Corrosion casting of the cardiovascular structure in adult zebrafish for analysis by scanning electron microscopy and X-ray microtomography.

Zebrafish have come to the forefront as a flexible, relevant animal model to study human disease, including cardiovascular disorders. Zebrafish are optically transparent during early developmental stages, enabling unparalleled imaging modalities to examine cardiovascular structure and function in vivo and ex vivo. At later stages, however, the options for systematic cardiovascular phenotyping are more limited. To visualise the complete vascular tree of adult zebrafish, we have optimised a vascular corrosion casting method. We present several improvements to the technique leading to increased reproducibility and accuracy. We designed a customised support system and used a combination of the commercially available Mercox II methyl methacrylate with the Batson's catalyst for optimal vascular corrosion casting of zebrafish. We also highlight different imaging approaches, with a focus on scanning electron microscopy (SEM) and X-ray microtomography (micro-CT) to obtain highly detailed, faithful three-dimensional reconstructed images of the zebrafish cardiovascular structure. This procedure can be of great value to a wide range of research lines related to cardiovascular biology in small specimens.

Constant morphological patterns in the hemolymph vascular system of crayfish (Crustacea, Decapoda).

We present a study of the hemolymph vascular system of the marbled crayfish, Procambarus fallax f. virginalis, the only crayfish species known to be parthenogenetic. To identify potential evolutionary patterns, we compared data from a total of 48 specimens of P. fallax with 22 specimens of Orconectes limosus. Visualizations (2D and 3D) were carried out using a combination of classical and modern morphological techniques. Our data were compared to the existing literature. Like all Decapoda, both P. fallax and O. limosus have a hemolymph vascular system, consisting of a globular heart with seven off-branching arteries. We were able to visualize in detail the heart of crayfish for the first time, i.e., the myocard with its clusters of muscles running through the lumen of the heart, the valves and flaps of ostia and arteries. Furthermore, the branching patterns of the seven artery systems were analyzed. Anatomical structures identified to be consistent in all specimens of both species were combined as ground pattern of hemolymph vascular system features for Astacida.

Automatic recognition of fundamental tissues on histology images of the human cardiovascular system.

Cardiovascular disease is the leading cause of death worldwide. Therefore, techniques for improving diagnosis and treatment in this field have become key areas for research. In particular, approaches for tissue image processing may support education system and medical practice. In this paper, an approach to automatic recognition and classification of fundamental tissues, using morphological information is presented. Taking a 40× or 10× histological image as input, three clusters are created with the k-means algorithm using a structural tensor and the red and the green channels. Loose connective tissue, light regions and cell nuclei are recognised on 40× images. Then, the cell nuclei's features - shape and spatial projection - and light regions are used to recognise and classify epithelial cells and tissue into flat, cubic and cylindrical. In a similar way, light regions, loose connective and muscle tissues are recognised on 10× images. Finally, the tissue's function and composition are used to refine muscle tissue recognition. Experimental validation is then carried out by histologist following expert criteria, along with manually annotated images that are used as a ground-truth. The results revealed that the proposed approach classified the fundamental tissues in a similar way to the conventional method employed by histologists. The proposed automatic recognition approach provides for epithelial tissues a sensitivity of 0.79 for cubic, 0.85 for cylindrical and 0.91 for flat. Furthermore, the experts gave our method an average score of 4.85 out of 5 in the recognition of loose connective tissue and 4.82 out of 5 for muscle tissue recognition.

Costameric proteins: from benchside to future translational cardiovascular research.

Costameres encircle the myocyte perpendicular to its long axis, and comprise two protein complexes: the dystrophin-glycoprotein complex (DGC) and the vinculin-talin-integrin system. They participate in signaling functions and protect muscle cells from damage induced by workload. The behaviour of those proteins has been a focus of study starting from skeletal and smooth muscle cells to cardiomyocytes, and still represents a topical subject for cardiovascular translational research. This review summarizes the past and present novel approaches of our and other groups of work on this subject of research.

Comparative morphology of the hemolymph vascular system in krill (Euphausiacea; Crustacea).

The phylogenetic position of Euphausiacea within Malacostraca is still under debate. Either they are seen as sister group to a taxon comprising Pancarida and Peracarida or closer related to Decapoda. Both hypotheses can be supported by characters of the circulatory system. Therefore, a comparative re-evaluation of the circulatory system seems to be feasible. Here we present the first three-dimensional data of the circulatory system of three euphausiacean species based on semi-thin sections and micro computer tomography in combination with corrosion casting. We were also able to study for the first time representatives of Bentheuphausia amblyops, the suggested sister taxon to all other euphausiaceans. The main pumping structure in the open circulatory system of Euphausiacea is the globular heart in the rear thoracic segments. From the anterior and posterior end of the heart two unpaired and four pairs of arteries emanate. The unpaired anterior aorta runs below the carapace from the anterior part of the heart into the anterior cephalothorax, where it supplies the first antennae, the brain and the eyes. The paired posterior aortae run into the pleon supplying the pleopods, uropods and the telson. The four pairs of cardiac arteries supply appendages in the cephalic region and viscera in the trunk. The unpaired descending artery connects to the subneural vessel supplying the thoracopods. A myoarterial formation of the anterior aorta is described in Bentheuphausia amblyops. The observed pattern of a globular heart situated in the posterior cephalothorax and comprising a meshwork of muscular strands (also running through its lumen) is highly likely to be homologous in Euphausiacea and Decapoda. The data are compared with the scarce literature present to date.

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis.

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

The circulatory system in Mysidacea--implications for the phylogenetic position of Lophogastrida and Mysida (Malacostraca, Crustacea).

The morphology of the circulatory organs in Mysida and Lophogastrida (traditionally combined as Mysidacea) is revisited investigating species so far unstudied. In addition to classical morphological methods, a newly developed combination of corrosion casting with micro computer tomography (MicroCT) and computer aided 3D reconstructions is used. Lophogastrida and Mysida show a highly developed arterial system. The tubular heart extends through the greater part of the thorax and is connected with the ventral vessel via an unpaired descending artery. It is suggested that a distinct ostia pattern supports the monophyly of Mysidacea. The cardiac artery system is more complex in Lophogastrida than in Mysida, consisting of up to 10 pairs of arteries that supply the viscera. In both taxa, an anterior and posterior aorta leads off the heart. In the anterior part of the cephalothorax the anterior aorta forms dilations into which muscles are internalized; these structures are called myoarterial formations. One of these myoarterial formations can also be found in all the other peracarid taxa but not in other Malacostraca.

Cooperation between VEGF and beta3 integrin during cardiac vascular development.

In the developing myocardium, vascular endothelial growth factor (VEGF)-dependent neovascularization occurs by division of existing vessels, a process that persists for several weeks following birth. During this remodeling phase, mRNA expression of beta3 integrin in the heart decreases significantly as vessel maturation progresses. However, in male mice lacking beta3, coronary capillaries fail to mature and continue to exhibit irregular endothelial thickness, endothelial protrusions into the lumen, and expanded cytoplasmic vacuoles. Surprisingly, this phenotype was not seen in female beta3-null mice. Enhanced VEGF signaling contributes to the beta3-null phenotype, because these vessels can be normalized by inhibitors of VEGF or Flk-1. Moreover, intravenous injection of VEGF induces a similar angiogenic phenotype in hearts of adult wild-type mice. These findings show a clear vascular phenotype in the hearts of mice lacking beta3 and suggest this integrin plays a critical role in coronary vascular development and the vascular response to VEGF.

Basement membrane heterogeneity during chick development as shown by tomato (Lycopersicon esculentum) lectin binding.

Basement membranes (BMs) constitute a distinct compartment of the extracellular matrix (ECM). All BMs show a similar structural appearance but differ in molecular composition. These variations have critical functional implications. The aim of this study is to establish the pattern of the tomato lectin (Lycopersicon esculentum agglutinin--LEA) binding sites in the BMs of the developing chick embryo (stages 4-21, Hamburger and Hamilton, 1951) in order to achieve a better understanding of the molecular heterogeneity of BMs. The study was performed with transmission electron microscopy (TEM) histochemistry, and confocal laser microscopy. TEM showed that LEA bound to the lamina densa and to the lamina fibroreticularis of the BMs. Through the period studied, most of the LEA binding appeared in the ectodermal BM and its derivatives. In the limb bud, LEA binding to the ectoderm BM was more intense in the ventral half than in the dorsal half. Furthermore, LEA allowed the early (HH16) detection of the transverse fibrillar tracts. In the lens and in the inner ear primordium, the BMs were LEA positive through the placode and cup stages. The binding was progressively reduced through the vesicle stage. The BMs of the olfactory primordium, and of the Rathke's pouch were positive. In contrast, the BMs of the developing central nervous system were negative. The BMs of both the paraxial and the lateral plates of the mesoderm were negative, whereas the notochord and the BM of the Wolffian duct were positive. The endodermal BM and its derivatives were negative. The ECM located between the fusing endocardial tubes, and the BM of the fusion zone of the paired aortae, were positive. This suggested an active role of the LEA-positive glycoproteins in the fusion of endothelia. Our results show the heterogeneity of the chick embryo BMs during development. In addition, LEA constitutes an excellent marker for the primordial germ cells.

The decapod crustacean circulatory system: a case that is neither open nor closed.

Historically, the decapod crustacean circulatory system has been classed as open. However, recent work on the blue crab, Callinectes sapidus, suggests the circulatory system may be more complex than previously described. Corrosion casting techniques were refined and used to map the circulatory system of a variety of crab species (order: Decapoda; family: Cancridae) to determine if the complexity observed in the blue crab was present in other species. Seven arteries arose from the single chambered heart. The anterior aorta, the paired anterolateral arteries, and the paired hepatic arteries exited from the anterior aspect of the heart. The small-diameter posterior aorta exited posteriorly from the heart. Exiting from the ventral surface of the heart, the sternal artery branched to supply the legs and mouthparts of the crab. These arteries were more complex than previously described, with arterioles perfusing all areas of the body. The arterioles split into fine capillary-like vessels. Most of these capillaries were blind ending. However, in several areas (antennal gland, supraesophageal ganglion) complete capillary beds were present. After passing through the capillary-like vessels, blood drained into a series of sinuses. However, rather than being arbitrary spaces as previously described, scanning electron micrographs showed the sinuses to be distinct units. Most of the sinuses formed a series of flattened membrane-bound lacunae. This complexity may qualify the decapod crustacean circulatory system as one that is "partially closed" rather than open.

Extracutaneous ultrastructural alterations in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is caused by mutations in the ABCC6 gene, encoding for the membrane transporter MRP6, whose physiological role is still unknown. PXE is characterized by skin, eye, and cardiovascular alterations mainly due to mineralization of elastic fibers. The ultrastructural alterations of a large number of tissues obtained at autopsy from 2 PXE patients were analyzed and compared to clarify the involvement of the various organs in PXE and to identify cell types responsible for clinical manifestations. Ultrastructural alterations typical of PXE were present in all organs examined and consisted mostly of fragmentation and mineralization of a number of elastic fibers, abnormalities of collagen fibril shape and size, and, less frequently, deposition of aggregates of matrix constituents in the extracellular space. The severity of alterations was more pronounced in the organs affected by the clinical manifestations of PXE. Interestingly, veins and arteries were similarly damaged, the adventitia and the perivascular connective tissue being the most affected areas. Therefore, alterations in PXE are systemic and affect all soft connective tissues, even in the absence of specific clinical manifestations. The localization of alterations suggests that fibroblasts and/or smooth muscle cells are very likely involved in the pathogenesis of the disorder. These findings may help in the diagnosis of PXE when clinical manifestations affect internal organs.

Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R.

As you like it, Part 3: A critique and historical review of calcification as seen with the electron microscope.

As You Like It, Part 3, is a continuation of the lectures given by the author (Ultra Path VIII and Ultra Path IX). It is a critical historical review of topics of interest to electron microscopists, attempting to show what went wrong and perhaps also why. The topics chosen this time demonstrate the prominent role electron microscopy has played in elucidating the diverse ways in which calcification can occur. The classic concept of dystropic and metastatic calcification is now inadequate to explain all observed phenomena. The electron microscope shows that calcification occurs in many different intracellular and extracellular sites and that each has its own morphology and etiology. Thus, a new classification based on ultrastructural morphology is born, but few seem to be aware of it. The author examines the ubiquitous but not too well-known phenomenon of cell remodeling, which often, but not always, leads to calcification. Topics are presented under the following headings: (1) matrical lipidic debris, (2) spherical microparticles, (3) matrix vesicles, (4) intramitochondrial calcification, (5) intralysosomal calcification, (6) calcification of collagen, (7) calcification of elastic fibers, and (8) calcification of secretory products.

Study of the effects of aging on macromolecular synthesis in various organ systems using microscopic radioautography.

In order to elucidate the effects of aging on macromolecular synthesis such as DNA, RNA, proteins, glucides and lipids in various organ systems of experimental animals and humans, systematic studies using light and electron microscopic radioautography in various organ systems including skeletal, muscular, digestive, respiratory, urinary, reproductive, endocrine, circulatory, nervous and sensory systems were studied after incorporation with macromolecular precursors. The experimental animals used were mainly ddY strain mice from embryo to postnatal days 1 and 3, weeks 1 and 2 or months 1, 2 and 6 months up to 1 and 2 years senescent stages. Animals were injected with [3H]-thymidine for DNA, [3H]-uridine for RNA, [3H]-amino acids for proteins, [3H]-glucose, [3H]-glucosamine and [35S]O4 for glucides, [3H]-glycerol for lipids and some low molecular target tracers such as hormones, inorganic substances and drugs. Results demonstrate that these precursors when incorporated into various cell types in various organs showed specific patterns of macromolecular synthesis as observed in perinatal to juvenile, mature and senescent stages. These effects of aging could answer some of the questions as to how but not why we get old.

Changes in cardiac electrophysiology, morphology, tissue biochemistry and vascular reactions in glutathione depleted animals.

The effects of acute and chronic glutathione depletion (single i.p. injection of 3 mmol/kg L-buthionine-S,R-sulphoximine and 2 mmol/kg for 4 days) on heart action potential (AP) characteristics, electronmicroscopy, cytochemistry and biochemistry and vascular contractility and nitric oxide-mediated relaxation were studied in rats and guinea pigs. In guinea pig cardiac preparations both acute and chronic glutathione depletion caused a significant decrease of maximum rate of rise of depolarization phase and duration of action potential AP(APD) at 25, 50, and 90% of repolarization but did not modify the other AP parameters. The contractile responses of helically cut aortic strips to norepinephrine were not altered by chronic glutathione depletion but the relaxing responses of precontracted preparations to acetylcholine were significantly reduced both in rats and guinea pigs. Morphologically there were indications of permeability changes, intracellular and interstitial edema and myofilament damage in the myocardium. There was also a decrease in cytochromoxydase and succinyl dehydrogenase activities both in rats and guinea pigs. The present data suggest that glutathione depletion may influence the Na+ and K+ channel activities, causes morphological and biochemical changes in cardiac preparations and may interfere with nitric oxide generation or its action in aortic strips.

Cardiorespiratory and renal responses to arterial chemoreceptor stimulation in early hypertension.

The peripheral arterial chemoreceptors (PAC) modify not only cardiorespiratory but also renal hemodynamic and excretory function. There is evidence that in hypertensive animals and humans the reflectoric actions of the PAC on ventilation and circulation differ from those of normotensive subjects. However, the influence of these receptors on kidney function of hypertensive subjects is poorly understood. Cardiorespiratory and renal responses to pharmacological stimulation of PAC by almitrine bismesylate during normoxia were measured in 16 normotensive (NT) and 13 age-matched borderline-hypertensive young men (BHT) undergoing water diuresis. Placebo experiments served as time controls in each subject. NT reacted to almitrine with significant rises in heart rate, minute ventilation, and filtration fraction. Renal vascular resistance tended to increase slightly. In BHT the drug caused a significant rise in heart rate and minute ventilation too, however, this reaction had a longer latency when compared to NT. In contrast to NT, filtration fraction, and renal vascular resistance decreased. Renal fractional sodium and lithium excretion did not show any clear response to almitrine in NT, but decreased in BHT. The results suggest that the weaker ventilatory response in BHT vs. NT might indicate a lower reactivity of their PAC to almitrine. The different reactions of the renal vascular bed to pharmacological chemoreceptor excitation in mild hypertensives might result from a different reactivity of the renal arterioles, whereas the enhanced proximal tubular sodium reabsorption could be due to an exaggerated increase in efferent renal nerve activity.

Long-term animal feeding trial of the refined konjac meal. II. Effects of the refined konjac meal on the aging of the brain, liver, and cardiovascular tissue cells in rats.

Rats of both sexes were fed on a basal feed containing 1% refined konjac meal (RKM) for 18 months and the effects of RKM on the cell aging were observed. A comparable group fed on the basic feed was used as the control. Results obtained demonstrate that the long-term feeding of RKM to rats can delay the course of cell aging of the gliocyte, cadiomyocyte, and the endothelial cell of the large and medium arteriases, hence it is likely to delay the occurrence of arteriosclerosis and improve the functions of the brain, heart and vascular system.

Cardiovascular and behavioral responses to nicotinic agents administered intrathecally.

We have examined the role of the spinal nicotinic receptors in mediating cardiovascular and behavioral responses in conscious rats. Intrathecal administration of nicotinic agonists to the lumbosacral region of the spinal cord caused a dose-dependent increase in systolic blood pressure, heart rate and a nociceptive (behavioral) response. The order of potencies for the pressor response was l-nicotine > or = cytisine > N-methylcarbamylcholine > or = dimethylphenylpiperazinium > d-nicotine. However, cytisine was the most potent in producing the heart rate increase and nociceptive response. Unlike the other agonists, cytisine also exhibited marked desensitization of the three responses upon repeated administration. The effects of nicotine were antagonized in a dose-dependent fashion by mecamylamine, hexamethonium, alpha-lobeline, dihydro-beta-erythroidine and methyllycaconitine. By contrast, cytisine-induced responses were blocked effectively by mecamylamine and methyllycaconitine, but not by alpha-lobeline or dihydro-beta-erythroidine. However, when alpha-lobeline or dihydro-beta-erythroidine antagonism of the pressor response to cytisine was monitored during the initial minute following intrathecal administration, both antagonists significantly inhibited the response. The competitive ganglionic blocker, trimethaphan, or the elapid alpha-toxin, alpha-bungarotoxin, when administered intrathecally, had no effect on nicotine- or cytisine-elicited responses. The cardiovascular responses to intrathecal nicotine and cytisine have two components. The first is likely mediated through direct sympathetic output and desensitizes rapidly to cytisine, the second is coupled indirectly to the nociceptive response and shows a diminished capacity for rapid desensitization. Agonist and antagonist specificities indicate that the spinal nicotinic receptor differs from those in ganglia and those characterized in brain to date. Although antagonist specificity of the blockade of nicotine and cytisine elicited responses differ, this may be due to the unique desensitization capacity of cytisine rather than an action mediated by distinct receptor subtypes.

Circulatory pattern and structure in the tail and tail fins of Xenopus laevis tadpoles.

This investigation was initiated with the intent of study capillary sprouting in the tadpole tail fin microcirculation of the African clawed frog, Xenopus laevis, using a combination of intravital video recordings and electron microscopy. The tadpoles were observed daily for periods up to one hour during one to two weeks. The capillary sprouts originated mostly from postcapillary venules, and within 24-48 h merged with other capillary sprouts, subsequently establishing a capillary loop with blood flow. As the circulatory patterns developed further, capillary regressions also occurred. As the electron microscope analyses of the capillary sprouts progressed, it became obvious that a thorough electron microscope study of the blood vessels of the tadpoles was required in order to explore structural characteristics of arterial and venous blood vessels. Thus, this article deals primarily with the general organization of the tadpole tail circulation, and the ultrastructural characteristics of the vascular walls. A subsequent article will deal with the role of endothelial cells, fibroblasts and pericytes in the process of capillary sprouting and regression, based on intravital recordings and electron microscope analyses.