Microvascular architecture of mouse urinary bladder described with vascular corrosion casting, light microscopy, SEM, and TEM.

The urinary bladder is a unique organ in that its normal function is storage and release of urine, and vasculature in its wall exhibits specialized features designed to accommodate changes in pressure with emptying and filling. Although we have previously described the fine details of the microvasculature of the urinary bladder of the rabbit and dog, information on the fine details of the microvasculature of the mouse bladder were deemed to be of value because of the increasing use of this species in developing genetic models for studying human disorders. The present study shows that many of the special features of the microvasculature of the mouse urinary bladder are similar to those described in the rabbit and dog, including vessel coiling, abundant collateral circulation, arterial sphincters, and a dense mucosal capillary plexus.

Three-dimensional analysis and computer modeling of the capillary endothelial vesicular system with electron tomography.

OBJECTIVE: We examined the three-dimensional organization of the endothelial vesicular system with TEM tomography of semi-thick sections. MATERIALS AND METHODS: Mouse abdominal muscle capillaries were perfused with terbium to label vesicular compartments open to the luminal surface. The tissue was prepared for TEM and semi-thick (250 nm) sections were cut. Dual axis tilt series, collected from +60° to -60° at 1° increments, were acquired in regions of labeled abluminal caveolae. These tomograms were reconstructed and analyzed to reveal three-dimensional vesicular associations not evident in thin sections. RESULTS: Reconstructed tomograms revealed free vesicles, both labeled and unlabeled, in the endothelial cytoplasm as well as transendothelial channels that spanned the luminal and abluminal membranes. A large membranous compartment connecting the luminal and abluminal surfaces was also present. Computer modeling of tomographic data and video animations provided three-dimensional perspectives to these structures. CONCLUSIONS: Uncertainties associated with other three-dimensional methods to study the capillary wall are remedied by tomographic analysis of semi-thick sections. Transendothelial channels of fused vesicles and free cytoplasmic vesicles give credence to their role as large pores in the transport of solutes across the walls of continuous capillaries.

High-resolution imaging of kidney vascular corrosion casts with Nano-CT.

A vascular corrosion cast of an entire mouse kidney was scanned with a modular multiresolution X-ray nanotomography system. Using an isotropic voxel pitch of 0.5 µm, capillary systems such as the vasa recta, peritubular capillaries and glomeruli were clearly resolved. This represents a considerable improvement over corrosion casts scanned with microcomputed tomography systems. The resolving power of this system was clearly demonstrated by the unique observation of a dense, subcapsular mat of capillaries enveloping the entire outer surface of the cortical region. Resolution of glomerular capillaries was comparable to similar models derived from laser scanning confocal microscopy. The high-resolution, large field of view and the three-dimensional nature of the resulting data opens new possibilities for the use of corrosion casting in research.

Microvasculature of the urinary bladder of the dog: a study using vascular corrosion casting.

The urinary bladder is an unusual organ in that its normal function includes filling and emptying with alternating changes in internal pressure. Although fluctuations in blood flow to the bladder wall are known to accompany these changes, detailed descriptions of the bladder microvasculature are sparse. The present study uses vascular corrosion casting and scanning electron microscopy to describe the three-dimensional anatomy of the microvasculature of the urinary bladder of the dog. Specialized features of that microvasculature, including collateral circulation, vessel folding, vessel orientation, the presence of valves and sphincters, and mucosal capillary density, that may enhance and control blood flow during normal bladder function, are described and discussed.

Bacterial activation of mast cells.

Mast cells often are found in a perivascular location but especially in mucosae, where they may response to various stimuli. They typically associate with immediate hypersensitive responses and are likely to play a critical role in host defense. In this chapter, a common airway pathogen, Moraxella catarrhalis, and a commensal bacterium, Neiserria cinerea, are used to illustrate activation of human mast cells. A human mast cell line (HMC-1) derived from a patient with mast cell leukemia was activated with varying concentrations of heat-killed bacteria. Active aggregation of bacteria over mast cell surfaces was detected by scanning electron microscopy. The activation of mast cells was analyzed by nuclear factor-kappaB (NF-kappaB) activation and cytokine production in culture supernatants. Both M. catarrhalis and N. cinerea induce mast cell activation and the secretion of two key inflammatory cytokines, interleukin-6 and MCP-1. This is accompanied by NF-kappaB activation. Direct bacterial contact with mast cells appears to be essential for this activation because neither cell-free bacterial supernatants nor bacterial lipopolysaccharide induce cytokine secretion.

Confocal microscopy, computer modeling, and quantification of glomerular vascular corrosion casts.

Corrosion-casted capillary systems of the kidney glomerulus were imaged with confocal microscopy because of the fluorescence properties of the casting plastic. Acquisition of a z-series through the glomerular capillaries provided three-dimensional data sets from which surface-rendered models were generated. These models could be rotated and viewed from any angle and also contained quantitative information allowing cast surface area and volume measurements to be calculated. The computer-generated models were also skeletonized to form a one-voxel-thick skeleton of the original model. The skeleton exhibited the three-dimensional topology and network of the capillary bed, and interior capillary relations could also be viewed. Quantitative information such as the total capillary length and number of capillary intersects was calculated from the skeletonized model. Extending this method to noncorroded kidney specimens revealed not only the casted vessels but also cellular features of the adjacent tissues surrounding the capillaries.

Regulation of eosinophil-active cytokine production from human cord blood-derived mast cells.

Human mast cells are multifunctional tissue-dwelling cells that play a crucial role in eosinophil-dependent disorders, such as asthma and parasitic diseases, by the secretion of eosinophil-active mediators. Mast cell-derived cytokines, generated in response to cross-linking of the high-affinity IgE receptor, can regulate eosinophil activation, survival, and chemotaxis. In this study, mast cells generated from human cord blood progenitors (stem cells) were studied for eosinophil-active inflammatory cytokine expression. Cord blood-derived mast cells (CBDMC) expressed typical intracellular scroll granules and microvilli-like structures on their cell surfaces, demonstrated the presence of tryptase, and elaborated prostaglandin D2 (PGD2) after cross-linkage of the high-affinity receptor for IgE (FcepsilonRI). CBDMC expressed tumor necrosis factor-alpha (TNF-alpha) and the eosinophil-active growth factors, interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after activation. (IL-1beta greatly enhanced IgE-dependent production of these cytokines in response to FcepsilonRI cross-linkage, suggesting a role for bystander/phagocytic cells in modulating mast cell function. In contrast, interferon-alpha (IFN-alpha) inhibited IL-5 and GM-CSF generation, and the glucocorticoid, dexamethasone (Dex), inhibited production of IL-5 and GM-CSF from CBDMC. A macrophage-mast cell-eosinophil axis may exist in vivo that may be susceptible to pharmacologic manipulation.

Na+,K+-ATPase activity and ultrastructural localization in the tegmentum vasculosum in the cochlea of the duckling.

The tegmentum vasculosum of the avian cochlear duct mimics the stria vascularis of the mammalian cochlear duct in both location and structure, and previous studies indicate that it may be its functional counterpart with regard to endolymph synthesis. In the present study, we report on the enzymatic activity and ultrastructural localization of the Na+,K+-ATPase in the tegmentum vasculosum of the duckling. Na+,K+-ATPase activity was determined by measuring K+-dependent, ouabain-sensitive p-nitrophenyl phosphatase (p-NPPase) activity in homogenates of dissected regions of the cochlear duct. The ultrastructural localization of the Na+,K+-ATPase was identified using K+-dependent, ouabain-sensitive, p-NPPase cytochemistry. Specific enzyme activity was localized primarily in homogenates of the tegmentum vasculosum (2.27 micromol p-nitrophenyl phosphate/mg protein/min) when compared to homogenates of the entire cochlear duct (0.69 micromol p-nitrophenyl phosphate/mg protein/min). Reaction product for p-NPPase was localized primarily along the basolateral plasma membrane folds of the dark cells. The cytochemical deposits appeared to be located exclusively on the cytoplasmic side of the plasma membrane. The light cells were devoid of reaction product. Biochemical and cytochemical localization of p-NPPase activity on the basolateral plasma membrane folds of the dark cells of the tegmentum vasculosum in conjunction with the ultrastructural morphology of these cells is compatible with a Na+,K+-ATPase-dependent ion transport function related to endolymph synthesis.

Ultrastructure and blood supply of the tegmentum vasculosum in the cochlea of the duckling.

The tegmentum vasculosum of the duckling consists of a highly folded epithelium which extends over the dorsal and lateral walls of the cochlear duct, separating the scala media from the scala vestibuli. This epithelium consists of two distinct cell types, dark cells and light cells, and is well vascularized. The surface of the epithelium is formed by a mosaic of alternating dark and light cells. The goblet-shaped dark cells have an electron-dense, organelle-rich cytoplasm, and are expanded basally by extensive basolateral plasma membrane infoldings, within which are numerous mitochondria. Dark cells are isolated from each other and from the capillaries within the epithelium by intervening light cells. In contrast, columnar light cells exhibit an electron-lucent, organelle-poor cytoplasm and may extend from the underlying capillaries to the endolymphatic surface. Light cells contain abundant, coated endocytic vesicles on their apical surfaces and are bound, apically, to other light cells or to dark cells by tight junctions and desmosomes. Laterally, light cells are linked to each other either by complex, fluid-filled membrane interdigitations or by extensive gap junctions. Plasma membrane interdigitations and obvious, fluid-filled intercellular spaces characterize the lateral borders between light and dark cells. Vascular corrosion casting reveals the three-dimensional anatomy of the cochlear vasculature. A continuous arteriolar loop fed by anterior and posterior cochlear arterioles encircles the cochlear duct. The rich capillary beds of the tegmentum vasculosum are supplied by arching arterioles arising from this loop. These capillaries are the continuous type and are situated primarily within the core of the epithelium or along its border with the scala vestibuli. The structure and blood supply of the tegmentum vasculosum are characteristic of an epithelium involved in active transport.

Vascular Corrosion Casting: Review of Advantages and Limitations in the Application of Some Simple Quantitative Methods.

Vascular corrosion casting has been used for about 40 years to produce replicas of normal and abnormal vasculature and microvasculature of various tissues and organs that could be viewed at the ultrastructural level. In combination with scanning electron microscopy (SEM), the primary application of corrosion casting has been to describe the morphology and anatomical distribution of blood vessels in these tissues. However, such replicas should also contain quantitative information about that vasculature. This report summarizes some simple quantitative applications of vascular corrosion casting. Casts were prepared by infusing Mercox resin or diluted Mercox resin into the vasculature. Surrounding tissues were removed with KOH, hot water, and formic acid, and the resulting dried casts were observed with routine SEM. The orientation, size, and frequency of vascular endothelial cells were determined from endothelial nuclear imprints on various cast surfaces. Vascular volumes of heart, lung, and avian salt gland were calculated using tissue and resin densities, and weights. Changes in vascular volume and functional capillary density in an experimentally induced emphysema model were estimated from confocal images of casts. Clearly, corrosion casts lend themselves to quantitative analysis. However, because blood vessels differ in their compliances, in their responses to the toxicity of casting resins, and in their response to varying conditions of corrosion casting procedures, it is prudent to use care in interpreting this quantitative data. Some of the applications and limitations of quantitative methodology with corrosion casts are reviewed here.

Comparative study of dentition among species of Poecilia (Pisces).

Many studies in the genus Poecilia have focused on reproductive and genetic characteristics of Poecilia formosa, the Amazon molly, and its sympatric species P. latipinna and P. mexicana. The research literature of Poecilia dentition has been limited to general tooth morphology. Essentially absent are comparative analyses of dentition patterns and total numbers of teeth. The current study uses dentition analysis as a method to compare species in the genus Poecilia and to address some taxonomic issues related to these fish. The study focused on fish from the areas of southern Texas and northeastern Mexico. Through the use of scanning electron microscopy, the lower jaws of Poecilia spp were examined to determine total numbers of outer and inner teeth. In addition, the differences in distribution patterns of the inner teeth were recorded and compared. Statistical analyses were performed to determine which comparisons were significant. This study reveals several observations: 1) variations in the numbers of outer and inner teeth exist in some of these fish with respect to site of collection; 2) differences in total teeth numbers and dentition patterns were found both interspecifically and intraspecifically; and 3) in addition, dentition analysis provided evidence regarding the origin of P. formosa. This study supports the current notion that P. latipinna, the proported paternal component, and P. mexicana limantouri, the purported maternal component, are the progenitor species of P. formosa. Two unresolved taxonomic questions were addressed through dentition analysis. First, the present study supports the exclusion of the triploid associate of P. formosa as a separate species from P. formosa. Second, this study shows a significant difference in the number of inner teeth and in dentition patterns between P. mexicana limantouri and P. mexicana mexicana. Such differences, in addition to previously known distinguishing characteristics, should prompt careful consideration of whether or not these taxa deserve specific status or retention of their current subspecific status. J. Morphol. 239:271-282, 1999. © 1999 Wiley-Liss, Inc.

The gill arch of the striped bass (Morone saxatilis). IV. Alterations in the ultrastructure of chloride cell apical crypts and chloride efflux following exposure to seawater.

Osmotically induced alterations in the ultrastructure of the apical crypts of chloride cells and changes in chloride efflux were studied in striped bass (Morone saxatilis). Striped bass were divided into three groups: fish adapted to freshwater, fish transferred directly from freshwater to 100% seawater (3% salt, w/v) for 24 hr or less, and fish adapted to 100% seawater for 7 days or more. Transmission electron microscopy studies revealed multicellular complexes of cells in both freshwater- and seawater-adapted fish. Cytoplasmic indigitations between cells in the complex were more numerous in seawateradapted bass. Scanning electron microscopy studies showed that the apical extensions in freshwater fish were uniform in size. Changes in ultrastructure and chloride efflux were observed within 3 hr after transfer to seawater. Initially the apical extensions of chloride cells become longer, more prominent, and branched. After 7 days in seawater some of the apical crypts develop into a deeper "pit" structure, while others remain like those of freshwater fish. An increase in the number of apical crypts is measured by 14 days after transfer. Chloride efflux increases to five times freshwater values after 24 hr and 17 times freshwater values after 7 days in seawater. Mitochondrial density is not significantly different between freshwater and seawater fish (7 or more days). The response of chloride cell apical crypts is not an all-or-none phenomenon as observed in other species. Striped bass are able to increase chloride efflux when osmotically stressed with little ultrastructural alteration.