New findings on 3-D microanatomy of cellular structures in human tissues and organs. An HRSEM study.

We present here findings obtained on a large number of human tissues over a period of more than ten years, by our modification of the Osmium maceration method for high resolution scanning electron microscopy (HRSEM). Data are documented by original pictures which illustrate both some 3-D intracellular features not previously shown in human tissues, and results obtained in our current studies on mitochondrial morphology and on the secretory process of salivary glands. We have demonstrated that mitochondria of cells of practically all human tissues and organs have usually tubular cristae, and that even the cristae that look lamellar are joined to the inner mitochondrial membrane by tubular connexions similar to the crista junctions later seen by electron tomography. Concerning salivary glands an important result is the development of a morphometric method that allows the quantitative evaluation of the secretory events.

Morphological alterations induced by cytochalasin D on serous cells of human submandibular gland in basal and stimulated conditions.

Cytochalasin D (CD) is a fungal toxin which binds to the faster growing end of actin microfilament and inhibits actin polymerization. By an in vitro incubation system of slices of human submandibular glands obtained at surgery, we investigated by light microscope (LM), transmission electron microscope (TEM), and high resolution scanning electron microscope (HRSEM) the morphological changes caused by CD on serous cells. We studied the effects of the drug on secretory events induced by isoproterenol (I) and carbachol (C). With LM, following CD incubation, canaliculi were enlarged and prominent vacuoles were seen throughout the cytoplasm. By TEM, the vacuoles, which in many cases were in continuity with the lumen, represented the distinctive feature of secretory cells. With HRSEM, intercellular canaliculi, seen from their cytoplasmic side, exhibited many small spherical bulges, corresponding to the coated pits seen with TEM and indicating that the retrieval of plasma membrane was arrested at an early phase by the disruption of the actin cytoskeleton. In specimens treated with secretagogues and CD, a consequence reported here for the first time was the presence of dense granules within the vacuoles. The protrusions seen by HRSEM on the cytoplasmic side of intercellular canaliculi, following secretagogues stimulations, appeared peculiar to each stimulants, even if combined with CD, suggesting that besides actin filaments, other components, unaffected by CD, also are involved in the process of exocytosis and related phenomena.

Serous and mucous cells of human submandibular salivary gland stimulated in vitro by isoproterenol, carbachol and clozapine: an LM, TEM, and HRSEM Study.

We have investigated by LM, TEM, and HRSEM the effects of D,L-isoproterenol (beta-adrenergic agent), carbachol (muscarinic agent) and clozapine on biopsy specimens of human submandibular gland stimulated in vitro in an inorganic oxygenated medium. Clozapine is a dibenzodiazepine derivative used in psychotic patients that provokes hypersalivation, a displeasing side effect that often causes discontinuance of therapy. Our findings demonstrate that clozapine acts on salivary mucous and seromucous (serous) cells of the gland as a muscarinic agonist. However, the induced secretory response seems to differ qualitatively and quantitatively from that resulting from carbachol. Thus, in agreement with published data resulting from therapeutic treatments and from experimental studies on rats, the mechanism of clozapine induced hypersialorrhea remains open to further investigation.

Morphofunctional studies on human labial salivary glands.

In this study, the first experimental investigation carried out at the ultrastructural level on mucous cells of human salivary glands, we have examined by light microscopy (LM), transmission electron microscopy (TEM), high resolution scanning electron microscopy (HRSEM), the secretory response of labial glands stimulated in vitro by the beta-adrenergic agent, D,L isoproterenol, and by the muscarinic agent carbachol. For comparison we have used identical methods to study samples of mixed portions of human submandibular glands. Morphological findings obtained here on both submandibular and labial glands mucous cells demonstrate that mucous droplets are released solely by muscarinic stimulation, and that cytological events occurring during secretory discharge are similar to those described by others, using TEM, on stimulated mucous cells of rat sublingual glands. Despite the fact that human labial glands are said to have a prominent cholinergic innervation with scanty adrenergic nerves, the response of seromucous cells in these organs to stimulation with carbachol and with isoproterenol was similar to that observed by us, (using LM, TEM and HRSEM), in serous cells of human major salivary glands.

A high resolution sem study of human minor salivary glands.

By SEM we have investigated the human minor salivary glands using the NaOH method for the visualization of endpieces and myoepithelial cells, and the osmium maceration technique that reveals membranous intracellular structures. With the former method all minor glands, including the posterior deep (Ebner's) lingual glands, consist of tubules sometimes dilated into alveoli, while true acini of the kind observed in human major salivary glands, are absent. Tubules of the posterior deep lingual gland exhibit stellate myoepitelial cells that leave a substantial part of the secretory cells uncovered. The latter cells, at variance with serous cells of major glands, do not show basal folds. In contrast, tubules of the other minor glands, like the mucous ones of major glands, are covered almost completely by band-like myoepithelial cells. The osmium maceration method clearly demonstrates that posterior deep lingual glands are serous in character and that all the other minor glands, together with the predominant mucous cells, possess a variable number of seromucous cells that, despite variations among individuals, increase in order from palatine and posterior superficial lingual (Weber's), to minor sublingual, labial, anterior lingual (Blandin and Nuhn's), and buccal glands.

Cell biology of human salivary secretion.

We treated surgical specimens of human parotid and submandibular glands in vitro to manipulate the receptor-signaling cascade pharmacologically and analyzed cellular responses by light microscopy on epoxy embedded sections. Treatment of specimens with the b-agonist, isoproterenol, and with the second messenger analog, dibutyryl cyclic AMP, stimulated serous acinar cells to engage in exocytosis and degranulation. The muscarinic agonist, carbachol, and the calcium ionophore, A23187, on the other hand, elicited formation of "vacuoles" in the cytoplasm of serous acinar cells. Taking previous in vivo human and animal studies into account, these changes are suggested as the morphological expression of enzyme release and fluid secretion, respectively. Specimens obtained from patients over 70 years old exhibited poor response even though their morphological appearance remained intact. Aged salivary glands are thus suggested to experience a decline in their secretory activity at the cellular level, probably by impairment of the signaling processes downstream to the receptor activation and second messenger production.

Fluid and amylase secretion by perfused parotid gland: physio-morphological approach.

Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly around 60 microL/g-min, whereas amylase secretion exhibited an initial peak, followed by a rapid decrease to reach a plateau. Isoproterenol (Isop 1 microM) alone did not induce fluid secretion although it evoked amylase secretion as measured in isolated perfused acini. Addition of Isop during CCh stimulation evoked a rapid and large rise in amylase secretion accompanied by small increase in oxygen consumption. Morphological observations carried out by HR SEM and TEM revealed exocytotic profiles following Isop stimulation. CCh stimulation alone seldom showed exocytotic profiles, suggesting a low incidence of amylase secretion during copious fluid secretion. Combined stimulation of CCh and Isop induced both vacuolation and exocytosis along intercellular canaliculi. These findings suggest that control of salivary fluid secretion is independent of the amylase secretion system induced by CCh and/or Isop.

A scanning and transmission electron microscope study of the human minor salivary glands.

All human minor salivary glands, apart from the posterior deep lingual (von Ebner's) glands which were serous, contained a minor population of seromucous cells that increased from palatine and posterior superficial lingual (Weber's) to labial, anterior lingual (Blandin and Nuhn's) and buccal glands, in that order. Unlike the predominant mucous cells, whose structure was uniform, serous and seromucous cells exhibited, in each gland, peculiar cytological and cytoarchitectural characters.

Historical note: Giuseppe Oronzo Giannuzzi and the discovery of the serous demilunes and of the intercellular canaliculi.

The tragic life and scientific achievements of Giuseppe Oronzo Giannuzzi are briefly outlined. Particular attention is focused on the discovery, made in Karl Ludwig's laboratory, of the serous demilunes and of the intercellular canaliculi of salivary glands.

Human salivary gland parenchymal cells seen by SEM from the cytoplasmic side using a new osmium maceration method.

By removing all or most organelles, we have exposed the cytoplasmic side of the plasmalemma and its specializations in serous cells and in cells of striated and excretory ducts of human major salivary glands. The areas of plasmalemma located beneath the lumen and those bordering the intercellular canaliculi are covered by evenly distributed particles arranged in a continuous band and, below it, in regularly spaced clusters. A similar pattern of particles is seen on the internal aspects of the juxtaluminal plasmalemma of cells of both striated and excretory ducts. Small isolated clusters of particles are seen in other regions of serous and ductal cells as well, being particularly numerous along the basal processes of cells of striated ducts. A distribution of particles resembling that present along intercellular canaliculi of serous cells also is seen on the plasmalemma bordering the biliary canaliculi where, however, the clusters look smaller and farther apart. Large clusters of particles, matching those seen on salivary glands and on liver, are present at the base of the short processes of cells of the stratum spinosum of squamous stratified epithelia. Since the sites of location of the clusters closely correspond to the areas where transmission electron microscopy (TEM) reveals the presence of desmosomes, we believe that the clusters may be related to these cellular junctions. Of more difficult interpretation are the particles present on the juxtaluminal band corresponding both to the zonula occludens and to the zonula adhaerens.

Cytoskeletal regulation of human salivary secretion studied by high resolution electron microscopy and confocal laser microscopy.

To study the cell regulation mechanisms of human salivary secretion, surgical specimens of human parotid and submandibular glands were treated in vitro with isoproterenol (beta-agonist), carbachol (muscarinic agonist), and cytochalasin D (microfilament disruptive agent), and morphological changes occurring in serous acinar cells were observed. Control acinar cells treated without secretagogues exhibited only occasional examples of exocytosis. Microfilaments, revealed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) of F-actin fluorescence stained by rhodamine-phalloidin, were localized underneath the luminal membrane to separate the secretory granules from the luminal membrane. Following isoproterenol treatment, secretory granules made direct contact with the luminal membrane and many omega-shaped exocytotic profiles appeared. TEM and scanning electron microscopy (SEM) showed these profiles to be of granule size or somewhat smaller and to be provided on their cytoplasmic surface with coated pits. Furthermore, CLSM detected the appearance of F-actin fluorescence around the exocytosed granule membranes. Carbachol treatment also evoked the formation in acinar cells of omega-shaped exocytotic profiles some of which were larger than the granules and which exhibited neither coated pits nor associated F-actin fluorescence. To determine if microfilaments regulate the post-exocytotic process of membrane retrieval, we combined isoproterenol treatments with cytochalasin D or carbachol. Following these treatments, F-actin fluorescence surrounding the exocytosed membrane was dispersed or diffused and the exocytotic profiles enlarged remarkably. These results led to the hypothesis that exo/endocytotic processes in human salivary serous acinar cells are regulated differently under autonomic receptor control mediated by microfilaments.

Exocytosis in human salivary glands visualized by high-resolution scanning electron microscopy.

The luminal membrane of salivary acinar cells creates a specialized cell surface area that accepts exocytosis and undergoes dynamic changes during secretion. These changes were visualized three-dimensionally from both the inside and outside of the cell in human parotid and submandibular glands, by application of in vitro secretory stimulation and then of OsO4 maceration to remove cytoplasmic organelles by varying degrees. In control glands treated without secretagogues, the luminal surface of serous acinar cells bore well-developed microvilli with only an occasional incidence of exocytotic profiles. Following treatment with the beta-adrenergic agonist, isoproterenol, considerable shortening and loss of microvilli occurred along the luminal membrane where, on its cytoplasmic side, many protuberances of sizes similar to or smaller than those of single secretory granules (approximately 1 micron in diameter) appeared. The cytoplasmic surface of these protuberances exhibited small vesicles (approximately 100-150 nm in diameter) that, by transmission electron microscopy, were shown to be coated pits or vesicles present on or around the exocytosed granule membranes. Treatment of tissues with the muscarinic agonist carbachol also caused a decrease of microvilli and the appearance of protrusions at the luminal membrane. However, unlike isoproterenol treatment, many of these protrusions were devoid of small pits or vesicles and were much larger than a single secretory granule. These results indicate that (1) secretory stimulation causes the dynamic transformation of microvilli at the luminal membrane, where granule docking and membrane fusion take place, and (2) after fusion, the exocytosed membranes are processed differently, by coated pit/vesicle mediated or non-mediated mechanisms, according to the autonomic receptor control.

Niels Stensen (Niccolò Stenone) and his first scientific offspring: the salivary glands.

Niels Stensen's (Niccolò Stenone) life and scientific achievements are briefly outlined and discussed. Particular attention is focused on his contribution to the understanding of salivary and exocrine secretions.

Further data on intracellular structures of human salivary glands. A SEM study.

Specimens of human salivary glands have been studied by our modification of the AODO maceration method which, carried out on sections of controlled thickness, allows the analytical study of human bioptical material. Lately, our technique has been further improved and simplified by omitting the treatment with dimethylsulfoxide and by using osmium-ferrocyanide as secondary fixative. Following maceration with diluted OsO4, some of the sections also were shaken for 10-15 min with a rotating agitator. Already at low magnification, all parenchymal cells were clearly distinguishable for their complement of cytoplasmic organelles. Serous cells and mucous cells at the beginning of their secretory cycle were characterized by well developed RER and Golgi apparatus, while mature mucous cells exhibited only scanty organelles compressed among the secretory droplets. Mitochondria were tubular, and often branched and convoluted. When sectioned, these organelles, besides the usual plate-like cristae, showed tubular cristae as well. The SER of striated and excretory duct cells was well developed and consisted of a network of smooth anastomosing tubules in the apical cytoplasm where it probably represented the transcellular pathway for ion transport. In specimens subjected to shaking, cytoplasmic organelles were occasionally removed allowing a nonobstructed view of the inner side of the plasmalemma and its specializations. With this technique the intercellular canaliculi of serous cells also became appreciable from their cytoplasmic side. They appeared as ribbon-like irregular protrusions with walls fenestrated by holes, corresponding to the interior of microvilli deprived of the cytoskeleton, and, sometimes, with lateral expansions possibly related to the mechanism of exocytosis. Results reported here clearly showed the usefulness of the maceration method in providing additional data on the cytoarchitecture of epithelial cells of salivary glands. Furthermore, by allowing the visualization of internal surfaces previously hidden to direct inspection, our technique may open new horizons in morpho/functional studies of human salivary glands.

The main excretory duct (Stensen's) of the human parotid gland: a transmission and scanning electron microscope study.

The epithelial cells of the human parotid main excretory duct (Stensen) were studied by transmission (TEM) and scanning (SEM) electron microscopy through a variety of procedures that allowed the visualization of their three-dimensional microanatomy. Stensen's duct in humans is lined, in its distal portion, with a pseudostratified epithelium with tall principal cells and smaller basal cells, while the epithelium becomes progressively stratified cylindrically toward the oral stoma. Goblet cells are scattered among the other epithelial cells. The principal cells exhibit, on their lateral surfaces, numerous flattened laminar folds probably involved in transporting processes. A well-developed smooth endoplasmic reticulum intermingled with mitochondria occupies the cellular apices. Some vesicles are recognized on the cytoplasmic surfaces of the apical and lateral plasmalemma when cytoplasmic organelles are removed. All these features are interpreted as being involved in the process of endocytosis. In both TEM and SEM, the principal cells show a relevant number of irregular apical protrusions that may represent a kind of apocrine secretion. Thus, with regard to function, the human Stensen's duct seems to modify the composition of saliva by processes of resorption and secretion, the latter coming from goblet cells as well. The basal cells have a surface microanatomy completely different from that of principal cells. They exhibit, in fact, only sparse microvillosities and smooth areas on their lateral aspect, while their stromal surface is greatly augmented by irregular thin ramified processes. The role of basal cells is also discussed.

3D-structure of cells of human salivary glands as seen by SEM.

To demonstrate by SEM the topography and cytoarchitecture of the different parenchymal components of human salivary glands, we have employed a number of techniques that allow either the exposure of internal and lateral cell surfaces or, following the removal of connective tissue, the visualization of endpieces, ducts, and myoepithelial cells. Serous glands consist of indented acini attached to the ducts in a grape-like fashion, whereas mucous and mixed glands are made up of smooth tubuli. Myoepithelial cells (mecs), which are abundant on the surfaces of acini, tubuli, and intercalated ducts, are sparse on striated ducts. They are star-shaped on acini, striated ducts, and most of the tubuli. Spindle-shaped mecs are seen, instead, on intercalated ducts and, occasionally, on mucous and mixed tubuli as well. Cells of striated ducts split into a number of large basal portions whose surface is covered by long laminated processes responsible for the striations seen with TEM. Excretory ducts are lined by small cup-shaped basal cells and by tall cylindrical cells, which are completely covered by short processes oriented at random. When observed from below, after removal of the basal lamina, the basal surfaces of cells of excretory ducts exhibit polygonal areas delimited by short reliefs. Those of striated ducts show, instead, long laminar processes arranged radially. Results presented here are discussed and put in relationship to the mechanism of saliva production.

3D microanatomy of human parotid gland.

We have performed a scanning electron microscope study on human parotid gland. By using a variety of techniques of maceration and digestion we have shown the 3D morphology of cells and of isolated endpieces.

Fine structure of excretory ducts of human salivary glands.

Excretory ducts of human major salivary glands are lined by an epithelium consisting of principal cells and by a discontinuous row of basal cells. The principal cells are tall and columnar with mitochondria, large lipofuscin granules and a central nucleus. Just beneath the plasmalemma bordering the lumen, their cytoplasm contains a number of small granules and vesicles similar to those observed in cells of striated ducts. Both in TEM and SEM, these cells also show large apical protrusions devoid of cytoplasmic organelles that may represent a kind of apocrine secretion. The cytoarchitecture of the principal cells seems to be at variance with that of cells of striated ducts. First, the cell body remains unique and does not split into major basal processes. Second, these cells usually lack the long laminated basal folds, housing vertically aligned mitochondria, that are typical of striated ducts. Instead, below the smooth area occupied by the junctional complexes, the lateral cell surfaces are completely covered by a great number of short irregular processes. These organelle-free folds are apparently involved in the mechanism of ion transport since, at their level, there is a strong reactivity for the transporting enzyme K(+)-pNPPase. The basal cells, which are small and cuboidal, have a dense and filamentous cytoplasm. Their functional role is still uncertain.

Ultrastructure of human bulbourethral glands and of their main excretory ducts.

The endpieces of human bulbourethral (BU) glands, studied with SEM after removal of the connective tissue, consist of short, coiled tubules often dilated into alveoli. Immunohistochemical studies at the EM level have shown that the mucous cells of these glands have mucous droplets, which react to blood group antigens, suggesting that BU glands participate in the secretion of these antigens into the seminal plasma. The main excretory duct is lined by a stratified columnar epithelium consisting of six-seven cellular layers. Cells of superficial layers, that are endowed with typical secretory granules, seem to contribute some unknown components to the secretions of these glands.

Ultrastructural observations on human sublingual gland.

That part of the human sublingual gland that corresponds in morphology to the conventional description of this organ presented in most histology texts (probably the major sublingual gland, in contradistinction to the aggregated small glands that compose the minor sublingual glands) was studied by electron microscopy. The gland is mixed, with slightly more mucous elements than seromucous ones. The mucous cells are arranged in tubules that usually are capped by seromucous demilunes. Seromucous cells also form occasional acini or may be scattered in the walls of the mucous tubules. The appearance of the mucous cells varies with the stage of the secretory cycle that they may be in. Their secretory droplets increase in number and progressively compress cytoplasmic organelles. Filamentous bodies also may be present. Based on secretory-granule substructure, four different kinds of seromucous cells can be recognized; these may be a morphological expression of asynchronous synthesis of different secretory proteins. The duct system is an abbreviated one compared to the other major salivary glands. The first duct segments, into which the mucous tubules drain, are similar to intercalated ducts. Larger ducts contain mitochondria-rich cells but lack the basal striations that characterize striated ducts. The paucity of typical striated ducts may be correlated with the elaboration of sodium-rich saliva by the human sublingual gland.