Nexuses between the smooth muscle cells of the guinea-pig ileum.

The circular musculature of the guinea-pig ileum has been studied by freeze-fracture to analyze quantitatively the gap junctions (nexuses) between its smooth muscle cells. The average cell surface area and cell volume are 5,074 micron 2 and 3,260 micron 3. The packing density of nexuses is 48/1,000 micron 2 of cell surface or approximately 244/muscle cell. Nexuses range in area from less than 0.1 to approximately 1.5 micron 2 and they occupy 0.212% of the cell surface. The average packing density of intramembrane particles or pits in nexuses is approximately 7,200/micron 2 of nexal surface, indicating that there may be approximately 77,000 intercellular channels in the full complement of nexuses of one muscle cell.

Plasticity of rat small intestine after removal of a chronic mechanical obstruction.

Chronic intestinal obstruction is associated with morphological changes and functional disorders clinically reported and experimentally documented in laboratory animals. In contrast, little is known about the properties of the hypertrophied intestine after removal of the obstruction. In the present study, we removed the ileal obstruction previously applied to the ileum of rats and, after 1 or 2 weeks, studied in vitro the motor responses of de-obstructed segments of intestine to pharmacological or electrical field stimulation (EFS). By 2 weeks after de-obstruction, maximal contractile responses to receptor (acetylcholine) and non-receptor (K(+)) mediated stimuli were comparable in operated and control tissues; furthermore, the loss of sensitivity to nitric oxide (NO) unmasked in obstructed tissues was, after de-obstruction, replaced by supersensitivity to exogenous NO and vasoactive intestinal polypeptide, probably acting through cyclic nucleotide-independent pathways. Despite the complete recovery of smooth muscle responses, neurogenic contractions remained impaired in de-obstructed tissue; however, the equal contribution of cholinergic/peptidergic components to EFS responses could represent a sign of gradual but delayed recovery of enteric neurotransmission.

Connection of smooth muscle cells to elastic lamellae in aorta of spontaneously hypertensive rats.

We have recently demonstrated that in large arteries of spontaneously hypertensive rats (SHR), there is no increase of stiffness despite the increase in wall thickness, a sign of mechanical adaptation of the arterial wall to the higher level of stress. Because the dense plaques of smooth muscle are a major site of anchorage between the muscle cells and extracellular matrix, we determined by electron microscopy the distribution of dense plaques and their connections to elastic lamellae in the abdominal aorta of 1-year-old SHR and control Wistar rats. In vivo echo-tracking measurement of aortic distensibility and elastic modulus indicates a reduction of arterial stiffness in SHR compared with Wistar rats when they are studied over a common range of blood pressure. The media thickness to body weight ratio was higher in SHR than in Wistar rats. In the media, the percentage of sectional area occupied by extracellular matrix was not different between Wistar rats and SHR. The average number of dense plaques per muscle cell was not different between Wistar rats and SHR. However, the percentage of cell surface occupied by dense plaques was increased in SHR, and the percentage of cell surface connected to the elastic lamellae was twice as high in SHR compared with Wistar rats (9.4+/-1.5% versus 3.8+/-1.1%). These results suggest that the elastin network plays a major role in the mechanical adaptation of the arterial wall in SHR, not through variations of its total amount but through variations of the extent of anchorage to the muscle cells.

Innervation of the guinea pig trachea: a quantitative morphological study of intrinsic neurons and extrinsic nerves.

The innervation of the guinea pig trachea was studied in wholemount preparations stained for acetylcholinesterase, catecholamines, and substance P immunoreactivity and by electron microscopy. The majority of parasympathetic and afferent nerve fibres arrive from the vagus via branches of the recurrent laryngeal nerves. The recurrent laryngeal nerves are composed of several fascicles comprising 600-700 small myelinated fibres (2-5 microns diameter) and about 1,000-2,000 unmyelinated fibres; both components exit from the nerve and project in fine branches to the trachea. A separate component of 200-250 large myelinated fibres (more than 5 microns diameter) runs the full length of the nerve and innervates the striated muscles of the larynx. The recurrent laryngeal nerves are slightly asymmetric in their origin, length, number, and composition of fibres, with the right nerve being shorter but with more numerous and thinner myelinated fibres. At the distal end of the recurrent nerve, a fine branch called the ramus anastomoticus connects it to the superior laryngeal nerve. In the tracheal plexus, there are on average 222 ganglion cells (range 166-327), distributed mostly in small ganglia of 12 or fewer neurons. The ganglionated plexus is situated entirely outside the tracheal wall, overlying the smooth muscle. Ligation experiments show that sympathetic nerve fibres reach the trachea with the recurrent nerves via anastomoses between the sympathetic chain and vagus nerves, or occasionally with recurrent nerves directly, the largest being at the level of the ansa subclavia. There are also perivascular sympathetic nerve plexuses. Substance P immunoreactive fibres enter the trachea from the vagus nerves and by pathways similar to those of sympathetic nerves. There are also paraganglion cells within the recurrent laryngeal nerve that contain catecholamines and are surrounded by substance P immunoreactive fibres. After cervical vagotomy, all the large myelinated fibres of the ipsilateral recurrent laryngeal nerve degenerate and so do all but 10 or 20 small myelinated fibres and all but a few unmyelinated fibres. Degenerating fibres are found within the entire tracheal plexus, indicating bilateral innervation. The small myelinated fibres that survive cervical vagotomy probably represent sympathetic or afferent nerves with their cell bodies located in sympathetic or dorsal root ganglia.

The number of neurons in the small intestine of mice, guinea-pigs and sheep.

A histochemical technique was used to stain the myenteric neurons in the intact wall of the small intestine of mice, guinea-pigs and sheep. The length and diameter of the small intestine and the total serosal surface area were also obtained. Myenteric neurons were counted on large whole-mount preparations of the muscularis externa. Counts were carried out also on the submucosal plexus, on a more limited scale. In the mouse a spatial density of 10,600 myenteric neurons per cm2 was found. The small intestine was 33 cm long and measured on average 11.5 mm in circumference, the total outer surface (serosal surface) amounting to about 38.0 cm2. The total number of myenteric neurons in the small intestine was calculated as about 403,000. In the guinea-pig the length of the small intestine was 145 cm, the average circumference 22 mm and the total outer surface area about 319 cm2. The neuronal packing density was 8600/cm2, and the total number of myenteric neurons about 2,750,000. In the sheep the small intestine was about 2100 cm long with an average circumference of 60 mm and a total surface area of about 12,600 cm2. The ganglion neuron density was about 2500/cm2, and the total number of myenteric neurons in the small intestine was calculated as about 31,500,000. Thus, in the sheep the small intestine contained about 11 times as many myenteric neurons as the guinea-pig and about 80 times as many as the mouse. The differences are in the same direction as, but not proportional to, the differences in body weight and in the length of the intestine. The neuronal spatial density was highest in the mouse and lowest in the sheep, and in the sheep the neurons were markedly larger, and gathered in ganglia that were larger and further apart from one another, than in the mouse, while they had intermediate values in the guinea-pig. A new way of expressing neuronal packing densities is presented together with the proposal of an arbitrary but reproducible unit of intestinal length (a segment whose length is equal to its diameter). In the submucosal plexus the neuronal density was about 3000/cm2 in the guinea-pig, about 8700/cm2 in the mouse and about 4500/cm2 in the sheep. In the mouse the submucosal neuron density decreased gradually along the length of the small intestine.

Homotransplant of pelvic ganglion into bladder wall in adult rats.

In these experiments a large portion of the pelvic ganglion of adult female rats was transplanted into the wall of the urinary bladder of the same animals. The morphology and fine structure of the transplants were studied in whole-mounts and in sections for light and electron microscopy, from two days up to four months after operation. The general architecture of the ganglion was preserved in all the transplants. The vascularization was re-established. Nerves grew out of the transplant and connections with the original intramural nerves of the bladder wall were established. All the synapses degenerated at the time of transplantation; new synapses began to reappear on the ganglion neurons in the oldest transplants. Although some neurons in the transplant degenerated during the first few days, the majority of neurons survived for the full length of the experiments (four months). Satellite glial cells and small intensely fluorescent cells had a similar structure and distribution as in control ganglia. The results show that the homotransplant of pelvic neurons into the bladder has a high rate of success, in terms of survival, maintenance of fine structure, growth and re-connections; these neurons of adult organisms display plastic and regenerative abilities.

Intramural neurones appear in the urinary bladder wall following excision of the pelvic ganglion in the rat.

The entire bladder of female rats was stained for acetylcholinesterase activity, in order to make visible all the intramural nerves. Ganglion neurones were never observed within the bladder wall of adult controls. In contrast, 2, 13 or 27 weeks after unilateral pelvic ganglion destruction a few intramural neurones were consistently observed along the remnants of nerves in the originally denervated half of the bladder. These neurones were often gathered into clusters of 5-15, inside a nerve or closely connected to it, with a faintly stained nerve leading to them and a more heavily stained nerve leading from them. The origin of the new intramural ganglion neurones is unknown, but they probably migrate after ganglionectomy, possibly from some accessory ganglion close to the bladder.

Nerve fibres in the uterine artery increase in number in pregnant guinea-pigs.

Perivascular nerves of the uterine artery were studied in four-month-old pregnant and non-pregnant guinea-pigs. In pregnancy, the uterine artery hypertrophies (more than two-fold growth in diameter and length and 50% increase in wall thickness), but its density of innervation remains high, because of the growth of many new axons. The axons in a complete transverse section of the vessel increase by 40%. The nerve bundles grow by 100%. Small nerve bundles increase more than large bundles: nearly 70% of the nerve bundles contain 10 or fewer axons in pregnant guinea-pigs (37% in non-pregnant animals). The increase in axon number is accompanied by formation of new varicosities, hence presumably of new neuromuscular junctions.

Motor responses of rat hypertrophic intestine following chronic obstruction.

The present work aims at investigating the changes in motor responsiveness of rat intestine hypertrophied by chronic mechanical obstruction. Motor responses to pharmacological agents and electrical field stimulation (EFS) were studied in hypertrophic ileal segments excised from rats subjected to experimental stenosis (n = 20) and compared with responses of control tissues from sham-operated animals (n = 20). Spontaneous motility and contractile responses to exogenous agents (KCl, acetylcholine and substance P) and EFS (10-s trains every minute, 120 mA, 0.5 ms, 1-10 Hz) were increased in hypertrophic longitudinal segments; however, normalization of motor responses to tissue wet weight revealed a remarkable reduction of contractile efficiency in hypertrophied tissues coupled with a loss of sensitivity to nitric oxide-mediated relaxation. Furthermore, EFS under non-adrenergic non-cholinergic (NANC) conditions unveiled a major role of the cholinergic component over the peptidergic one in the neurogenic contraction of hypertrophic intestine. On the whole, hypertrophic intestinal growth emerges as a dynamic process entailing adaptation of smooth muscle and neuronal structures to the increased functional load imposed by lumen obstruction.

Glial fibrillary acidic protein (GFAP) immunoreactivity in enteric ganglia of the chick embryo.

We examined by immunohistochemistry the expression of glial fibrillary acidic protein (GFAP) in enteric ganglia of the chick embryo, using a polyclonal antibody. The morphology of enteric ganglion cells was examined by electron microscopy. Faint GFAP immunoreactivity was detected in ganglion cells and cell processes from around day 7 in ovo. Later in development the intensity of the immunofluorescence increased and it became more evident that immunoreactive small ganglion cells (interpreted as primitive glial cells), and their processes, surrounded larger negative cell profiles (interpreted as primitive neuronal cells); GFAP immunofluorescence was also evident in intramuscular and mucosal nerve trunks. In colocalization experiments, GFAP immunoreactivity was detected in a proportion of HNK-1/N-CAM immunoreactive ganglion cells, in both the myenteric and submucosal plexus. In addition, we observed GFAP immunoreactive nerves in wholemount preparations of chick gut from as early as day 4.5 in ovo. In the ganglionated nerve of Remak, GFAP immunoreactive satellite and Schwann cells were in evidence from day 5 of incubation. Neuronal markers, such as neurofilament, have been detected very early in development in neural crest cell populations in chick enteric ganglia. In contrast, the expression of markers of the glial phenotype has previously been observed only in the late stages of embryonic development. From our experiments, we conclude that neuronal and glial phenotypes are immunohistochemically distinct from as early as day 4.5 of incubation, even if by ultrastructural criteria glial cells are clearly distinguishable from neurons only after day 16 in ovo.

Decrease and disappearance of intramural neurons in the rat bladder during post-natal development.

While confirming previous results that the bladder of adult female rats is devoid of intramural neurons, we show that during postnatal development some intramural neurons are present. There is about 200 of them per bladder at birth, and their number progressively decreases during post-natal life. In this strain of rats some neurons are still present at 12 weeks of age, and in one animal (out of five) there were still 25 neurons at 20 weeks of age.

Tracheal parasympathetic neurons of rat, mouse and guinea pig: partial expression of noradrenergic phenotype and lack of innervation from noradrenergic nerve fibres.

Noradrenergic nerves were studied in whole-mount preparations of the rat, mouse and guinea pig trachea by means of glyoxylic acid-induced catecholamine fluorescence and dopamine beta-hydroxylase immunoreactivity. In an effort to raise tissue levels of catecholamines, some specimens were also treated with the monoamine oxidase inhibitor pargyline, and with L-DOPA, a precursor of noradrenaline. Noradrenergic nerve fibres were detected around blood vessels, within the tracheal smooth muscle and in the mucosa, but never around or in the proximity of neurons of the tracheal ganglia, even after amine precursor loading. These parasympathetic ganglion cells did not show catecholamine fluorescence under control conditions. In the rat and mouse, but not in the guinea pig, some tracheal neurones were dopamine beta-hydroxylase immunoreactive and showed uptake and metabolism of amine precursors, thus expressing aspects of the catecholaminergic phenotype.

Some intrinsic neurons of the guinea-pig heart contain substance P.

Whole-mount preparations of the posterior wall of the atria of the guinea pig heart containing intrinsic ganglion cells and nerve plexuses were stained for substance P-like immunoreactivity by the peroxidase-antiperoxidase method. Substance P-like nerve fibres are present as pericellular baskets around most, but not all, of the neuronal cell bodies, and are also found in the connecting nerve bundles, as perivascular nerve plexuses and in the myocardium and pericardium. The majority of ganglion cell bodies are negative for substance P, as reported previously, but we describe for the first time, a small subpopulation of intrinsic neuronal cell bodies which show immunoreactivity for substance P. Therefore, not all cardiac substance P nerves are extrinsic afferent fibres. At present, the physiological role of intrinsic substance P neurones is not clear.

The taenia of the rabbit colon, an elastic visceral muscle.

The longitudinal musculature (taeniae) of the rabbit proximal colon was studied by phase contrast and electron microscopy. The taenia coli of the rabbit has several structural features which are not found in other visceral muscles of this or other species, and which, on the other hand, are common in certain vascular muscles. Among the characteristics of taenia coli are: highly corrugated muscle cell profiles (even when the muscle is fixed in a resting condition), very high surface-to-volume ratio (1 micron m2/0.5 micron m3), large percentage volume of the extracellular space (about 40%), absence of intramuscular septa, and very large number of elastic fibres, which run mainly longitudinally in broad meshworks parallel to the serosal surface. Several points of contact between elastic fibres and muscle cell membrane occur at all levels along the length of the cells, often involving 11-nm microfibrils; these contacts, and others involving collagen fibrils, are regarded as cell-to-stroma junctions. The taenia coli is virtually devoid of intramuscular blood vessels, including capillaries. The possibility that the lack of capillaries is related to the abundance of elastic material is discussed.

Chicken gizzard. The muscle, the tendon and their attachment.

The fine structure and the organization of muscle and connective tissue in the middle portion of the chicken gizzard (muscular stomach) has been studied by light and electron microscopy. The musculature is divided into long, well-defined bundles arranged circularly and concentrically and extending between the two tendons (tendinous aponeurosis). The muscle bundles are inserted onto the inner surface of the tendon at an angle of about 45 degrees. In addition to muscle cells (which are ultrastructurally similar to those of the small intestine) the musculature contains fibroblasts and interstitial cells and a small number of nerve bundles and capillaries. The gizzard tendons are very compact, made of parallel fascicles of collagen fibrils with interposed stellate tendon cells; ultrastructurally they are very similar to the tendon of skeletal muscles of this and other species. Their collagen fibrils range in size from 30 to 160 nm. The muscle cells that approach the tendon develop longitudinal invaginations of the cell membrane and then break into finger-like terminal processes heavily encrusted with dense bands. The membrane of the invaginations and the terminal processes are surrounded by a basal lamina material which embeds a conspicuous web of small collagen fibrils. The boundary between tendon and muscle is sharp, without interpenetration of the two tissues. A novel type of cell is found at the interface of muscle and tendon (junctional cells), filled with intermediate filaments and some rough endoplasmic reticulum and displaying a trace of a basal lamina.

Structure of the musculature of the chicken small intestine.

The small intestine of the chicken was studied by light and electron microscopy. The musculature, measuring about 180 microns in thickness in the distended intestine, consists of four layers (outer longitudinal, outer circular, inner circular and inner longitudinal) which are directly apposed to one another. There is no layer of connective tissue equivalent to the submucosa of mammalian intestine, and the intestinal glands lie close to the inner longitudinal muscle. Mucosal folds are not formed during isotonic contraction of the intestine. The muscle cells of the chicken small intestine are characterized by large, numerous and sharply outlined dense bodies, by the presence of an extremely thin basal lamina, by prominent dense bands at the cell surface but relatively few intermediate junctions. There are many areas of direct apposition between cell membranes of adjacent cells and little collagen between the muscle cells. The four muscle layers have each distinctive structural features. Gap junctions between muscle cells occur only in the outer circular layer. The outer circular and outer longitudinal layers are closely apposed and numerous junctions of the adherens type link cells of the two layers. Intramuscular blood capillaries are rare and are found virtually only in the outer circular layer; their endothelial cells are joined by tight junctions. In the outer circular layer (but not in the other layers) there are two further cell types, fibroblasts and interstitial cells, which can be clearly distinguished from one another. The latter cells are intimately related to nerve bundles and are connected by gap junctions to some muscle cells.

Ultrastructure of the tracheal muscle in developing, adult and ageing guinea-pigs.

The musculature of the trachea of adult guinea-pigs is ultrastructurally similar to other visceral muscles. However, tracheal muscle cells have irregular outlines, large accumulations of glycogen, a small number of gap junctions, and many small elastic fibres and collagen fibrils (50 nm in diameter). Fibroblasts, mast cells, Schwann cells and axons are found within the muscle, but no interstitial cells. Capillaries run in the connective tissue septa. The tracheal muscle is well differentiated at the end of the fetal life. At this time, muscle cells have an appearance similar to that of a mature muscle. However, the cells are smaller, especially in length, and their orientation is less regular. There are no undifferentiated cells; a few muscle cells are seen in mitosis. There is a limited amount of stroma, mainly small collagen fibrils not exceeding 30 nm in diameter. Within the muscle there are many nerves, but no fibroblasts or mast cells. Many structural contacts link together the muscle cells, but there are no gap junctions. Gap junctions develop in the first few days after birth and are distinct in 5-day-old animals. In ageing guinea-pigs (30-36 months old) the muscle cells are slightly larger than in the young adult. They display deep invaginations of the cell membrane and a very irregular profile. There is an increase in the number of glycogen granule clusters, and the basal lamina is particularly prominent. The amount of stroma has increased, and is made of large elastic fibres and collagen fibrils up to 105 nm in diameter.

Hypertrophy of visceral smooth muscle.

Smooth muscles of viscera undergo a large increase in volume when there is a chronic, partial obstruction impairing the flow of lumenal contents. Hypertrophy of smooth muscle occurs in various medical conditions and several methods are available for inducing it experimentally in laboratory animals, especially in urinary bladder, small intestine and ureter. The hypertrophic response differs somewhat with the type of organ, the animal species, the age of the subject, and the experimental procedure. Ten- to fifteen-fold increases in muscle volume develop within a few weeks in the urinary bladder or the ileum of adult animals, a growth that would not have occurred in the lifespan of the animal without the experimental intervention. The general architecture of the muscle and the boundaries with adjacent tissues are well preserved. In intestinal hypertrophy, muscle cells increase in number: mitoses are found in mature, fully differentiated muscle cells. Cell division by full longitudinal splitting of muscle cells may also occur. Enlargement of muscle cells accounts for most of the muscle hypertrophy. The hypertrophic muscle cell has an irregular profile with deep indentations of the cell membrane, bearing caveolae and dense bands; however, the cell surface grows less than the cell volume (reduction of surface-to-volume ratio). The nucleus is crenated and is much less enlarged than the cell (reduction of the nucleo-plasmatic ratio). Mitochondria grow in number but in some muscles their spatial density decreases; intermediate filaments increase more than myofilaments. The spatial density of sarcoplasmic reticulum is generally increased. In the hypertrophic intestine, gap junctions increase in number and size; in the bladder, gap junctions are absent both in control and in hypertrophy. Thus the hypertrophic muscle cell is not only larger than a control cell, but has a different pattern of its structural components. Extensive neo-angiogenesis maintains a good blood supply to the hypertrophic muscle. The density of innervation is much decreased in the hypertrophic intestine, whereas it appears well maintained in the bladder. Neuronal enlargement is found in the intramural ganglia of the intestine and in the pelvic ganglion. The mechanisms involved in hypertrophic growth are unknown. Three possible factors, mechanical factors, especially stretch, altered nerve discharge, and trophic factors are discussed.

Development of smooth muscle: ultrastructural study of the chick embryo gizzard.

The growth and differentiation of smooth muscle in the chicken gizzard were studied by electron microscopy from the 10th day in ovo to 6 months after hatching; during this period the organ grows 1000-fold in weight. At the earliest stage studied, smooth muscle cells, interstitial cells, and fibroblasts are immature but can already be clearly distinguished. The structural components of muscle cells develop in a characteristic sequence. Mitochondria are more abundant in immature muscle cells (8% in 14 days embryos and 7% in 19 days embryos) than in the adult (5%). Caveolae are virtually absent in the 11 day embryo; they become more common at the end of embryonic life, but continue to increase in relative frequency after hatching. Gap junctions appear around the 16th day in ovo as minute aggregates of connexons, which then grow in size, probably by addition of new connexons. In the earliest stages studied, myofilaments occupy 25% of the cell profile and are assembled into bundles accompanied by dense bodies and surrounded by loosely arranged intermediate filaments. By contrast, membrane-bound dense bands are scarce until the latter part of embryonic life, an observation suggesting that myofilament formation and alignment is not a process initiated near the cell membrane or directed by the cell membrane, and that only late in development bundles of myofilaments become extensively anchored to dense bands over the entire cell surface: at that time myofilaments occupy more than 75% of the cell volume. The muscle cells increase about four-fold in volume over the period studied; the 1000-fold increase in muscle volume is mainly accounted for by an increase in muscle cell number. Mitoses are found in the gizzard musculature at all embryonic ages with a peak at 17-19 days; they occur in muscle cells with a high degree of differentiation. These cells divide at a stage when they are packed with myofilaments and form junctions with neighbouring cells: the mitotic process affects the middle portion of the cell, which takes up an ovoid shape and eventually divides, whereas the remaining portions of the cell do not differ in appearance from the surrounding muscle cells. At all stages of development the population of muscle cells has a uniform appearance (apart from the cells in mitosis), and the growth and differentiation seem to proceed at the same pace in all the cells. There are no undifferentiated cells left behind in the tissue for later development.

The costo-uterine muscle of the guinea-pig: a smooth muscle attaching the uterus to the last rib.

A conspicuous smooth muscle (of transverse sectional area comparable to that of the taenia coli) situated in the suspensorium ovarii and in the ligamentum ovarii proprium of the guinea-pig is described. The muscle is attached to the last rib, it reaches the medial side of the ovary, to which it is loosely attached; it passes beyond the caudal pole of the ovary (from which it receives additional muscle bundles), it approaches the oviduct, passing ventral to it, then it spreads around it and eventually reaches the distal end of the uterus; this muscle (which is here indicated as costo-uterine muscle) is in direct continuation with, or transforms itself into, the longitudinal outer coat of the uterus.