Microcirculation in mouse spleen (nonsinusal) studied by means of corrosion casts

Corrosion casts of mouse spleen, examined by scanning electron microscopy, enabled vascular pathways of the arterial, intermediate, and venous circulations to be traced over considerable distances. The arterial tree is surrounded by white pulp immediately upon entering at the hilus, and relatively few arterioles extend into red pulp. A profusion of capillaries is present in both periarterial lymphatic sheaths and lymphatic nodules, arranged as bifurcating systems (rather than anastomosing networks) terminating in the marginal sinus (MS) and marginal zone (MZ). The MS, which is situated between white pulp and MZ, consists of a discontinuous layer of flattened anastomosing spaces which are up to six times as large as those in rat spleen. Extensive filling of the entire MZ took place before appreciable filling of surrounding red pulp occurred. Capillary terminations in red pulp are always continuous with reticular meshwork, i.e., no evidence for a “closed” circulation was found. Casts of the venous origins support the classification “pulp venules” rather than “venous sinuses” and show major morphological differences from the richly anastomosing system of sinuses in rat. In the subcapsular region of mouse spleen large anastomosing veins ramify over the surface, with reticular meshwork occupying extensive areas between adjacent veins. For in vivo microscopy this arrangement offers advantages over that found in rat spleen (accompanying paper), where almost the entire surface is densely covered with venous sinuses.     

Microvascularization and histomorphology of lateral line organs in adult Xenopus laevis

The microvasculariaztion of the lateral line organs (LLOs) of the adult pipid frog, Xenopus laevis was studied by scanning electron microscopy of vascular corrosion casts (VCCs) and correlative light microscopy of paraplast embedded tissues sections. Scanning electron micrographs of VCCs revealed that each neuromast within the LLO rests on a distinct bowl‐like capillary network (vascular bowl). One to three vascular bowls were supplied by an ascending arteriole and drained by a descending venule towards the skin deep dermal vascular network. Blood flow regulation mechanisms in form of intimal cushions were present at the origin of ascending arterioles supplying LLOs, microvenous valves were present at the confluence of deep dermal venules and veins. This together with sprouting and nonsprouting angiogenesis (intussusceptive microvascular growth) found in vascular bowls demonstrate that in adult Xenopus the capillary bed of LLO's still can be adjusted to changing energetic needs. J. Morphol. 275:497–503, 2014. © 2013 Wiley Periodicals, Inc.     

The intermediate circulation in the nonsinusal spleen of the cat, studied by scanning electron microscopy of microcorrosion casts

Scanning electron microscopy of microcorrosion casts was used to visualize circulatory pathways of the intermediate circulation in nonsinusal spleen of cat. The marginal sinus (MS) around lymphatic nodules is a distinct vascular space which fills preferentially before the filling of the marginal zone (MZ) and surrounding red pulp occurs. The MS, which has a plentiful vascular supply, does not usually enclose the nodule completely. From the MS, flow occurs radially outwards into the MZ. Corrosion casts and histological sections both showed that a diversity of forms of the MZ exists: The thickness of MZ and the arrangement of its reticulum vary among nodules and between different areas of the same nodule, from a complete absence to a region of up to 50 microns in width. No direct arteriovenous connections were found (in contrast to dog spleen: Schmidt et al., '83b). Aside from capillary endings in the MS and MZ, all arterial capillaries terminate in the reticular spaces of the red pulp, i.e., the circulation appears to be entirely "open." From each capillary termination a great variety of flow pathways through the reticular meshwork to the pulp venules is available; some of these routes are quite long but others may involve distances as short as 15-25 microns. Evidence of flow into ellipsoid sheaths was abundant in casts from dilated spleens, but scarce in contracted spleens. In contrast to the extensive system of interconnected venous sinuses in dog spleen, the pulp venules found in cat spleen are nonanastomosing, shorter, and much smaller in caliber, and all receive flow freely from the reticular meshwork via open ends and fenestrations in their walls.     

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.     

Rapid and selective removal of larval erythrocytes from systemic circulation during metamorphosis of the bullfrog, Rana catesbeiana

Mechanisms of hemoglobin transition during bullfrog metamorphosis were investigated by labeling red blood cells from larvae (L-RBC) and from froglets (A-RBC) with a fluorescent dye, PKH26. The life span of the labeled L-RBC in systemic circulation was significantly shorter when they were injected into the animals at the metamorphic climax, compared to injection into pre- or postmetamorphic animals. The A-RBC had a long life span regardless of the metamorphic stage of the recipient animal. Therefore, L-RBC were selectively removed from the systemic circulation at the time of metamorphic climax. During climax, the labeled L-RBC were ingested by hepatic and splenic macrophages, indicating that macrophages are involved in the specific elimination of L-RBC.     

Phenotypic differences between red pulp capillary and sinusoidal endothelia help localizing the open splenic circulation in humans

The distribution of capillaries, sinuses and larger vessels was investigated by immunohistology in paraffin sections of 12 adult human spleens using a panel of antibodies. Double staining for CD34 and CD141 (thrombomodulin) revealed that capillary endothelia in the cords of the splenic red pulp and at the surface of follicles were CD34⁺CD141⁻, while red pulp sinus endothelia had the phenotype CD34⁻CD141⁺. Only in the direct vicinity of splenic follicles did sinus endothelial cells exhibit both antigens. Thus, splenic sinuses do not replace conventional capillaries, but exist in addition to such vessels. The endothelium in arterioles, venules and larger arteries and veins was uniformly CD34⁺CD141⁺. Anti-CD34 and anti-CD141 both additionally reacted with different types of splenic stromal cells. Differential staining of capillaries and sinuses may permit a three-dimensional reconstruction of serial sections to unequivocally delineate the “open” and “closed” splenic circulation in humans.     

Lymphoid organ development in Xenopus thymectomized at eight days of age

This paper examines the effect of early thymectomy on the subsequent development of lymphoid tissues in the toad, Xenopus laevis. At the time of thymic removal (8 days post-fertilization) all the lymphoid organ anlagen are at a rudimentary state of differentiation and contain few, if any, small lymphocytes. Despite the absence of any thymic tissue all thymectomized animals grew normally. Thymectomized larvae developed relatively normal lymphoid organs. However, lymphoid depletion was apparent in the splenic red pulp and in the pharyngeal ventral cavity bodies. Examination of the lymphoid organs of post-metamorphic Xenopus revealed reduction in spleen size following thymectomy. Lymphoid depletion was evident in the splenic red pulp of many thymectomized toadlets and reduction in proportion of white to red pulp was also noted in a few of these animals. Absence of the thymus had no apparent effect on the histology of the other lymphoid organs examined.     

Splenic white pulp as a thymus-independent area in the African clawed toad,Xenopus laevis

Summary An indirect immunofluorescence study of the frozen sections of the spleen of an anuran amphibian, Xenopus laevis, showed that lymphocytes bearing a small amount of immunoglobulin (Ig) were localized mostly in the white pulp of non-immunized toads. There were fewer fluorescent cells in the red pulp. In the toads hyperimmunized with human gamma globulin (HGG), cells with strong cytoplasmic fluorescence increased significantly in the outer part of the white pulp. Electron microscopy of spleens from these toads showed that plasma cells at different stages of maturation were abundant in the white pulp, whereas in the red pulp, a smaller number of maturer plasma cells were observed. These results indicate that, in contrast with its mammalian counterpart, the splenic white pulp of this anuran is the site where thymusin-dependent lymphocytes commence blast formation and transformation into plasma cells.     

Microscopy of vascular architecture and arteriovenous communications in the spleen of two odontocetes

The red pulp of the spleens of the short-finned pilot whale (Globicephala macrorhynchus) and the Pacific bottle-nosed dolphin (Tursiops truncatus gilli) (Odontoceti) were examined by light and electron microscopy and found to comprise two venous layers, an inner and an outer. The inner layer is homologous to the intermediate zone (IZ) of primitive-type mammalian spleens and contains sinusoids consisting of endothelial cells and a thin layer of extracellular deposits. Its vascular structure is unclear. The venous vessels of this layer eventually communicate with veins of the perivenous outer layer. The perivenous layer contains veins of various sizes, interstitial elements, and trabeculae. It is filled with blood cells, particularly plasma cells, but no myeloid cells. The perivenous layer (PVL) is homologous to the red pulp of common mammalian spleens but shows signs of involution. The white pulp gives origin to arterial terminals that end in the red pulp, where they communicate directly with the sinusoidal veins producing a closed circulation. The arterial terminals do not show ellipsoids. The presence of an IZ with a closed circulation and the involution of the red pulp makes the spleen of Odeontoceti another example of a mammalian spleen of the primitive type that has been altered by the evolutionary process. Vascular remodelling of the spleen of Odontoceti seems to follow the pattern noted in the spleens of nonmammalian vertebrates. © 1994 Wiley-Liss, Inc.     

Aspects of the ultrastructure of the alimentary canal and respiratory ducts in Xenopus laevis larvae

The pharynx, intestine and respiratory duct of Xenopus laevis were examined by light and electron microscopy, at different stages of the metamorphic cycle, through climax. It is well known that preclimactic larvae are suspension feeders and that after climax specimens feed on solid food. It is shown that the histology of the pharynx and the alimentary canal changes in adaptation to the change in the mode of feeding. Suspension feeding utilises ciliary activity but after climax cilia have disappeared from the pharynx and alimentary canal, when new neuromuscular mechanisms are utilised in feeding. Other morpho-histological changes in various intestinal and respiratory tissues are likewise considered in terms of functional activity.     

Microvascularization of the interhyoid muscle in larval Xenopus laevis (Daudin): Scanning electron microscopy of vascular corrosion casts and correlative light microscopy

The interhyoid muscle in tadpoles of Xenopus laevis (Daudin) is an important part of the buccal pump, a functional unit that provides unidirectional flow of water through mouth and pharynx. In anuran tadpoles, this flow is crucial in both respiration (gas exchange) and food intake (ingestion). The microvascular anatomy of the interhyoid muscles of 43 tadpoles of X. laevis from developmental stages 49–60 was examined by scanning electron microscopy of vascular corrosion casts and correlative light microscopy of paraplast embedded Goldner stained serial tissue sections. Analysis of vascular corrosion casts of the interhyoid muscle showed that several descending branches of external carotid arteries supplied the interhyoid muscle. Arteries splitted into many arterioles at the dorsal surface of the interhyoid muscle and formed sheaths of longitudinally orientated capillaries around muscle fibers. Postcapillary vessels formed perpendicularly orientated arrays of collecting venules (mean diameter: 15.6 μm), which drained the interhyoid muscle from the ventral surface into external jugular veins. Cast analyses revealed sprouting angiogenesis at the capillary level and nonsprouting angiogenesis at distal domains of the venous system. Both means of angiogenesis that persisted throughout the developmental periods examined are thought to represent a superposition of concurrent developmental and physiological processes. The dense microvascular bed of the interhyoid muscle reflects its high demand for supply with oxygen and nutrients. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Renal microvasculature in the adult pipid frog,Xenopus laevis: A scanning electron microscope study of vascular corrosion casts

We studied the opisthonephric (mesonephric) kidneys of adult male and female Xenopus laevis using scanning electron microscopy (SEM) of vascular corrosion casts and light microscopy of paraplast embedded tissue sections. Both techniques displayed glomeruli from ventral to mid-dorsal regions of the kidneys with single glomeruli located dorsally close beneath the renal capsule. Glomeruli in general were fed by a single afferent arteriole and drained via a single thinner efferent arteriole into peritubular vessels. Light microscopy and SEM of vascular corrosion casts revealed sphincters at the origins of afferent arterioles, which arose closely, spaced from their parent renal arteries. The second source of renal blood supply via renal portal veins varied interindividually in branching patterns with vessels showing up to five branching orders before they became peritubular vessels. Main trunks and their first- and second-order branches revealed clear longish endothelial cell nuclei imprint patterns oriented parallel to the vessels longitudinal axis, a pattern characteristic for arteries. Peritubular vessels had irregular contours and were never seen as clear cylindrical structures. They ran rather parallel, anastomosed with neighbors and changed into renal venules and veins, which finally emptied into the ventrally located posterior caval vein. A third source of blood supply of the peritubular vessels by straight terminal portions of renal arteries (vasa recta) was not found.     

Neurotrophin receptors and enteric neuronal development during metamorphosis in the amphibian<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

During metamorphosis, the frog intestine goes through a dramatic shortening with extensive apoptosis and regeneration in the epithelial layer and connective tissue. Our aim was to study changes in the enteric nervous system represented by one inhibitory (vasoactive intestinal polypeptide; VIP) and one excitatory (substance P, neurokinin A; SP/NKA) nerve population and concomitant changes in neurotrophin receptor occurrence during this development in the gut of Xenopus laevis adults and tadpoles at different stages of metamorphosis (NF stages 57–66). Sections were incubated with antibodies against the neurotrophin Trk receptors and p75^NTR, and the neurotransmitters VIP and SP/NKA. Trk-immunoreactive nerves increased dramatically but transiently in number during early metamorphic climax. Nerves immunoreactive for p75^NTR were present throughout the gut, decreased in number in the middle intestine during climax, and increased in the large intestine during late metamorphosis. The percentage of VIP-immunoreactive nerves did not change during metamorphosis. SP/NKA-immunoreactive nerves were first apparent at NF stages 61–62 in the middle intestine and increased in the stomach and large intestine during metamorphosis. Endocrine cells expressing SP/NKA increased in number in stomach, proximal, and middle intestine during metamorphic climax. Thus, neurotrophin receptors are expressed transiently in neurons of the enteric nervous system during metamorphosis in Xenopus laevis and SP/NKA innervation is more abundant in the intestine of the postmetamorphic frog than in the tadpole.This study was supported by grants from the Swedish Research Council to S. Holmgren     

Microvascular anatomy and histomorphology of extrapulmonary bronchi in adult Xenopus laevis Daudin (Lissamphibia; Anura) point to a role in aerial gas exchange – histomorphology of tissue sections and scanning electron microscopy of vascular corrosion casts

Studies on the amphibian respiratory tract microvascular anatomy are few. Using scanning electron microscopy (SEM) of vascular corrosion casts (VCCs) and light microscopy of perfusion‐fixed tissue sections, we studied the bronchial microvascular anatomy in the adult South African Clawed Toad, Xenopus laevis Daudin. Histomorphology showed that the bronchial wall consists (from luminal to abluminal) of squamous epithelium, subepithelial capillary bed, cartilage rings or cartilage plates, a layer of dense connective tissue, a layer of smooth muscle cells, and squamous epithelium (serosa). SEM of VCCs reveals that bilaterally a ventral, a dorsal (Ø 77.21 ± 7.61 μm), and a caudal bronchial artery supply the bronchial subepithelial capillary bed. The ventral bronchial artery has 3–4 branching orders (interbranching distances: 506.3 ± 392.12 μm; branching angles of first‐ and second‐order bifurcations: 24.60 ± 10.24° and 29.59 ± 14.3°). Casts of bronchial arteries display imprints of flow dividers and sphincters. Cranial and caudal bronchial veins (Ø 154.78 ± 49.68 μm) drain into pulmonary veins. They lack microvenous valves. The location of the dense subepithelial capillary meshwork just beneath the thin squamous bronchial epithelium and its drainage into the pulmonary veins make it likely that in Xenopus, bronchi assist in aerial gas exchange.     

Microvascular anatomy of the esophagus in larval and adult Xenopus laevis Daudin: a scanning electron microscope study of microvascular corrosion casts and correlative light microscopy

Larval to adult microvascular anatomy of the esophagus was studied in the South African Clawed Toad, Xenopus laevis (Daudin) by scanning electron microscopy of vascular corrosion casts and correlative light microscopy of paraplast embedded stained tissue sections. Analyses of vascular corrosion casts of tadpole esophaguses at premetamorphosis revealed a wide‐meshed, but mature looking capillary bed which during following prometamorphosis increased in density and gained the adult‐like pattern during late metamorphic climax by sprouting and nonsprouting angiogenesis. In adult Xenopus, the esophageal mucosa possessed a dense subepithelial capillary bed fed by one or two esophageal arteries that originated from right and/or left thoracic aorta just distal to the origin of the subclavian arteries. In the adult undistended esophagus, esophageal arteries revealed an undulating course, a pattern that guarantees a continuous blood supply when the esophagus is extremely wide expanded as it is the case when adult Xenopus swallows large prey.     

DNA SYNTHESIS AND CELL TURNOVER IN RANA PIPIENS TADPOLE LIVER DURING SPONTANEOUS METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during metamorphosis of R. pipiens liver was investigated. Average DNA/cell was constant at 11.6 pg/ nucleus through stage XXV; but increased during juvenile growth; during metamorphosis stages, changes in total DNA content must correspond to changes in cell number. Rates of DNA synthesis were estimated by rates of ³H-thymidine incorporated into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. DNA content increased steadily from premetamorphosis until late prometamorphosis; at preclimax stages XVIII and XX there were two successive decreases in DNA content of approximately 30%. Fluctuations in synthesis rates preceded corresponding fluctuations in content; DNA synthesis was maximal at stages XVI and XVIII, decreased nearly ten-fold at metamorphic climax, and then gradually rose again during late climax stages. The size of the endogenous thymine pool increased transitorily during spontaneous metamorphosis corresponding to a stage of maximal DNA synthesis. These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthesis rates and content during metamorphosis. Metamorphosis of the tadpole liver appears to be associated with both proliferation and cellular death, perhaps a replacement of “larval” by “adult” cells. Metamorphosis of the liver cannot be occuring in a “fixed population of cells” as is commonly assumed. An interpretation of the population dynamics of the metamorphic liver is presented.     

Macrophages and Reticulum Cells in the Spleen of the Dogfish,Scyliorhinus canicula

The presence and ultrastructural features of reticulum cells and macrophages were studied in the spleen of the dogfish Scyliorhinus canicula. Three morphologically distinguishable regions of the spleen were identified: the white pulp, the red pulp and the ellipsoids. In all three, the splenic parenchyma was a meshwork supported by reticulum cells and fibres. Reticulum cells in both the white and the red pulp are irregular elements, the processes of which are joined by cell junctions and embrace developing reticular fibres. The ellipsoids of the dogfish spleen are terminal branches of the splenic arteries of the white pulp, with a sheath consisting of reticulum cells, reticular fibres, ground substance, macrophages and occasional lymphocytes. Isolated melanomacrophages also occur in the ellipsoid walls as well as in the red pulp. In both the white and the red pulp phagocytic reticulum cells, and macrophages appear frequently forming cell associations with surrounding blood cells, mainly lymphocytes. The functional significance of the ellipsoids and the cell-cell clusters of the white and the red pulp is discussed in relation to the immune capacities demonstrated in elasmobranchs.     

Energetics of metamorphic climax in the southern toad (<Emphasis Type="Italic">Bufo terrestris</Emphasis>)

During metamorphic climax, anuran larvae must rely on stored energy because changes in oral and digestive morphology prevent foraging and efficient assimilation. Thus, the time required to store adequate energy for metamorphic climax may set a lower limit on age at which it can occur. Therefore, the amount and type of energy used during metamorphic climax must be determined. To quantify the energetic costs of metamorphic climax in Bufo terrestris, oxygen consumption during climax was measured. Wet mass, dry mass, and lipid mass for a group of individuals at the initiation of climax (forelimb emergence, FL) and for another group at the end of climax (complete tail resorption, TR) were also measured to determine whether lipids were used to fuel metamorphic climax. The total amount of energy used, maintenance costs, and development costs during metamorphic climax varied considerably among individuals. Variation in energy metabolism during climax was not related to differences in energy metabolism during larval development or body mass at initiation of climax. TR individuals were significantly lighter in terms of wet mass and had less body water than FL individuals. However, the two groups did not differ in dry mass or lipid mass. Therefore, lipid catabolism is not a major source of energy during metamorphic climax in B. terrestris. As a result, decreases in age at metamorphosis may not be constrained by the need to store energy in the form of lipids.     

Microvascular anatomy of the brain of the adult pipid frog,Xenopus laevis (Daudin): A scanning electron microscopic study of vascular corrosion casts

To demonstrate the 3D microvascular anatomy of the brain of the model organism Xenopus laevis Daudin scanning electron microscopy of vascular corrosion casts was correlated with light microscopy of stained 7 µm thick serial tissues sections. Results showed that supplying arteries descended from the leptomeningeal surface without remarkable branchings straight to the subventricular zone where they branched and capillarized. Capillaries showed few H‐ and/or Y‐shaped anastomoses during their centrifugal course toward the leptomeningeal surface where they drained into cerebral venules and veins. Apart from the accessory olfactory bulb and the vestibule‐cochlear nucleus where capillaries were densely packed, capillaries formed a wide‐meshed 3D network throughout the brain parenchyma and thus contrasted to urodelian brains where hairpin‐shaped capillaries descend from the leptomeningeal vessels into varying depths of the brain parenchyma. In about two‐third of specimens, a closed arterial circle of Willis was found at the base of the brain. If this circle in Xenopus might serve the same two functions as in men is briefly discussed. Choroid plexuses of third and fourth ventricle were found to have a high venous, but a low arterial inflow via one small choroidal artery only. Findings are compared with previous studies on the vascularization of the anuran brain and discrepancies in respect to presence or absence of particular arteries and/or veins in Ranids, Bufonids, and Pipids studied so far are discussed with particular emphasis on the techniques used in the various studies published so far.     

Migration pathways of recirculating murine B cells and CD4+ and CD8+ T lymphocytes

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.