Enteric Glia and Enteric Neurons, Associated.

Peripheral neurons are never found alone and are invariably accompanied by glial cells, with which they are intimately associated in compact, highly deformable structures.Myenteric ganglia of the guinea-pig, examined in situ by electron microscopy, show that in their neuropil (axons and dendrites, and glial cells and processes) the glia constitutes almost half of the volume and almost half of membrane extent.In the glia, the expanse of the cell membrane predominates over that of their cytoplasm, the opposite being the case with the neural elements.The profile of the glial elements is passive and is dictated by the surrounding elements, mainly the axons, and hence it is predominantly concave.The enteric glia is widely developed; however, it is not sufficient to form a full wrapping around all neurons and around all axons (unlike what is found in other autonomic ganglia).Glial processes are radially expanding laminae, irregularly tapering, branching, and penetrating between axons.Some processes have a specialized termination attached to the basal lamina of the ganglion.Mitochondria are markedly more abundant in neural element that in the glia (up to a factor of 2).Many expanded axons, laden with vesicles clustered beneath membrane sites, abut on glial cells and processes, while these show no matching structural specializations.

Enteric glia: extent, cohesion, axonal contacts, membrane separations and mitochondria in Auerbach's ganglia of guinea pigs.

Studied by electron microscopy and morphometry, Auerbach's ganglia comprise nerve cell bodies that occupy ~ 40% of volume; of the neuropil, little over 30% is neural processes (axons, dendrites) and little less than 30% is glia (cell bodies, processes). The amount of surface membrane of neural elements only marginally exceeds that of glia. Glial cells extend laminar processes radially between axons, reaching the ganglion's surface with specialized membrane domains. Nerve cells and glia are tightly associated, eliminating any free space in ganglia. Glia expands maximally its cell membrane with a minimum of cytoplasm, contacting a maximal number of axons, which, with their near-circular profile, have minimal surface for a given volume. Shape of glia is moulded by the neural elements (predominantly concave the first, predominantly convex the second); the glia extends its processes to maximize contact with neural elements. Yet, a majority of axons is not reached by glia and only few are wrapped by it. Despite the large number of cells, the glia is not sufficiently developed to wrap around or just contact many of the neural elements. Mitochondria are markedly fewer in glia than in neurons, indicating a lower metabolic rate. Compactness of ganglia, their near-circular profile, absence of spaces between elements and ability to withstand extensive deformation suggest strong adhesion between the cellular elements, holding them together and keeping them at a fixed distance. Many axonal varicosities, with vesicles and membrane densities, abut on non-specialized areas of glia, suggesting the possibility of neurotransmitters being released outside synaptic sites.

Spatial lay-out of various smooth muscles.

The characteristic mechanical activities of the smooth muscles found in all organs of the body are highly variable and depend mainly on the spatial arrangement of the muscle cells and the stroma: mass, orientation, relationships, links, constraints, which are deployed in various configurations. These structural features are examined here for their mechanical relevance, in light and electron microscopic views of several muscles of viscera and blood vessels, in a selection of mammalian species. Smooth muscles are incompressible and therefore maintain constant volume. They do not have available space and any movement of a part requires displacement of another part. Most of them have no terminations or points of attachment, and in hollow organs such as intestines, blood vessels and uro-genital tract they usually form structures closed onto themselves, such as rings or bag-like containers In these situations, changes in the size of the lumen is achieved very efficiently by a concentric inward enlargement that accompanies muscle contraction. The longitudinal arrangement of collagen blocks an elongation of small blood vessels upon contraction, further enhancing the efficiency of lumen reduction. In other muscles, links between layers and special arrangements of the stroma allow both shortening and elongation of a tubular organ to occur. The mechanics of smooth muscles has many characteristic features (some unique, some shared with those of hydrostats, some at variance with other muscles) and histological data are a contribution to our understanding of these properties.

Muscle cells, nerves, fibroblasts and vessels in the detrusor of the rat urinary bladder.

All the cells of rat detrusor muscle fall into one of five ultrastructural types: muscle cells, fibroblasts, axons and glia, and vascular cells (endothelial cells and pericytes). The tissue is ~79% cellular and 21% non-cellular. Muscle cells occupy 72%, nerves ~4% (1/3 axons, 2/3 glia), and fibroblast >3% of space. Muscle cells (up to 6 µm across and ~600 µm long, packed to almost 100,000 per mm2) have surface-to-volume ratio of 2.4 µm2/µm3 ~93% of cell volume is contractile apparatus, 3.1% mitochondria and 2.5% nucleus. Cell profiles are irregular but sectional area decreases regularly towards either end of the cell. Muscle cells are gathered into bundles (the mechanical units of detrusor), variable in length and size, but of constant width. The musculature is highly compact (without fascia or capsule) with smooth outer surfaces and extensive association and adhesion between its cells. Among many types of intercellular contact and junction, digitations are very common, each muscle cell issuing minute finger-like processes that abut on adjacent cells. Sealed apposition are wide areas of specialized contact, possibly forming a chamber between two muscle cells, distinct from the extracellular space at large (stromal space). The innervation is very dense, virtually all intramuscular axons being varicose (including afferent ones). There are identifiable neuro-muscular junctions on each muscle cell, often several junctions on a single cell. There are also unattached terminals. Fibroblasts (involved in the production of collagen), ~1% of the total number of cells, do not make specialized contacts.

Lamina propria: The connective tissue of rat urinary bladder mucosa.

To describe and illustrate the structure of the propria, the bladder of adult rats was fixed in controlled conditions of distension and examined by light and electron microscopy. The lamina propria, ~50 µm thick in the distended bladder, consists of a superficial part (the cellular component), adjacent to the urothelium, rich in nerves, capillaries, fibroblasts and thin bundles of collagen, and a deep, thicker part (the fibrous component), adjacent to the detrusor, rich in large collagen fibres and with few fibroblasts. In the cellular part there is an extensive plexus of afferent nerve fibers and a dense capillary network (with numerous pericytes), lying close to the urothelium, that is unique to the bladder. The main resident cells are fibroblasts, adhering to each other at the end of laminar extensions without forming specialized junctions. The deep part of the lamina propria is made of thick collagen fibers, interwoven and crisscrossing each other, with a few fibroblasts in the interstitial spaces between them. In summary, the superficial part of the lamina propria has most of the bladder afferent nerves, contains many fibroblasts and has a network of suburothelial capillaries. The deep part as a whole forms an ovoid balloon of woven fibrous material that is acted upon by the detrusor musculature attached to its outer surface. The lamina propria is a strong fibrous barrier between urothelium and musculature. The abundance of collagen points to the main role for its fibroblasts, that is, the production of collagen fibrils, assisting the mechanical role of the lamina propria.

Afferent nerve fibres in the wall of the rat urinary bladder.

Structure and distribution of afferent nerve fibres in the rat bladder were studied by fluorescence microscopy after selective staining with antibodies against neuropeptide CGRP. Afferent fibres are very abundant (by comparison with other viscera) and interconnected in all bladder parts: muscle, urothelium, connective tissue, blood vessels, serosa. Their highest concentration is beneath the urothelium in equatorial and caudal regions, where they form a plexus, while individually maintaining a tree-like structure with innumerable branches running without preferential orientation. In cranial regions, mucosal afferent fibres become rare or absent. Abundant fibres are found in the detrusor, within each muscle bundle, with long strings of varicosities parallel to muscle cells. Afferent fibres, invariably varicose over hundreds of micrometres of their terminal parts and while still branching, comprise chains of hundreds of varicosities. Varicosities are irregular in size, frequency and separation, without specialised terminal structures around them, or within or around the fibre's ending. The possibility that varicosities are transduction points for sensory inputs is discussed, with the implication of a process taking place over considerable length in each branch of each fibre. Interconnectedness of afferent nerves of various bladder tissues, distribution of varicosities over hundreds of micrometres along axonal branches, absence of clear target structures for the fibres, apparent irregularity in the size and sequence of varicosities suggest an innervation that is not rigidly wired with distinct sensory pathways. In fact, the structural evidence suggests extensive afferent integration at the periphery, with wide distribution of source points and broad range of physical detectors.

Mitochondria in smooth muscle cells of viscera.

The spatial density of mitochondria was studied by thin-section electron microscopy in smooth muscles of bladder, iris and gut in mice, rats, guinea-pigs and sheep. Morphometric data included areas of muscle cell profiles (~6,000 muscle cells were measured) and areas of their mitochondria (more than three times as many). The visual method delivers accurate estimates of the extent of the chondrioma (the ensemble of mitochondria in a cell), measuring all and only the mitochondria in each muscle cell and no other cells. The digital records obtained can be used again for checks and new searches. Spatial density of mitochondria varies between about 2 and 10% in different muscles in different species. In contrast, there is consistency of mitochondrial density within a given muscle in a given species. For each muscle in each species there is a characteristic mitochondrial density with modest variation between experiments. On the basis of data from serial sections in the rat detrusor muscle, mitochondrial density varies very little between the muscle cells, each cell having a value close to that for the whole muscle. Mitochondrial density is different in a given muscle, e.g., ileal circular muscle, from the four mammalian species, with highest values in mouse and lowest in sheep; in mice the mitochondrial density is nearly three time higher that in sheep. In a given species there are characteristic variations between different muscles. For example, the bladder detrusor muscle has markedly fewer mitochondria than the ileum, and the iris has markedly more.

Increased stiffness and cell-matrix interactions of abdominal aorta in two experimental nonhypertensive models: long-term chemically sympathectomized and sinoaortic denervated rats.

RATIONALE: Sinoaortic denervated (SAD) and chemically sympathectomized (SNX) rats are characterized by a decrease in arterial distensibility without hypertension and would, thus, be relevant for analyzing arterial wall stiffening independently of blood pressure level. The fibronectin network, which plays a pivotal role in cell-matrix interactions, is a major determinant of arterial stiffness. We hypothesized that in SAD and SNX rats, arterial stiffness is increased, due to alterations of cell-matrix anchoring leading to spatial reorganization of the extracellular matrix. METHODS: The intrinsic elastic properties of the arterial wall were evaluated in vivo by the relationship between incremental elastic modulus determined by echotracking and circumferential wall stress. The changes of cell-extracellular matrix links in the abdominal aorta were evaluated by studying fibronectin, vascular integrin receptors, and ultrastructural features of the aorta by immunochemistry. RESULTS: In both experimental conditions, wall stiffness increased, associated with different modifications of cell-extracellular matrix adhesion. In SAD rats, increased media cross-sectional area was coupled with an increase of muscle cell attachments to its extracellular matrix via fibronectin and its α5-ß1 integrin. In SNX rats, reduced media cross-sectional area was associated with upregulation of αv-ß3 integrin and more extensive connections between dense bands and elastic fibers despite the disruption of the elastic lamellae. CONCLUSION: In aorta of SNX and SAD rats, a similar arterial stiffness is associated to different structural alterations. An increase in αvß3 or α5ß1 integrins together with the already reported increase in the proportion of less distensible (collagen) to more distensible (elastin) components in both models contributes to remodeling and stiffening of the abdominal aorta.

Nerve distribution in rat urinary bladder after incorporation of acellular matrix graft or subtotal cystectomy.

OBJECTIVE: In the treatment of reduced bladder capacity, matrix grafts have been used as a scaffold into which cell elements from the native bladder grow, eventually forming a new bladder segment. Functioning motor nerve endings in such segments in the rat have been demonstrated, although little is known about nerve distribution. We compare the pattern of nerve distribution in scaffold-augmented rat bladders with that in bladders regrown after subtotal cystectomy and that in control bladders. MATERIAL AND METHODS: Female Sprague-Dawley rats were either subtotally cystectomized (n=7) or had a part of the bladder dome replaced by an acellular collagen (small intestinal submucosa) matrix graft (n=10). Fourteen age-matched, unoperated animals were used as controls. Two and a half to 10 months after surgery the bladders were stained for acetylcholinesterase and studied in wholemounts. RESULTS: No ganglion neurons were observed in any of the bladders. On their ventral side the control bladders showed longitudinal nerve trunks, running in parallel along the longitudinally oriented muscle bundles, while on the lateral and dorsal aspects the nerves were thinner, more irregularly arranged and frequently branched. In the bladders regrown after subtotal cystectomy, the ventral nerves were seen running obliquely to the still longitudinally oriented muscle bundles, resembling the pattern of the normal bladder base; the pattern of the dorsolateral nerves was the same as that in the controls. In the matrix bladders, the muscle and nerve patterns in the native part were the same as those in controls. Muscle bundles were growing into the matrix, accompanied by nerves, which showed limited branching when entering the matrix, usually running in parallel to the muscle, but then branching within the matrix. CONCLUSIONS: The nerves in the matrix grafts and the regrown parts of the subtotally cystectomized bladders derive from preexisting nerves in the bladder. In neither case does the nerve trunk or muscle bundle arrangement fully attain the pattern found in normal bladders.

Estimate of size and total number of neurons in superior cervical ganglion of rat, capybara and horse.

The superior (cranial) cervical ganglion was investigated by light microscopy in adult rats, capybaras (Hydrochaeris hydrochaeris) and horses. The ganglia were vascularly perfused, embedded in resin and cut into semi-thin sections. An unbiased stereological procedure (disector method) was used to estimate ganglion neuron size, total number of ganglion neurons, neuronal density. The volume of the ganglion was 0.5 mm3 in rats, 226 mm3 in capybaras and 412 mm3 in horses. The total number of neurons per ganglion was 18,800, 1,520,000 and 3,390,000 and the number of neurons per cubic millimetre was 36,700, 7,000 and 8,250 in rats, capybaras and horses, respectively. The average neuronal size (area of the largest sectional profile of a neuron) was 358, 982 and 800 microm2, and the percentage of volume occupied by neurons was 33, 21 and 17% in rats, capybaras and horses, respectively. When comparing the three species (average body weight: 200 g, 40 kg and 200 kg), most of the neuronal quantitative parameters change in line with the variation of body weight. However, the average neuronal size in the capybara deviates from this pattern in being larger than that of in the horse. The rat presented great interindividual variability in all the neuronal parameters. From the data in the literature and our new findings in the capybara and horse, we conclude that some correlations exist between average size of neurons and body size and between total number of neurons and body size. However, these correlations are only approximate and are based on averaged parameters for large populations of neurons: they are less likely to be valid if one considers a single quantitative parameter. Several quantitative features of the nervous tissue have to be taken into account together, rather than individually, when evolutionary trends related to size are considered.

Development of visceral smooth muscle.

The development of the smooth musculature of viscera has attracted the interest of only relatively few investigators, and thus the field appears somewhat underexplored. The major emphasis on histochemical evidence--at the expense of ultrastructural and functional studies--may have limited the progress in this area. Mature tissue is formed through the differentiation of precursors into muscle cells and through the organization of these cells into a complex tissue where distribution and orientation of muscle cells, deployment of abundant extracellular materials and addition of other cellular elements (interstitial cells, fibroblasts, nerves, blood vessels) are characteristic and specific features. The precursor cells are found at sites where a muscle develops, and they derive predominantly from the mesoderm, but also from the neuroectoderm and from the endoderm. The process starts at different times in different organs. The earliest stages of differentiation are characterized by the precursor cells aggregating and becoming elongated; their longitudinal axis lies in a position similar to the one they will have in the mature muscle. Both the cytological and the histochemical differentiation follow distinct patterns in various muscles, with characteristic temporal sequences in the appearance of key features. This process must impart distinct functional properties to a muscle cell at each stage of its development. However, the chronological correspondence between ultrastructural and histochemical development is poorly understood. Histochemical studies have detected gradients of maturation of the muscle cells, for example, across the thickness of the gizzard musculature and along the length of the small intestine; ultrastructural studies have not yet confirmed the existence of these gradients. Muscle growth is accounted for by muscle cell enlargement (without nucleus duplication) and an increase in muscle cell number by mitosis of pre-existing differentiated muscle cells. De-differentiation and division of muscle cells, migration of muscle cells and late development of muscle cell precursors have all also been considered as possible mechanisms for muscle growth. Several authors have described the presence of precursor cells within developing smooth muscles, and they have described late differentiation of some muscle cells or waves of differentiation that would give rise to phenotypic heterogeneity of the mature muscle cell population. In contrast, other studies, mainly by electron microscopy, have suggested that, within large visceral muscles, the muscle cells differentiate synchronously. There are interesting data on the influence of adjacent tissues on the development of a smooth muscle, but the interplay of these and other factors has not been fully investigated. Smooth muscles contract from early in their development, hence mechanical factors are likely to influence development: on the one hand, passive stresses imposed on the muscle by other tissues, such as adjacent muscles or the contents of the viscera and, on the other hand, active forces generated by the muscle itself. The very attraction of visceral smooth muscles in the study of cellular morphogenesis--an attraction that has not yet been highlighted or exploited in scientific studies, either descriptively or experimentally--is that, onto a single type of cell, a large range of factors interact, such as the genetic expression, chemical influences (from other muscles, endocrine glands, nerves, other intramuscular cells) and mechanical factors.

Killing activity of neutrophils is mediated through activation of proteases by K+ flux.

According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.