopy (SEM). Six Wistar rats were used for the vascular casting method with Mercox resin. Following the resin polymerization, the tongue of each animal was dissected and corroded in a 5% sodium hydroxide solution. Once the corrosion and drying of the specimens were completed, the specimens were mounted on aluminum stubs, coated with carbon and gold and analyzed under SEM. The results showed that the ventral surface of the tongue presents simple, even and abundant vasculature constituted by a vascular plexus consisting of a superficial vascular network and by the ranine veins. The superficial vascular network, made up of the ascending and descending branches, presents as a loose network, with little morphological variation between the capillary loops.     

New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy

Hexamethyldisilizane (HMDS) and tetramethylsilane are organic compounds that are volatile at ambient temperature and which can therefore be used for air-drying biological samples for SEM studies. The techniques using these compounds provide results that are comparable with those obtained by critical point drying, but which involve a very simple process that saves time and money. Both techniques were applied to SEM studies of Ephemeroptera and Plecoptera eggs in order to assess their suitability as alternative methods to critical point drying for these kinds of biological material. The results show no morphological differences between eggs HMDS air-dried and critical point-dried.     

Distinct vascular zones in the canine prostate

The vascular bed of canine prostate was studied and detailed distinct vascular zones were visualized on corrosion casts by scanning electron microscopy. This study was performed because of scarce information about the zonal vascularization of the prostate gland in dogs. There are no studies for three-dimensional microvascular distribution of the capsular vessels and the capsular microvascular trabeculae. SEM (vascular corrosion casting method) was used to show 3D angioarchitecture of the prostate gland. The lobules on the dorsal and lateral surface of the gland were numerous but small. Their small size is probably due to the abundant blood supply in the region. Few but large ventral lobules were observed. Three prostatic zones were clearly defined: capsular, parenchyma, and urethral. The diameter of the venous blood vessel compared to arterial vessels of the capsule was smaller. Two types of arteries were observed in trabeculae: direct and branched. The direct arteries were straight, with only a few branches. The branched arteries contained many bifurcations, with the vessel's diameter decreasing gradually. The trabeculae capillary network formed loops, with frequent sphincter-like constrictions and pouch-like protuberances.     

3D analysis of SEM images of corrosion casting using adaptive stereo matching

The corrosion casting method represents one of the most widely used technique to study the 3D microvascularization of many tissues, both in their normal and pathological conditions. For a long time this technique was used only to perform a qualitative evaluation of the images obtained by scanning electron microscopy (SEM). A quantitative evaluation of vascular parameters (e.g., interbranching and intervascular distances, angle measurements, lengths and diameters) was lacking, mainly because of the difficulties found in the measurement performed on 2D SEM images. Then, some authors reported a quantitative method based on the analyses of stereo-pair images that allowed precise morphometric measurements. To visualize the specimens in 3D, it was necessary to use red-green glasses. In this article we describe a new approach by which we can automatically obtain a 3D reconstruction of vascular cast specimen's surface directly from stereo-images. Moreover, we developed a software that performed micrometric measurements on the 3D construct generated from the stereo-pictures. In conclusion, implementing together these two softwares and applying them to corrosion casting samples made it possible to render in 3D the surface of corrosion cast as well as make quantitative measurements on the corrosion casts.     

Microvasculature of the buffalo (Bubalus bubalis) choroid plexuses: structural, histochemical, and immunocytochemical study

The choroid plexuses (CPs) in mammals produce the cerebrospinal fluid (CSF). In the literature, the morphology of CPs and the process that regulates the production of CSF are virtually nonexistent for domestic ruminants. Thus this study has two aims: 1. to investigate the morpho-structure of the buffalo CP microvasculature utilizing light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques, and 2. to investigate the relationship between the blood vessels and both the elongated cells and the cells with multiple protrusions located in the CPs. SEM and TEM analyses of the CPs from buffalo brain showed morphological and structural features similar those reported in other mammalian species. Moreover the blood microvasculature is the major component responsible for the formation of the CSF, secreted by the encephalic CPs. In addition the chemical composition of this fluid depends on several morpho-functional characteristics of the vascularization of the CPs. These characteristics are as follows: two shapes of the vascular organization: lamina-like and ovoid-like elongated cells of the CPs, which connect the ventricular cavities to the blood capillaries; and the CP capillaries have diverse forms. In the present study the employment of NADPHd and NOS I was taken as indirect evidence for the presence of NO for investigation their specific role in CPs. Then NOS I immunoreactivity is found in the walls of CP blood vessels demonstrating indirectly the presence of NO with a vaso-dilatatory and autoregulation function of vascular tone by cholinergic nerve stimulation of blood vessel smooth muscle.     

New corrosion cast media and its ability for SEM and light microscope investigation

SEM of corrosion casts (CC) provides the opportunities to study the vessels and ducts in the phyllogenetic and ontogenetic (age‐related) settings, as well as the pathogenesis, compensation, and sanogenesis in different diseases and experimental models. Along with the refinement of SEM CC, the requirements toward casting media (CM) as nontoxicity, low viscosity, quick polymerization, resistance to corrosion solutions, availability, and so on, gradually has developed. We aimed to adapt the sets widely used in dental practice toward the modern requirements to the CC. The following ratio of the components of Protacryl‐M and Aycryl‐C sets were used for the preparation CM—0.25 g MAYCRYL Powder +0.08 g Benzoyl Peroxide +5.0 ml Protacryl‐M liquid component +0.2 Redont Colour (dye concentrate). The obtained solidifying mass was injected in the blood vessels and biliary ducts of the adult Wistar white rats. The SEM of CC of different organs’ vascular networks, as well as a biliary tract, reveals that offered CM excellently replicates the forms and branching features of studied tubular structures of all sizes and gives the adequate imprinting of their luminal surfaces. Besides, CM may provide the replication of perivascular spaces and give the casts having no analogous in the appropriate literature. The CM prepared by us perfectly reproduces all possibilities of famous rubbers widely used for the casting of different vascular–ductular structures. Besides, it presents the new implications, which should be implemented in the profound research of the connective‐tissue skeleton of different organs.     

Vascular corrosion casting technique steps

The vascular corrosion casting technique produces a replica of vascular beds of normal or pathological tissues. Once associated with scanning electron microscopy (SEM), this technique provides details of the three-dimensional anatomic arrangement of the vascular replica, which is the main advantage of this method. The present study is intended to describe the steps of the vascular corrosion casting technique and the different ways to perform them. them.     

Traditional coating techniques in scanning electron microscopy compared to uncoated charge compensator technology: Looking at human blood fibrin networks with the ZEISS ULTRA Plus FEG‐SEM

Scanning electron microscopy (SEM) studies surface morphology. Biological material needs to be coated to render the material conductive, and gold coating is traditionally used, although other coating material like carbon and ruthenium vapors may also be used. With modern SEM technology (e.g., ZEISS ULTRA Plus FEG‐SEM), we are able to work at very low kilovolts and also view fine surface structure in much better detail than with previous older technology. Some machines also allow for the study of uncoated material, although this is usually not done with biological material. This study focuses on surface clarity by comparing gold, ruthenium vapor, and carbon coating techniques for biological material. Human fibrin networks are used as example. Uncoated specimens are also viewed with a ZEISS ULTRA Plus FEG‐SEM because of its unique nitrogen charge compensator, and here, the first micrographs for uncoated human fibrin networks versus carbon, gold, and ruthenium coating are shown. We conclude that gold coating for biological material is not preferable with the latest SEM machines, as this method forms gold islands on top of the biological material and therefore produces a false surface morphology. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Scanning electron microscopic observation of microvasculature in periodontium.

In this study, vascular resin cast models in the periodontium of beagle dogs were prepared and three-dimensional observation of the relationship between the gingiva and periodontal ligament (PDL) vascular network was performed. After the perfusion of Ringer's solution and fixative, synthetic resin was injected from the inferior alveolar arteries. Soft tissue was digested by proteinase solution and specimens were examined under scanning electron microscope (SEM). The gingival vascular network (GVN) in the region facing the teeth consisted of sulcular and junctional epithelium. The vascular network of the sulcular epithelium (SE) had a renal glomerulus-like form and the junctional epithelium (JE) consisted of squamous mesh. The gingival sulcular fluid exudated from the vascular network directly beneath the JE, and leukocytes permeated from the vascular network beneath the epithelium. Thus, we considered that the GVN performs an important function in the protection against the inflammation. Periodontal ligament had a polygonal mesh vascular network that was anastomosed to the venous plexus of alveolar bone through Volkmann's canals (VC). When occlusal force was applied, the blood in the periodontal vessels flowed out through VC into the bone marrow, and when the force was removed, it flowed backward into the PDL. This blood transfer acted as an absorber against occlusal force. Our findings suggest that the blood vessels of the gingiva perform an important function in defending against inflammation, while the blood vessels of the PDL play a key role in absorbing occlusal force.     

A quantitative evaluation of the glomerular capillary endothelium in rat and human kidneys: Utilization of vascular perfusion,freeze-cracking of tissue,and scanning electron microscopy

Modern morphological investigation requires the use of a variety of technological approaches and the employment of rigorous morphometric analysis for an adequate evaluation of the structural and ultrastructural features of a tissue or organ. The introduction of the technique of freeze-cracking of tissue to expose new surfaces has made it possible to quantitate the normal surface characteristics of the glomerular capillaries of the mammalian kidney. This report describes the techniques used for the preparation and quantitative assessment of normal glomerular endothelial morphology. The techniques of in vivo and in vitro vascular perfusion of kidneys as a method of fixation and the freeze-cracking of tissue are outlined in detail. In addition, a morphometric analysis of the endothelial surface characteristics are described and values are reported for the control rat and human kidneys from transplant donors.     

Scanning electron microscopic study of immunogold-labeled human leukocytes

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25°C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.     

Identification of nerve fibres in the scanning electron microscope.

The use of silver as a "stain" for nerve fibres in scanning electron microscopy (SEM) preparations has been investigated. Samples of cardiac tissues were treated according to the Bielschovsky silver impregnation method. Following embedding in paraffin, successive sections were selected for light microscopy and for SEM studies, respectively. The silver impregnated fibres, when examined in SEM preparations, appeared nearly white against a greyish background of cardiac tissue. They were therefore easy to localize even at a low magnification. These nerve fibres were identified in the same tissue, but different blocks by means of transmission electron microscopy (TEM) studies and by fluorescence microscopy.     

Effects of steroid hormones on vascular functions

The purpose of this review is to present reported findings of effects of steroids on vascular function and to discuss the biological significance in vascular physiology and pathology. Steroid hormones play various roles in vascular functions through the specific receptor localized in the endothelium and underlying vascular smooth muscle cells (VSMCs). Estrogen replacement therapy in postmenopausal women is in general associated with a reduction in mortality from cardiovascular disease and the beneficial effects of estrogens are caused by modulation of lipid metabolism, expression of leukocyte adhesion molecules, and migration of VSMCs in the vascular wall. Estrogens also play a role as vasodilators through an increment in nitric oxide (NO) production in endothelium or modulation of calcium homeostasis in VSMCs. Some progestin molecules partially oppose the effects of estrogens on vascular functions, but striking differences exist among the types of molecules. Testosterone shows a vasorelaxant response, but detailed actions of androgens on vascular functions remain unknown. Glucocorticoids act on vasoconstriction through reduced prostacyclin production, increased alpha-adrenoceptor numbers, and inhibition of NO synthase, while mineralocorticoids induce hypertrophy and hyperplasia of VSMCs and perivascular fibrosis and cause peripheral vascular resistance, in addition to changes in vascular electrocyte permeability. Some rapid actions of steroids are mediated by "nongenomic" responses in vascular tissues. Recent studies have also demonstrated that steroid-producing or -metabolizing enzymes are expressed in the vascular wall, suggesting the local regulation of steroids in the vascular system. Thus, steroid hormones greatly influence vascular functions and an understanding of the mechanisms may provide new means to prevent vascular diseases.     

Light microscopy evaluation of polyurethane vascular grafts porosity by Sudan Black B staining

In small‐diameter vascular grafts, the porosity of the internal surface plays an important role because it affects initial thrombus deposition and therefore the graft’s patency. As well as many other studies reported in the literature we have carried out a study of the relationship between porosity and the manufacturing parameters of polyurethane (PU) grafts by standard scanning electron microscopy (SEM) analysis. However, SEM was not completely satisfactory for evaluating the ‘sponge‐like’ uptake of water by the graft due to the unavoidable water loss and metal coating during preparation. In fact this preparation produces artefacts of the three‐dimensional porous structure. To avoid this problem we investigated the possibility of observing the graft’s internal surface through a stereomicroscope after it had absorbed water. We looked into a simple staining procedure which preferentially colours the PU graft fibres with respect to the void areas. After testing different kinds of stains, we eventually found that Sudan Black B, which usually stains for all kinds of lipid, turned out to be an excellent stain for the water‐loaded PU grafts when diluted with ethanol. This staining procedure, coupled with a computerized image analysis system, allowed us to evaluate the degree of void and average void size of the graft internal surface and to correlate these data with graft density and manufacturing parameters.     

Study of intussusceptive angiogenesis in inflammatory regional lymph nodes by scanning electron microscopy

The aim of the present study was to verify the occurrence of intussusceptive angiogenesis in blood vessels from submandibular lymph nodes responsible for lymphatic drainage of the tongue. A surgical wound inflicted on the ventral tongue of male Wistar rats and submandibular regional lymph nodes were evaluated at different postoperative periods. Scanning electron microscopy (SEM) was used to observe 123 lymph nodes at times 2, 3, 7, 10, 14, and 21 postoperative days. During the analysis of the vascular models with SEM, intussusceptive angiogenesis was observed in all groups evaluated. This was more extensive on the second and third postoperative days (83.33% and 80%, respectively), representing in these groups the expansion of the vascular chain of lymph nodes. At 21 postoperative days, intussusceptive angiogenesis (42.85%) was suggestive of vascular remodeling. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Three-dimensional fine structure of the biliary tract: Scanning electron microscopy of biliary casts

The three-dimensional structure of the biliary tract was studied by scanning electron microscopy (SEM) of biliary casts. The replica of the biliary tract was successfully prepared by retrograde injection of low viscosity resin into the common bile duct. Bile canaliculi are intricate networks in which hexagonal and pentagonal meshworks are interconnected. Each hexagonal or pentagonal meshwork is on a plane, but adjoining meshworks are on different planes. Bile canalicular networks connect with bile ductules at the periphery of the portal tract. The intrahepatic bile duct showed considerable interspecies variation. The human bile duct has plexiform side branches and periductal sacculi, which are most numerous near the liver hilum and fewest in the smaller portal tracts. The hilar plexus and sacculi are present on opposite sides of the bile duct. The plexus formed at the bifurcation of the bile ducts exhibits a plane. Periductal sacculi were also observed in the monkey and pig bile ducts, particularly the latter, while rat bile ducts possess a peculiar portal bile ductular plexus situated between the portal tract and the surrounding liver parenchyma. No such structures were observed in either the dog or rabbit bile ducts. SEM of the biliary casts showed that the biliary tract was not a simple draining tube but had additional structures, such as periductal sacculi and plexiform side branches. These structures, together with the peribiliary vascular plexus, may be implicated in the modification of bile.