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.     

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.     

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.     

Bottomless Bowls: Why Visual Cues of Portion Size May Influence Intake**

Objective: Using self‐refilling soup bowls, this study examined whether visual cues related to portion size can influence intake volume without altering either estimated intake or satiation. Research Methods and Procedures: Fifty‐four participants (BMI, 17.3 to 36.0 kg/m²; 18 to 46 years of age) were recruited to participate in a study involving soup. The experiment was a between‐subject design with two visibility levels: 1) an accurate visual cue of a food portion (normal bowl) vs. 2) a biased visual cue (self‐refilling bowl). The soup apparatus was housed in a modified restaurant‐style table in which two of four bowls slowly and imperceptibly refilled as their contents were consumed. Outcomes included intake volume, intake estimation, consumption monitoring, and satiety. Results: Participants who were unknowingly eating from self‐refilling bowls ate more soup [14.7 ± 8.4 vs. 8.5 ± 6.1 oz; F(1,52) = 8.99; p < 0.01] than those eating from normal soup bowls. However, despite consuming 73% more, they did not believe they had consumed more, nor did they perceive themselves as more sated than those eating from normal bowls. This was unaffected by BMI. Discussion: These findings are consistent with the notion that the amount of food on a plate or bowl increases intake because it influences consumption norms and expectations and it lessens one's reliance on self‐monitoring. It seems that people use their eyes to count calories and not their stomachs. The importance of having salient, accurate visual cues can play an important role in the prevention of unintentional overeating.     

Scanning electron microscopy of vascular architecture in the gastric mucosa of the golden hamster

Summary The three-dimensional architectures and the regional differences of the vascular system in the mucosa of the hamster stomach were revealed by scanning electron microscopy of corrosion casts. In the forestomach, the vascular network spreads two-dimensionally in a thin lamina propria. In the corpus and the antrum, the capillaries in the thick lamina propria are well developed, extending three-dimensionally along the gastric pits and glands. In the corpus, the submucosal arteries enter the lamina propria to become ascending capillaries, which project toward the top of the lamina propria and anastomose to create a capillary network beneath the mucosal epithelium. A subepithelial capillary is much wider in diameter than an ascending capillary and is, therefore, a sinusoid capillary. Subepithelial capillaries join descending venules, which are less numerous than the ascending capillaries. Near the gastric lumen, the capillaries in the corpus can be classified into two types: arched type in the cephalic (upper) region and honeycomb type in the caudal (lower) region. In the antrum, the submucosal arterial plexus is less well developed than that in the corpus. The mucosal aspect of the corrosion cast shows many clumps, formed by a unit of capillary network. Functional significances of different vascular architectures in the gastric mucosa of the forestomach, corpus, and antrum are discussed.This study was supported in part by grants from the Research Fund of the Ministry of Education, Science, and Culture, Japan     

An injection-corrosion study on the angioarchitecture of rat skin]

The angioarchitecture of the skin in the SD strain rat was investigated by scanning electron microscopy using resin cast. The neck part of rat skin was composed of 3 layers of vascular networks: the first layer (superficial layer), the second layer (middle layer), and the third layer (deep layer). Two types of capillary architectures were present around the hair follicle: small architectures composed of random network and large architectures composed of 3 parts distinguished by the construction of network. The large architecture, the capillary of hair papilla and the capillary network of sebaceous gland was thought to compose one circulation unit. The afferent vessels into such units branched from Kandelaberarterie-Petersen occurring from the 3rd layer, ascended to the 2nd layer, then descended to the bottom of the network and composed the capillary architecture. There were 2 types of efferent vessels from such circulation unit. They occurred from superficial and deep part, respectively, of the vascular architecture around the hair follicle and descended straight to the 3rd layer. The presence of straight vessels between the 2 nd and 3 rd layer, and the simple arrangement of vascular layers were thought to be characteristic of the angioarchitecture of the rat skin.     

The Different Vascular Patterns of Slime Glands in the Hagfishes,Myxine glutinosa Linnaeus and Eptatretus stouti Lockington A Scanning Electron Microscope Study of Vascular Corrosion Casts*

The vascular patterns of the epidermally derived slime glands of the Atlantic hagfish, Myxine glutinosa, and of the Pacific hagfish, Eptatretus stouti, have been studied by scanning electron microscopy of vascular corrosion casts. In Myxine a simple two-dimensional vascular network sheaths the slime glands, while in Eptatretus there is also a great number of capillary loops of different lengths arising from the sheathing network and extending into the interior of the glands. These basic differences in slime glands vascular patterns are thought to reflect substantially different physiological behaviour of the slime glands in Myxine and Eptatretus.     

Peripheral vascularization of the dermal laminae of the equine hoof

The vascular architecture of the dermal laminae was studied by scanning electron microscopy of vascular corrosion casts. Ultrastructurally, the laminar vasculature consisted of arterioles, capillaries, venules and veins, arranged in a sheet-like network. Through the laminae, arterioles ran parallel to the solar surface and branched at two levels to form a continuous arteriolar arcade, parallel to the hoof wall. Capillaries originating from these arcades formed hairpin loops joining the marginal vein prior to forming an axially situated venous network. Additional capillaries were also given off by the arterioles, forming an abaxially arranged capillary plexus.     

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.     

Critical role for the kinesin KIF3A in the HIV life cycle in primary human macrophages

Macrophages are long-lived target cells for HIV infection and are considered viral reservoirs. HIV assembly in macrophages occurs in virus-containing compartments (VCCs) in which virions accumulate and are stored. The regulation of the trafficking and release of these VCCs remains unknown. Using high resolution light and electron microscopy of HIV-1–infected primary human macrophages, we show that the spatial distribution of VCCs depended on the microtubule network and that VCC-limiting membrane was closely associated with KIF3A+ microtubules. Silencing KIF3A strongly decreased virus release from HIV-1–infected macrophages, leading to VCC accumulation intracellularly. Time-lapse microscopy further suggested that VCCs and associated KIF3A move together along microtubules. Importantly, KIF3A does not play a role in HIV release from T cells that do not possess VCCs. These results reveal that HIV-1 requires the molecular motor KIF3 to complete its cycle in primary macrophages. Targeting this step may lead to novel strategies to eliminate this viral reservoir.     

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

The three-dimensional microvascular anatomy of the testes of adult Xenopus laevis was studied by scanning electron microscopy of vascular corrosion casts. Results showed that testicular arteries branch off from urogenital arteries and approach the testes via the mesorchium. At the dorsal surface of testes, arteries course towards the medial and lateral surfaces of the ovoid testes, pierce the tunica albuginea and run in the subalbugineal space and then penetrate the testicular parenchyma. In the parenchyma, they branch in all directions. At sites where larger branches arise, sphincters are present. Intimal cushions are found at the origin of smaller branches. Terminal arterioles capillarize and form a wide-meshed capillary network around convoluted seminiferous tubules. Inter- and peritubular capillaries cannot be differentiated reliably. Arterio-venous transition distances are short. Arterio-arterial anastomoses and veno-venous anastomoses are present. Testicular venules empty into a venous subalbugineal plexus which shows veno-venous anastomoses of different lengths and calibres. Signs of the ongoing non-sprouting angiogenesis, particularly intussusceptive branch remodelling, reflect the dynamics of the testicular parenchyma. The present study is the first that demonstrates the testicular microvascular anatomy in an anuran species and shows that X. laevis and men share the intricate convoluted seminiferous tubules with associated vascular patterns as a feature in common.     

Adhesive interactions between CD34<Superscript>+</Superscript>-derived dendritic cell precursors and dermal microvascular endothelial cells studied by scanning electron microscopy

Dendritic cells are migratory cells. Before they extravasate from the circulation into the skin across capillary blood vessel walls, they have to interact with endothelial cells. Using a fluorimetric adhesion assay, we have recently shown that CD34⁺-derived dendritic cell precursors are able to bind to resting and stimulated dermal microvascular endothelial cells. In the present study, we attempted to visualize this process at an ultrastructural level. CD34⁺ progenitor cells were purified from human cord blood samples by means of immunomagnetic beads, and dendritic cells were generated by culture in the presence of GM-CSF, TNF- and hSCF for 5 days. Immature CD83^– CD86^low dendritic cells were added to human dermal microvascular endothelial cells grown to confluence on membrane chambers. After 2 h, unbound dendritic cell precursors were removed, and bound cells were prepared for routine scanning electron microscopy. We found that (1) dendritic cell precursors firmly adhere to microvascular endothelial cells, enveloping them with their surface processes; (2) dendritic cell precursors are extremely deformable as they squeeze through the dense network of microvascular endothelial cells; (3) microvascular endothelial cells form, in part, a multi-layered network rather than the typical cobblestone pattern as seen by phase-contrast microscopy. The morphology of dendritic cell precursors and of human dermal microvascular endothelial cells was examined here, for the first time, by scanning electron microscopy. These data further emphasize that CD34⁺-derived dendritic cells efficiently adhere to dermal microvascular endothelial cells.     

Self-reinforced Behaviour Does not Explain Contra-freeloading in the Mongolian Gerbil

In various situations animals prefer to work for food even when free food is available. Several hypotheses have been put forward to explain this phenomenon, most of them either suggesting that a learned behaviour may be self-reinforcing or that the animal has to perform a certain species specific behaviour. Mongolian gerbils (Meriones unguiculatus) were used in the present study. Half of them were given access to a cage with one bowl with sunflower seeds, and another bowl with sand and 30 seeds, 20 min/d for 5 d. The amount of seeds eaten from each bowl and the time spent digging in the bowls, on the floor, and in the corners was recorded. The animals were then put in the same cage with one bowl with seeds but the other bowl now contained only sand (20 min/d for 5 d). For the other group the treatment was reversed. Almost all digging took place in the 30 seed bowl, and most seeds eaten were taken from this bowl. When this bowl contained only sand the amount of digging went down drastically for that bowl. Nor was there any compensatory digging on the floor or in the corners of the cage. The present results indicate that the self-reinforcing hypothesis and the obligatory species specific response hypothesis are not enough to explain the contra-freeloading phenomenon. A possible alternative hypothesis, that the behaviour is a way of exploring and reducing uncertainty is discussed.     

Extraverted Children Are More Biased by Bowl Sizes than Introverts

Extraverted children are hypothesized to be most at risk for over-serving and overeating due to environmental cues – such as the size of dinnerware. A within-subject field study of elementary school students found that extraverted children served themselves 33.1% more cereal in larger bowls (16-oz) than in smaller (12-oz) bowls, whereas introverted children were unaffected by bowl size (+5.6%, ns). However, when children were asked by adults how much cereal they wanted to eat, both extraverted and introverted children requested more cereal when given a large versus small bowl. Insofar as extraverted children appear to be more biased by environmental cues, this pilot study suggests different serving styles are recommended for parents and other caregivers. They should serve extraverts, but allow introverts to serve themselves. Still, since the average child still served 23.2% more when serving themselves than when served by an adult, it might be best for caregivers to do the serving whenever possible – especially for extraverted children.     

Brain-capillary architecture of the newt,Triturus cristatus carnifex (Caudata: Salamandridae): Scanning electron-microscopical study of vascular corrosion casts

The brain-microvascular architecture of the newt (Triturus cristatus carnifex) was studied by scanning electron-microscopical examination of microvascular corrosion casts. It is characterized at the leptomeningeal level by a superficial network of single vessels that leave the meningeal sheath to form hairpin-shaped capillary loops. These loops penetrate the nervous parenchyma to varying depths and are variously bent, inclined, and twisted. Each hairpin-like loop consists of descending and ascending limbs connected by a distal U-shaped segment. The limbs are of equal diameter and lack physical differentiation. Some loops branch in a secondary loop, but there are no intraparenchymal connections with analogous surrounding structures. On vascular casts we observed detailed imprints of endothelial cell nuclei. This microvascular architecture is present in the whole brain except in leptomeninx and choroidal plexuses. The vascular pattern of T. cristatus carnifex resembles that of apodans. However, it differs from the anuran condition, which is characterized by a three-dimensional parenchymal network and from that of Ambystoma mexicanum which displays an intermediate condition between a single-capillary network and a capillary-loop model. At the level of paired capillaries, a counter-current exchange system may occur between the two limbs of the same loop, an arrangement that could promote uniformity of extracellular fluid composition in parenchyma.     

CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

Myeloid dendritic cells (DCs) can capture HIV-1 via the receptor CD169/Siglec-1 that binds to the ganglioside, GM3, in the virus particle membrane. In turn, HIV-1 particles captured by CD169, an I-type lectin, whose expression on DCs is enhanced upon maturation with LPS, are protected from degradation in CD169⁺ virus-containing compartments (VCCs) and disseminated to CD4⁺ T cells, a mechanism of DC-mediated HIV-1 trans-infection. In this study, we describe the mechanism of VCC formation and its role in immune evasion mechanisms of HIV-1. We find HIV-1-induced formation of VCCs is restricted to myeloid cells, and that the cytoplasmic tail of CD169 is dispensable for HIV-1 trafficking and retention within VCCs and subsequent trans-infection to CD4⁺ T cells. Interestingly, introduction of a di-aromatic endocytic motif in the cytoplasmic tail of CD169 that results in endocytosis of HIV-1 particles, suppressed CD169-mediated HIV-1 trans-infection. Furthermore, super-resolution microscopy revealed close association of CD169 and HIV-1 particles in surface-accessible but deep plasma membrane invaginations. Intriguingly, HIV-1 particles in deep VCCs were inefficiently accessed by anti-gp120 broadly neutralizing antibodies, VRC01 and NIH45-46 G54W, and thus were less susceptible to neutralization. Our study suggests that HIV-1 capture by CD169 can provide virus evasion from both innate (phagocytosis) and adaptive immune responses.     

Splenic angioleiomyoma in an owl monkey (Aotus nancymae)

Background An adult male owl monkey (Aotus nancymae) underwent a splenectomy. When the spleen was removed, a small, nodular mass slightly bulging over the splenic surface was noted. Methods The mass was examined by light and transmission electron microscopy and by immunohistochemistry. Results On light microscopy, the mass was well‐circumscribed, non‐encapsulated, and composed of haphazardly arranged smooth muscle bundles admixed with numerous small capillary‐like structures containing blood. Immunohistochemical (IHC) staining revealed the tumor was strongly positive for smooth muscle actin yielding vascular smooth muscle bundles, and for Factor VIII, staining endothelial cells within the smooth muscle bundles. Transmission electron microscopy (TEM) showed a large portion of the cells to be atypical appearing smooth muscle and a few cells had structures resembling Weibel–Palade bodies indicating endothelial cells. Conclusions Based on cell morphology, by light and TEM, and IHC a final diagnosis of splenic angioleiomyoma was made. This is, to our knowledge, the first report of an angioleiomyoma in a non‐human primate.     

Glomerular bypass shunts in the kidney of the Atlantic hagfish,Myxine glutinosa

Summary The vascular pathways associated with the glomerulus of the Atlantic hagfish, Myxine glutinosa have been studied by scanning electron microscopy of corroded resin casts of the vasculature. Although the overall pattern of the renal vasculature did not differ from earlier reports, a previously unreported vascular pathway which arose from the renal artery and bypassed the glomerular capillaries in 28% of glomeruli was clearly demonstrated. Glomerular bypass shunts either ran to join the loose capillary network around Bowman's capsule and thereby drain into the network of vessels associated with the mesonephric duct (ureter), or ran directly into the ureteral system of vessels and subsequently into the posterior cardinal veins. Glomerular bypass shunts which theoretically permit renal arterial blood to bypass the process of filtration may play a role in the regulation of body fluid volume.     

Model of skin vascularization in Rana esculenta L.: Scanning electron microscopy of microcorrosion casts

Summary Microcorrosion casts of blood vessels in the skin of Rana esculenta L. were examined by means of scanning electron microscopy with particular reference to the subepidermal network of respiratory capillaries. Due to the fact that arteries and veins lie in the deeper layers of the stratum spongiosum of the corium, the respiratory vessels form a morphologically homogeneous network. Functionally, however, this network is subdivided into small areas with a centripetal direction of blood flow. The deep capillary net, situated at the base of the stratum compactum of the corium, is not so dense as the respiratory network and does not directly communicate with it. Alveolar glands of the skin have no effect on the distribution of capillaries in the two networks.     

FIB‐SEM tomography of human skin telocytes and their extracellular vesicles

We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non‐epithelial spaces (stroma) of many organs (see www.telocytes.com ). TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms (containing mitochondria, endoplasmic reticulum and caveolae) and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB‐SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB‐SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon‐like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) – the podoms. FIB‐SEM tomography also showed numerous extracellular vesicles (diameter 438.6 ± 149.1 nm, n = 30) released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc.