Microvascularization of the spleen in larval and adult Xenopus laevis: Histomorphology and scanning electron microscopy of vascular corrosion casts

Microvascular anatomy and histomorphology of larval and adult spleens of the Clawed Toad, Xenopus laevis were studied by light microscopy of paraplast embedded serial tissue sections and scanning electron microscopy (SEM) of vascular corrosion casts (VCCs). Histology showed i) that white and red pulp are present at the onset of metamorphic climax (stage 57) and ii) that splenic vessels penetrated deeply into the splenic parenchyma at the height of metamorphic climax (stage 64). Scanning electron microscopy of VCCs demonstrated gross arterial supply and venous drainage, splenic microvascular patterns as well as the structure of the interstitial (extravasal) spaces representing the “open circulation routes.” These spaces identified themselves as interconnected resin masses of two distinct forms, namely “broccoli‐shaped” forms and highly interconnected small resin structures. Arterial and venous trees were clearly identified, as were transitions from capillaries to interstitial spaces and from interstitial spaces to pulp venules. Venous sinuses were not diagnosed (nonsinusal spleen). The splenic circulation in Xenopus laevis is “open.” It is hypothesized that red blood cells circulate via splenic artery, central arteries, penicillar arteries, and red pulp capillaries primarily via “broccoli‐shaped” interstitial spaces, pulp venules and veins into subcapsular veins to splenic veins while lymphocytes circulate also via the interstitial spaces represented by the highly interconnected small resin structures in vascular corrosion casts. In physiological terms, the former most likely represent the fast route for blood circulation, while the latter represent the slow route. J. Morphol. 277:1559–1569, 2016. © 2016 Wiley Periodicals, Inc.     

Scanning electron microscopy of pulmonary vascular endothelium in rats with hypoxia-induced hypertension

Scanning electron microscopy was used to study the endothelial surface of the pulmonary trunk, artery, and vein in normobaric control rats as well as in rats exposed to hypobaric hypoxia for 7 and 21 days. The individual endothelial cells of the normobaric pulmonary trunk and hilar artery were flat and slightly elongated with elevated nuclear regions, and those of the intermediate-sized artery were more elongated and had more microvilli than the large arteries studied. Their endothelial cell boundaries were outlined by beaded cytoplasmic projections. The surfaces of the normobaric hilar and intermediate-sized veins were smooth and demonstrated numerous longitudinal streaks. These venous endothelial cells were elongated and their cell boundaries were outlined by low discontinuous marginal folds. Exposure to hypobaric hypoxia caused the following changes on the arterial surface: elevation of the endothelial cells; formation of microvilli-rich cell clusters; formation of hollow defects; and the attachment of leukocytes. Hypobaric hypoxia also caused the disappearance of the longitudinal streaks and the occurrence of microvilli-rich cells in the hilar veins. The endothelial surface modifications in the hypobaric rats could be related to thickening of the endothelium, intimal edema, increased intimal connective tissue, luminal invasion of leukocytes, and increased endothelial cell proliferation, known to occur in systemic arteries of hypertensive animals.     

Specialized veins in the submucosa of the equine ileocecal junction.

Spirally arranged bundles of sub-endothelial smooth muscle enfold the small to medium-sized submucosal veins in the equine ileocecal junction. The muscle bundles, accompanied by the endothelial lining, bulge into the lumen of the vessels, partly occluding the latter. Transmission electron microscopy of the muscle cells reveals features consistent with vascular smooth muscle ultrastructure. It is proposed that the throttling effect of the muscle bundles causes engorgement of the submucosal venous plexus, which then assists in the closing of the ileocecal orifice.     

Three-dimensional organisation of the vasculature of the rat spermatic cord and testis

Summary The vascular architecture of the rat testis and spermatic cord was studied by a corrosion cast technique combined with scanning and transmission electron microscopy, and light microscopy. The casts preserve the endothelial impressions of the vessels and enable them to be differentiated into the various vascular components. Frequent arterio-arterial anastomotic arcades and occasional arterio-venous anastomotic channels are seen. A well defined hexagonal pattern of intertubular and peritubular vessels surround the seminiferous tubules. Prominent large endothelial nuclei protrude into the arterial lumina at branching sites, but their functional significance is not known. The outermost vascular layer of the testis consists of large veins, venules, and capillaries, but lacks any arterial branches; it also contains loosely arranged veno-venous anastomotic networks. We have named this vascular layer the sub-albugineal venous plexus. The testicular artery increases in luminal diameter as it approaches the testis. The periarterial capillary plexus, which lies between the pampiniform plexus and the testicular artery, is drained by two types of venules.     

Ultrastructural organization of the endotheliocytes of iliac lymph node capillaries in pregnancy (morphometric study)

Using light and electron microscopy and morphometry, the morphological changes in the lymph nodes of arterial and venous parts of capillaries were studied on the 11th, 17th and 21st days of pregnancy in rats. Ultrastructural changes in endothelial cells of blood vessels in the uterine lymph nodes during normal pregnancy are of adaptive nature and are possibly responsible for the relief of the blood congestion in the system of the inferior vena cava and for the improvement of the utero-placental circulation.     

Arterial versus venous endothelial cells

Vascular endothelial cells (ECs) form the inner lining of all blood vessels from the largest artery and veins, viz., the aorta and venae cavae, respectively, to the capillaries that connect the arterial and venous systems. Because these two major conducting systems of the cardiovasculature differ functionally, it is not surprising that the physical makeup of arteries and veins, including the ECs that line their lumina, are also distinct. Although few would argue that the local environment contributes to the differences between arteries and veins, recent evidence has shown that the specification of arterial and venous identity is largely genetically determined. The authors are supported by NIH EY05318 and EY015435 (P.A.D.). Dr. dela Paz is supported by NRSA Institutional Research Training Grant T32 HL076115. Dr. D’Amore is a Research to Prevent Blindness Senior Scientific Investigator.     

Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization

The transmembrane ligand ephrinB2 and its receptor tyrosine kinase EphB4 are molecular markers of embryonic arterial and venous endothelial cells, respectively, and are essential for angiogenesis. Here we show that expression of ephrinB2 persists in adult arteries where it extends into some of the smallest diameter microvessels, challenging the classical view that capillaries have neither arterial nor venous identity. EphrinB2 also identifies arterial microvessels in several settings of adult neovascularization, including tumor angiogenesis, contravening the dogma that tumor vessels arise exclusively from postcapillary venules. Unexpectedly, expression of ephrinB2 also defines a stable genetic difference between arterial and venous vascular smooth muscle cells. These observations argue for revisions of classical concepts of capillary identity and the topography of neovascularization. They also imply that ephrinB2 may be functionally important in neovascularization and in arterial smooth muscle, as well as in embryonic angiogenesis.     

Microvascularization of the thyroid glands in larval and adult Xenopus laevis‐ histomorphology and scanning electron microscopy of vascular corrosion casts

This study demonstrates gross arterial supply, venous drainage and microvascular patterns of larval and adult thyroid glands in the African Clawed Frog, Xenopus laevis by scanning electron microscopy of vascular corrosion casts and light microscopy of stained serial tissue sections. Results confirm published findings gained by microscopical dissections with respect to gross arterial supply. However, in adult frogs one rather than two thyroid veins are found. This study reveals for the first time that bilaterally located thyroid glands in premetamorphosis have immature capillary networks, lack a clear hierarchy of blood vessels, and show many signs of sprouting angiogenesis. During metamorphic climax, blood vessels gain a clear hierarchy and capillaries form closed networks around thyroid follicles. From climax onwards, non‐sprouting angiogenesis (intussusceptive microvascular growth) becomes the prevailing mode of angiogenesis intensifying follicle capillarization. Due to narrow interfollicular spaces, thyroidal arterioles remain superficial while draining venules are located interfollicularly. In contrast with the mammalian thyroid gland where most thyroid follicles have their own capillary beds, most thyroid follicles in Xenopus share their capillary beds with neighbouring follicles. Consequently, the concept of individual morphological and functional angiofollicular units applicable to adult mammalian and human thyroid glands is not applicable to larval and adult amphibian thyroid glands.     

Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.     

Artery and vein size is balanced by Notch and ephrin B2/EphB4 during angiogenesis

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. Here, we demonstrate that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-of-function mutations show small aortae and large cardinal veins. Notch does not affect the overall number of endothelial cells but balances the proportion of arterial to venous endothelial cells, thereby modulating the relative sizes of both vessel types. Loss of ephrin B2 or its receptor EphB4 also leads to enlarged aortae and underdeveloped cardinal veins; however, endothelial cells with venous identity are mislocalized in the aorta, suggesting that ephrin B2/EphB4 signaling functions distinctly from Notch by sorting arterial and venous endothelial cells into their respective vessels. Our findings provide mechanistic insight into the processes underlying artery and vein size equilibration during angiogenesis.     

Morphology and tyrosine-hydroxylase immunohistochemistry of the systemic secondary vessel system of the blue catfish,Arius graeffei

Fish have a secondary vessel system which emerges from the primary vasculature via large numbers of coiled origins. The precise role of this vessel system is unknown. Vascular casting techniques and scanning electron microscopy reveal that the secondary vessels of the blue catfish, Arius graeffei, originate from dorsal, lateral, and ventral segmental primary arteries and from the caudal dorsal aorta. These vessels anastomose with each other to form larger secondary arteries which parallel the primary vessels for their entire length. Secondary vessels do not appear to form a capillary bed in the skin in A. graeffei as they do in some fish species. Coiled secondary vessel origins are abundant within the tunica media and adventitia of the primary vessels from which they emerge. The origins of the secondary vessels are surrounded by the extensive cytoplasmic processes of specialized endothelial cells. These processes extend for up to 6 μm into the lumen of the primary vessel. Ultrastructurally the coiled secondary capillaries consist of an endothelial cell tube which is surrounded by a single layer of pericytes. These endothelial cells extend large numbers of microvilli into the lumen of the coiled secondary capillary. Nerve terminals are commonly associated with the coiled secondary capillaries. Immunohistochemistry has revealed the presence of tyrosine-hydroxylase, an enzyme involved in catecholamine synthesis in nerve varicosities close to secondary vessels in A. graeffei. This vessel system could therefore be regulated by adrenergic nerves. © 1996 Wiley-Liss, Inc.     

Morphology and vascular anatomy of the accessory respiratory organs of the air-breathing climbing perch, Anabas testudineus (Bloch)

The vascular organization and endothelial cell specialization of the air-breathing organs of Anabas testudineus were examined by light and scanning electron microscopy of fixed tissue and vascular corrosion replicas. The vessels supplying blood to the lining of paired suprabranchial chambers and the plicated labyrinthine organs within the chambers are tripartite, having a median artery and paired, lateral veins. Hundreds of respiratory islets, the functional units of gas exchange, cover the surfaces of both the chamber and labyrinthine organ. A median islet artery supplies the central aspect of each islet and gives rise to numerous short arterioles from which the transverse channels are formed. Transverse channels are parallel capillary-sized vessels that extend in two rows away from the medial arterioles and drain laterally into one of two lateral islet veins. Basally situated single rows of endothelial cells lining the transverse channels form thick, evaginated, tongue-like cytoplasmic processes that project freely into the lumen from the tissue side of the channel. Other thin, septate, cytoplasmic extensions of the same cells form valve-like septa that extend across the channel. Both the septa and tongue-like processes appear to direct the red blood cells to the epithelial side of the channel and thus decrease the diffusion distance between the air and red cell. A large sinusoidal space lies under the transverse channels and may support the channels and even elevate them during increased oxygen demand. The epithelium covering the transverse channels is smooth, which enhances air convection and minimizes unstirred layer effects. The epithelium between the channels contains microvilli that may serve to trap bacteria or particulates and to humidify the air chambers.     

Extra-alveolar veins are contiguous with, and leak fluid into, periarterial cuffs in rabbit lungs.

We previously observed physiological evidence that arterial and venous extra-alveolar vessels shared a common interstitial space. The purpose of the present investigation was to determine the site of this continuity to improve our understanding of interstitial fluid movement in the lung. Orange G and Evans blue dyes were added to the arterial and venous reservoirs, respectively, of excised rabbit lungs as they were placed 20 cmH2O into zone 1 (pulmonary arterial and venous pressures = 5 cmH2O, alveolar pressure = 25 cmH2O). After 10 s or 4 h the lungs were fixed by immersion in liquid N2, freeze-dried, cut into 5-mm serial slices, and examined by light macroscopy. Serial sections of 0.25-0.5 mm were subsequently examined by scanning electron microscopy. In the animals subjected to the zone 1 stress for 4 h, arterial and venous extra-alveolar vessels were surrounded by cuffs of edema. The edema ratio (cuff area divided by vessel lumen area) was greater around arteries than veins and decreased with increasing vessel size. Periarterial cuffs usually contained orange dye and frequently contained both orange and blue dye. Lymphatics containing orange or blue dye were frequently seen in periarterial cuffs. Scanning electron microscopy demonstrated that extra-alveolar veins of approximately 100 microns diameter were anatomically contiguous with arterial extra-alveolar vessel cuffs. In rabbit lungs, both arterial and venous extra-alveolar vessels (and/or alveolar corner vessels) leak fluid into perivascular cuffs surrounding arterial extra-alveolar vessels, and lymphatics located in the periarterial cuff contain fluid that originates from both the arterial and venous extra-alveolar vessels.     

Early stages of trophoblastic invasion of the maternal vascular system during implantation in the macaque and baboon.

Trophoblastic invasion and remodeling of the uteroplacental (spiral) arteries in primates are well-documented, but virally nothing is known of the early stages of these phenomena. Therefore, we examined invasion of the maternal vasculature in macaques and baboons at, and immediately following, implantation. Following penetration of the uterine epithelium (day 9), trophoblast spreads along the residual epithelial basal lamina. By day 10, cytoplasmic processes penetrate the epithelial and endothelial basal laminae, and syncytial trophoblast insinuates itself between maternal endothelial cells. As lacunae develop, both syncytial and cytotrophoblast are exposed to maternal blood. Endovascular cytotrophoblast was first observed in subepithelial dilated capillaries and venules. These vessels are lined by increasingly hypertrophied endothelial cells. The spiral arterioles are unmodified at this time. Particularly interesting was the observation that there is rapid extensive endovascular trophoblast invasion of the spiral arterioles immediately beneath the implantation site. By day 14-16 nearly all of the small arterioles directly beneath the site are completely occluded. There is no invasion of the veins in this region. Somewhat later, the deeper arterioles in the principal zone are invaded. Rather than a continuous stream of cells invading the deeper arterioles, these endovascular cells occur in clusters ranging from a few cells to groups of cells that completely plug the lumen. Our results indicate that trophoblastic invasion of maternal vessels occurs very early; and, at least initially, trophoblast can migrate between and along endothelial cells without causing their lysis. The endovascular cells eventually interrupt the endothelial lining of the arterioles and penetrate the walls of the vessels. The occlusion of arterioles underneath the site suggests that circulation through the lacunae at this stage is indirect. Corresponding stages of human development were examined, and no invasion of arterioles could be observed prior to formation of an extensive cytotrophoblastic shell.     

The formation of capillary-like tubes by calf aortic endothelial cells grown in vitro

Cloned, large vessel endothelial cells derived from fetal bovine and bovine calf aortas formed three-dimensional structures in vitro without tumor-conditioned medium or special substrata. Transmission electron microscopy showed the structures to be hollow tubes composed of typical endothelial cells with overlapping and interdigitating cytoplasmic processes typical of those seen in in vivo capillaries. The putative lumen of these tubes generally contained abundant electron-dense fibrous material, which by ruthenium red and indirect immunofluorescent staining appeared to be extracellular matrix. This suggests that the endothelial cell orientation in the tubes is the reverse of that normally found in in vivo vessels.     

Choose your fate: artery, vein or lymphatic vessel?

The specification of cell fate is integral to embryonic development. Recent research has identified several molecules that are involved in the development of the embryonic vasculature. Their combined actions are required for the specification and development of the arteries, veins and lymphatic vessels; vascular networks that are vital for embryonic and adult survival, and whose malfunction causes major pathological disorders. Recent discoveries have impacted our understanding of the embryonic origins of arterial, venous and lymphatic endothelial cells and the signals that mediate their navigation into mature, functional circulatory systems.     

Chorioallantoic placenta formation in the rat: II. Angiogenesis and maternal blood circulation in the mesometrial region of the implantation chamber prior to placenta formation.

Rat gestation sites were examined on days 7 through 9 of pregnancy by light microscopy and transmission and scanning electron microscopy to determine the extent of vascular modifications in the vicinity of the mesometrial part of the implantation chamber (mesometrial chamber). At a later time, the mesometrial chamber is, in conjunction with the uterine lumen, the site of chorioallantoic placenta formation. On day 7, in the vicinity of the mesometrial chamber, vessels derived from a subepithelial capillary plexus and venules draining the plexus were dilating. By early day 8, this network of thin-walled dilated vessels (sinusoids) was further enlarged and consisted primarily of hypertrophied endothelial cells with indistinct basal laminas. Sinusoids were frequently close to the mesometrial chamber's luminal surface which was devoid of epithelial cells but was lined by decidual cell processes and extracellular matrix. By late day 8, cytoplasmic projections of endothelial cells extended between healthy-appearing decidual cells and out onto the mesometrial chamber's luminal surface, and endothelial cells were sometimes found on the luminal surface indicating that endothelial cells were migrating. The presence of maternal blood cells in the mesometrial chamber lumen suggested that there was continuity between the chamber and blood-vessel lumens. On day 9, the mesometrial chamber was completely lined with hypertrophied endothelial cells, and sinusoid lumens were clearly continuous with the lumen of the mesometrial chamber. Mesometrial sinusoids and possibly the mesometrial chamber lumen were continuous with vessels in vicinity of the uterine lumen that were fed by mesometrial arterial vessels. Clearing of the mesometrial chamber lumen during perfusion fixation via the maternal vasculature indicated the patency of this luminal space and its confluence with mesometrial arterial vessels and sinusoids. The conceptus occupied an antimesometrial position in the implantation chamber on days 7 through 9, and it was not in direct contact with uterine tissues in the vicinity of the mesometrial chamber. These observations suggest that angiogenesis, not trophoblast invasion or decidual cell death, plays a major role in the opening of maternal vessels into the mesometrial chamber lumen before the formation of the chorioallantoic placenta.     

Structure of the Rete Mirabile in the Kidney of the Rat as Seen with the Electron Microscope

Electron micrographs of the rete mirabile in the medulla of the rat have revealed that the endothelium of the afferent and efferent vessels are markedly different in fine structure. The venous capillaries returning blood from the papilla are lined with a fenestrated endothelium much like that in the peritubular capillaries of the kidney. The arterial capillaries delivering blood to the papilla have an unperforated lining of overlapping endothelial cells with extremely irregular tapered margins. It is pointed out that the organization of particularly the latter vessels suggests that the functional capabilities of these retia go beyond those of a simple diffusion countercurrent exchanger.     

Endothelial cell lineages of the heart

During early gastrulation, vertebrate embryos begin to produce endothelial cells (ECs) from the mesoderm. ECs first form primitive vascular plexus de novo and later differentiate into arterial, venous, capillary, and lymphatic ECs. In the heart, the five distinct EC types (endocardial, coronary arterial, venous, capillary, and lymphatic) have distinct phenotypes. For example, coronary ECs establish a typical vessel network throughout the myocardium, whereas endocardial ECs form a large epithelial sheet with no angiogenic sprouting into the myocardium. Neither coronary arteries, veins, and capillaries, nor lymphatic vessels fuse with the endocardium or open to the heart chamber. The developmental stage during which the specific phenotype of each cardiac EC type is determined remains unclear. The mechanisms involved in EC commitment and diversity can however be more precisely defined by tracking the migratory patterns and lineage decisions of the precursors of cardiac ECs. Work carried out by the authors is supported in part by the NIH.     

Scanning electron microscopy of normal rat liver: the surface structure of its cells and tissue components.

Scanning electron microscopy (SEM) allows the surface ultrastructure of intrahepatic cells and other tissue components of liver to be delineated. Excellent depth of focus of the SEM makes it possible to visualize surfaces of intact cells in their native configurations. This report details the surface characteristics and inter-relationships of hepatocytes and hepatic plates, sinusoidal endothelial cells and sinusoids, presumed Kupffer cells, vessels, bile ducts, connective tissue, and the capsule of rat liver. Hepatocytes present three structurally distinctive faces--the intercellular face containing flat surfaces and bile canaliculus, the sinusoidal face, and the connective tissue face which abuts portal tracts and hepatic veins. Sinusoidal endothelium is penetrated by large (1 to 3 mum) and small (0.1 mum) fenestrae, the latter occurring in clusters of up to 50. The width of bile canaliculi and distribution of large fenestrae vary proximodistally along hepatic plate or sinusoid. The cells of portal bile ductules contain microvilli located in linear rows and sparse cilia. Endothelium of hepatic artery and of portal vein is sparsely fenestrated.