Giorgio Gabella

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.

Innervation of the Gastrointestinal Tract

Publisher Summary This chapter discusses the innervation of the gastrointestinal tract. It examines the structures involved in nervous control of the activities of the gastrointestinal tract. They can be classified as (1) nerve pathways reaching the gastrointestinal tract, including afferent and efferent pathways, (2) ganglia and plexuses located within the wall of the stomach and the intestine, and (3) terminal parts of nerve fibers making contact with the effectors (muscular and glandular) or forming sensory endings. The effectors include secretory cells and smooth-muscle cells of the intramural blood vessels and of the muscle layers proper. The presence of highly developed intramural plexuses is a characteristic feature of the gastrointestinal tract. It is probably related to the complexity of functions of the vertebrate gut, notably regulated propulsive activity. The efferent pathways leading to the gastrointestinal tract can be divided into three major groups: (1) the vagus nerves, (2) the splanchnic nerves, with the abdominal plexus and its perivascular nerves reaching the stomach and intestine, and (3) the pelvic plexus and its nerves.

Fall in the number of myenteric neurons in aging guinea pigs

The neurons of the myenteric plexus of the entire small intestine were stained in young adult (3-4 mo old) and aging guinea pigs (26-30 mo old). Total length and circumference of the intestine were measured in the same experiments. The small intestine of the aging guinea pigs was longer, and (in the conditions of distention used) had a total serosal surface approximately 70% greater than in young adult animals. The spatial density of myenteric neurons per unit of serosal surface fell dramatically in aging animals, and the total number of myenteric neurons in the small intestine ranged between 1.1 and 1.6 million, i.e., it was only 40%-60% of the value obtained in young adult guinea pigs (2.75 million). The light microscope appearance of the neurons of the two groups of animals was markedly different and the suggestion is put forward that in aging guinea pigs the substantial reduction in neuron number is accompanied by structural changes and reorganization of the neurons that are left.

Urinary bladder of rat: fine structure of normal and hypertrophic musculature

SummaryThe fine structure of the muscle of the urinary bladder in female rats is similar to that of other visceral muscles, although it is arranged in bundles of variable length, cross-section and orientation, forming a meshwork. When distended, the musculature is 100–120 μm thick, with some variation and occasional discontinuity. Extended areas of cell-to-cell apposition with uniform intercellular space occur between muscle cells, whereas attachment plaques for mechanical coupling are less common than in other visceral muscles. There are no gap junctions between muscle cells. Many bundles of microfilaments and small elastic fibres run between the muscle cells. After chronic partial obstruction of the urethra, the bladder enlarges and is about 15 times heavier, but has the same shape as in controls; the growth is mainly accounted for by muscle hypertrophy. The outer surface of the hypertrophic bladder is increased 6-fold over the controls; the muscle is increased 3-fold in thickness, and is more compact. Mitoses are not found, but there is a massive increase in muscle cell size. There is a modest decrease in percentage volume of mitochondria, an increase in sarcoplasmic reticulum, and no appreciable change in the pattern of myofilaments. Gap junctions between hypertrophic muscle cells are virtually absent. Intramuscular nerve fibres and vesicle-containing varicosities appear as common in the hypertrophic muscle as in controls. There is no infiltration of the muscle by connective tissue and no significant occurrence of muscle cell death.

DISTRIBUTION OF AFFERENT AXONS IN THE BLADDER OF RATS

The distribution of afferent axons in the bladder of rats was studied by means of immunohistochemistry for calcitonin gene-related peptide (CGRP), in frozen sections and in wholemount preparations of mucosa and muscle coat. Synaptophysin-immunofluorescence was used for the general detection of all intramural axons. The afferent axons were distributed over four distinct targets: at the base of the epithelium, inside the epithelium, on blood vessels (both arteries and veins) and along muscle bundles. In the mucosa, all the afferent axons, except the perivascular ones, lay either inside the epithelium or in a subepithelial plexus very close to the basal surface of the epithelium. The plexus was thickest in the neck of the bladder and in the initial portion of the urethra, and it became progressively less dense in the adjacent regions; it did not extend beyond the equatorial region, and therefore the mucosa of the cranial region of the bladder had no afferent axons. Most of the axons in the subepithelial plexus were terminal axons and included conspicuous varicosities arranged in very long chains; branching points were numerous, usually at right angles and located at the level of a varicosity; some axons split and then rejoined, forming closed axonal loops. The afferent innervation of the musculature was more diffuse, and appeared uniform throughout the bladder. After unilateral surgical denervation (by excision of the pelvic ganglion 5–7 days earlier) areas of complete denervation were observed, but there were large areas where the innervation was only reduced. The results showed that there is a bilateral innervation of many regions of the mucosa and the musculature, including individual muscle bundles. A substantial number of fibres crossed the midline into the contralateral side of the bladder. CGRP-immunofluorescence in mucosal afferent axons is enhanced in the surviving axons 5 days after contralateral denervation, a change which is interpreted as an early sign of regeneration.

Presence and Distribution of Sensory Nerve Fibers in Human Peritoneal Adhesions

ObjectiveTo assess the distribution and type of nerve fibers present in human peritoneal adhesions and to relate data on location and size of nerves with estimated age and with clinical parameters such as reports of chronic pelvic pain. Summary Background DataPeritoneal adhesions are implicated in the cause of chronic abdominopelvic pain, and many patients are relieved of their symptoms after adhesiolysis. Adhesions are thought to cause pain indirectly by restricting organ motion, thus stretching and pulling smooth muscle of adjacent viscera or the abdominal wall. However, in mapping studies using microlaparoscopic techniques, 80% of patients with pelvic adhesions reported tenderness when these structures were probed, an observation suggesting that adhesions themselves are capable of generating pain stimuli. MethodsHuman peritoneal adhesions were collected from 25 patients undergoing laparotomy, 20 of whom reported chronic pelvic pain. Tissue samples were prepared for histologic, immunohistochemical, and ultrastructural analysis. Nerve fibers were characterized using antibodies against several neuronal markers, including those expressed by sensory nerve fibers. In addition, the distribution of nerve fibers, their orientation, and their association with blood vessels were investigated by acetylcholinesterase histochemistry and dual immunolocalization. ResultsNerve fibers, identified histologically, ultrastructurally, and immunohistochemically, were present in all the peritoneal adhesions examined. The location of the adhesion, its size, and its estimated age did not influence the type of nerve fibers found. Further, fibers expressing the sensory neuronal markers calcitonin gene-related protein and substance P were present in all adhesions irrespective of reports of chronic abdominopelvic pain. The nerves comprised both myelinated and nonmyelinated axons and were often, but not invariably, associated with blood vessels. ConclusionsThis study provides the first direct evidence for the presence of sensory nerve fibers in human peritoneal adhesions, suggesting that these structures may be capable of conducting pain after appropriate stimulation.

Neurogenic inflammation in the rat trachea. II. Identity and distribution of nerves mediating the increase in vascular permeability

SummaryThis study addresses the question of whether increased vascular permeability, which is a prominent feature of neurogenic inflammation in the respiratory tract, is mediated by sensory axons that end near venules in the airway mucosa. In these experiments, neurogenic inflammation was produced in the tracheal and bronchial mucosa of atropine-treated Long-Evans rats by electrical stimulation of the left or right superior laryngeal nerve and/or cervical vagus nerve. The particulate tracer Monastral blue was injected intravenously to localize the sites of increased vascular permeability, and microspectrophotometry was used to measure the amount of extravasated Monastral blue in the trachea and thereby quantify the increase in vascular permeability. In some rats, selective denervations were made to locate the cell bodies of neurons that mediate the increase in vascular permeability; in others, fluorescence immunohistochemistry and quantitative electron microscopic methods were used to determine which structures in the tracheal mucosa are innervated by these neurons. The study revealed that the vagally mediated increase in vascular permeability was sudden, transient (half-life=2.4 min) and restricted to venules. Stimulation of the left or right superior laryngeal nerve increased the permeability of venules in the extrathoracic trachea, whereas stimulation of either vagus nerve increased vascular permeability in the intrathoracic trachea and bronchi. All nerves had bilateral effects in the trachea, but the vagus nerves had largely unilateral effects in the bronchi. Neurons that mediated the increase in venular permeability had their cell bodies in the jugular (superior sensory) ganglion of the vagus nerve or rostral portion of the nodose (inferior sensory) ganglion. Preganglionic autonomic vagal neurons in the brain stem were not essential for this increase in venular permeability. Few nerves identifiable by substance P-immunohistochemistry or electron microscopy were located near the affected venules, and no nerves were within 1 μm of the walls of venules. However, the epithelium and arterioles of the airway mucosa were densely innervated. All intraepithelial nerves were within 0.1 μm of epithelial cells, and at least two-thirds of nerves near arterioles were within 1 μm of the vessel walls. We conclude that the increase in venular permeability associated with neurogenic inflammation in the trachea and bronchi of rats is mediated by sensory axons that travel in the vagus nerves and superior laryngeal nerves. We question whether tachykinins from the sensory nerves mediate the increase in vascular permeability through a direct action on venules, and raise the possibility that these nerves evoke the release from epithelial cells of mediators that contribute to the increase in vascular permeability.

Hypertrophy of intestinal smooth muscle

SummaryProximal to an experimental stenosis of the small intestine of rats and guinea-pigs a remarkable hypertrophy of the muscle coat develops 3–5 weeks after the operation. There is no increase in the length of the intestine but an overall increase in volume of the muscularis externa up to 10 times. This increase is accounted for by an increase in size and in number (by mitosis) of smooth muscle cells of both the longitudinal and circular layers. Bundles of newly-formed smooth cells appear in the serosa and are circularly arranged. In the hypertrophic smooth muscle cells of the circular layer the ratio of surface to volume is 0.80 (0.80 μm2 of cell surface for every μm3 of cell volume) as against 1.4 in the control muscle. The hypertrophic muscle cells have a highly developed sarcoplasmic reticulum and show a large number of nexuses. The density of innervation (number of axons per given number of smooth muscle cells) is smaller than in controls. Few collagen fibrils are visible in the extracellular space.

Quantitative morphological study of smooth muscle cells of the guinea-pig taenia coli

SummaryA quantitative study of muscle cells of the guinea-pig taenia coli is reported. Stereological methods were used on electron micrographs and phase contrast micrographs. Smooth muscle cells of taeniae fixed under 1 gram load were about 515 μm long. Muscle cell volume was about 3,500 μm3 and cell surface 5,300 μm2. About 168,000 caveolae were found at the surface of each muscle cell, covering about 29 percent of its surface. They produced a 73 percent increase of the cell membrane compared to a smooth-surfaced cell. The ratio surface-to-volume is about 1∶0.67 if the geometrical surface is considered, or 1∶0.39 if the total surface of the cell membrane (including the caveolae) is considered. Mitochondria constituted 3.5–4 percent of the cell volume. A few nexuses were observed, both between two muscle cells and between a muscle cell and an interstitial cell. In serial sections septa of connective tissue and groups of muscle cells were found to disappear within few tens of microns or to merge with other septa, and the taenia did not appear to be divided into clear-cut muscle cell bundles. Bundles of smooth muscle cells were seen passing from the taenia to the underlying circular muscle. The transverse sectional area of the taenia ranged between 0.14 and 0.39 mm2; it showed about 526 blood vessels · mm-2.

Size of neurons and glial cells in the intramural ganglia of the hypertrophic intestine of the guinea-pig

SummaryA quantitative light microscopic study has been carried out on the myenteric and submucosal ganglia of the guinea-pig ileum, after inducing hypertrophy of the wall with an experimental stenosis. The area of the profiles of nerve cells, of nerve cell nuclei and of glial nuclei, and the percentage area of the neuropil were measured, and the relative numbers of neurons and glial cells were estimated. The average size of neurons in both plexuses was greatly increased over the control values. Nearly half of the neuronal profiles measured over 500 μm2 in sectional area (less than 3% in the controls), and less than 1% measured less than 150 μm2 (21% in controls). The average size of glial nuclei (and presumably that of the glial cells too) was increased in the hypertrophic ganglia. The number of glial cells (relative to the number of neurons) was unchanged or slightly decreased in the hypertrophic myenteric ganglia; in contrast, it was markedly increased in the submucosal ganglia. In both ganglia the hypertrophy was accompanied by a decrease in the percentage volume of neuropil.