High Glucose Reduces the Paracellular Permeability of the Submandibular Gland Epithelium via the MiR-22-3p/Sp1/Claudin Pathway.

Tight junctions (TJs) play an important role in water, ion, and solute transport through the paracellular pathway of epithelial cells; however, their role in diabetes-induced salivary gland dysfunction remains unknown. Here, we found that the TJ proteins claudin-1 and claudin-3 were significantly increased in the submandibular glands (SMGs) of db/db mice and high glucose (HG)-treated human SMGs. HG decreased paracellular permeability and increased claudin-1 and claudin-3 expression in SMG-C6 cells. Knockdown of claudin-1 or claudin-3 reversed the HG-induced decrease in paracellular permeability. MiR-22-3p was significantly downregulated in diabetic SMGs and HG-treated SMG-C6 cells. A miR-22-3p mimic suppressed claudin-1 and claudin-3 expression and abolished the HG-induced increases in claudin-1 and claudin-3 levels in SMG-C6 cells, whereas a miR-22-3p inhibitor produced the opposite effects. Specificity protein-1 (Sp1) was enhanced in diabetic SMGs and HG-treated SMG-C6 cells, which promoted claudin-1 and claudin-3 transcription through binding to the corresponding promoters. A luciferase reporter assay confirmed that miR-22-3p repressed Sp1 by directly targeting the Sp1 mRNA 3'-untranslated region (3'-UTR). Consistently, the miR-22-3p mimic suppressed, whereas the miR-22-3p inhibitor enhanced, the effects of HG on Sp1 expression. Taken together, our results demonstrate a new regulatory pathway through which HG decreases the paracellular permeability of SMG cells by inhibiting miR-22-3p/Sp1-mediated claudin-1 and claudin-3 expression.

Spatiotemporally differential expression of HER (human epithelial growth factor receptor)-2 with sexual dimorphism in submandibular gland of mice

SUMMARY: Considering that the submandibular gland (SMG) of postnatal mice performs active cell proliferation, apoptosis and differentiation which are regulated by proto-oncogene products in cancerous cells, the expression and localization of a proto-oncogene product HER (human epidermal growth factor receptor)-2 was examined in SMG of postnatal mice. In Western blot analysis, the expression for HER-2 was high until pre-puberty, and it decreased from puberty to young adult stages with male SMG more dominant. In immunohistochemistry, the immunoreactivity was positive in acinar and ductal cells of newborn SMG with distinct localization at the intercellular apposition sites. The immunoreactivity in acinar cells progressively decreased to negligible levels by pre-pubertal stage, while it remained positive in most ductal cells throughout the postnatal time-course. The immunoreactivity in cells of terminal tubules and intercalated ducts, both of which have a high potential to produce cells, were seen at levels similar to those of more proximal ducts, while the immunoreactivity in ductal basal cells was significantly high, but the granular convoluted tubule cells were seen at negligible levels in male and at faint levels in female. In immuno-electron microscopy of excretory ducts, the immunoreactivity was dominantly localized on the basal infolding membranes as well as vesicles and vacuoles of various sizes, but rarely in Golgi apparatus and mitochondria. The immunoreactivity without association to any membranous structures were also seen, though not numerous. The relation of expression levels of HER-2 in various portions of normal SMG to those in their cancerous ones is briefly discussed.

RESUMEN: Considerando que la glándula submandibular (GSM) de ratones postnatales realiza la proliferación celular activa, apoptosis y diferenciación que están reguladas por productos protooncogénicos en células cancerosas, la expresión y localización de un producto protooncogénico HER (receptor del factor de crecimiento epidérmico humano) - 2 se examinó en GSM de estos ratones. En el análisis de Western blot, la expresión de HER-2 fue alta hasta la prepubertad, y disminuyó desde la pubertad hasta las etapas de adultos jóvenes con GSM macho más dominante. En inmunohistoquímica, la inmunorreactividad fue positiva en las células acinares y ductales de GSM de recién nacido con una localización distinta en los sitios de aposición intercelular. La inmunorreactividad en las células acinares disminuyó progresivamente a niveles insignificantes en la etapa prepuberal, mientras que permaneció positiva en la mayoría de las células ductales durante el transcurso del tiempo posnatal. La inmunorreactividad en las células de los túbulos terminales y los conductos intercalados, los cuales tienen un alto potencial para producir células, se obser- vó a niveles similares a los de los conductos más proximales, mientras que la inmunorreactividad en las células basales ductales fue significativamente alta, pero en el túbulo contorneado granular las células se observaron en niveles insignificantes en los machos y en niveles débiles en las hembras. En la microscopía inmunoelectrónica de los conductos excretores, la inmunorreactividad se localizó de manera predominante en las membranas de pliegues basales, así como en vesículas y vacuolas de varios tamaños, pero raramente en el aparato de Golgi y en las mitocondrias. También se observó la inmunorreactividad sin asociación a ninguna estructura membranosa, aunque no numerosa. Se discute brevemente la relación de los niveles de expresión de HER-2 en varias porciones de GSM normal con aquellos en sus cancerosos.

Effects of aflatoxin B1 on the submandibular salivary gland of albino rats and possible therapeutic potential of Rosmarinus officinalis: a light and electron microscopic study.

Background: Aflatoxin B1 (AFB1), a highly toxic mycotoxin, is one of the contaminants of food items such as corn, rice, nuts, and flour. This study aimed to evaluate the effect of AFB1 on the histology and ultrastructure of the submandibular salivary glands (SMSG) of albino rats and examine the possible therapeutic effect of Rosmarinus officinalis extract. Methods: This study used 21 adult male albino rats equally divided into three groups as follows: Group C (saline-treated control group); Group A (AFB1 treated group) subjected to intraperitoneal injection of AFB1 (2 mg/kg) once daily for four weeks; Group R (rosemary-treated group) subjected to AFB1 as in Group A followed by two weeks of intraperitoneal injection of Rosmarinus officinalis extract (400mg/kg) once daily. At the end of the experimental periods, SMSGs were excised and fixed for histological and ultrastructural examinations. Results: SMSGs of the AFB1 group presented atrophied serous acini with numerous cytoplasmic vacuolations; their granular convoluted tubules, striated ducts and excretory ducts presented signs of degeneration in their cell lining with the presence of abundant cytoplasmic vacuolations. In addition, dilated blood vessels engorged with red blood cells were frequently seen. Ultrastructural findings of the AFB1 group showed some acinar cells with degenerated mitochondria presenting loss of cristae and vacuolations as well as irregular, shrunken nuclei with condensed  chromatin. Dilated  rough endoplasmic reticulum were observed in granular convoluted tubules and striated ducts. The glands of animals that received rosemary extract almost regained their normal architecture. Conclusions: It can be concluded that rosemary extract has an ameliorative effect on the deleterious histological and ultrastructural changes induced by chronic AFB1 intake in rat SMSGs.

[MICRO- AND ULTRASTRUCTURAL RECONSTRUCTION OF THE RAT SUBMANDIBULAR GLAND IN THE LATE STAGES OF EXPERIMENTAL TYPE 2 DIABETES MELLITUS].

Electron microscopic investigations of the animals' submandibular gland, conducted in 6 weeks of the experiment, established that ultrastructural changes increase in glandular cells of terminal secretory units in comparison with early period of the experiment. Serocytes have osmiophilic, rather small or picnotic nuclei. Perinuclear spaces of karyolemma are uneven, external nuclear membrane forms local protrusions. Electron density of the karyoplasm is significant, appearing homogenous, nuclei are not observed. Evident submicroscopic changes in blood capillaries of the submandibular gland in experimental diabetes mellitus indicate the impairment of blood-tissue barrier and transcapillary exchange. Deep destructive modifications of all branches of microcirculatory blood flow of the submandibular gland are observed in 8-week course of experimental diabetes mellitus. As compared with the 6th week of investigation, a reliable slight dilation of organ artery diameter, dilation of the diameter of interlobular arterioles, dilation of the diameter of intralobular (precapillary) arteriole and dilation of the capillary diameter were observed. Dilation, as compared to 6th weeks of the experiment, of postcapillary venules was observed. Compared to the indices of the 6th weeks of the experiment, an index of trophic activity of the submandibular gland tissuereaches its maximum meaning and an index of packing density of the capillaries reaches its minimum meaning. Capillary network loses delicate, tortuous pattern and often breaks due to destruction of the capillary component. Arteriovenous anastomoses dilate and blood from the arterioles flows into the venous bed avoiding destructed capillaries. Venules are dilated; thin-walled, retained fragments of the capillaries are significantly dilated in some areas. Swelling of connective tissue stroma and significant swelling of the interstitium are observed. Walls of the capillaries and venules are deformed. The walls of the arterioles are thickened due to plasmorrhagia, sclerosis and hyalinosis.

Expression and localization of endogenous phospholipase Cß3 confined to basal cells in situ of immature ducts and adult excretory ducts of submandibular gland of mice.

Our previous study demonstrated that, different from the parotid and sublingual glands, the submandibular glands of adult mice did not show an immunoblot band for PLCß3 which is critical in the secretion mechanism by muscarinic cholinergic signaling. Therefore, the submandibular glands of mice at various stages of postnatal development were examined for this enzyme molecule in immunoblot and immunohistochemistry. In immunoblot, a weak band for PLCß3-expression was detected only at early postnatal stages. In immunohistochemistry, PLCß3-immunoreactivity was distinctly found in most basally located cells of immature ducts, while the immunoreactivity was weakly seen in terminal tubule cells without significant immunoreactivity in adjacent acinar cells. In contrast, the immunoreactivity was distinctly found in some basal cells of adult excretory ducts, and it was ultrastructurally localized densely in close association with bundles of tonofilaments in the cells. The present finding suggests the possibility that Ca2+ signaling governed by phospholipase Cß3 is involved in the differentiation of ductal basal cells into apical cells through control of keratin molecule(s) in the cells.

Localization of phospholipase C ß3 in the major salivary glands of adult mice.

Phospholipase C (PLC)ß has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCß isoforms in mouse salivary glands in situ. A single major band was detected for PLCß3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCß1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCß3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCß3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.

Phenylephrine Alleviates 131I Radiation Damage in Submandibular Gland Through Maintaining Mitochondrial Homeostasis.

PURPOSE: The impairment of the salivary glands is a permanent side effect of 131I ablation therapy for patients with differentiated thyroid cancer. Effective and safe treatments for protecting the salivary glands against 131I are currently not available. Mitochondria are susceptible to ionizing radiation, but alterations after 131I exposure are unknown. Here, we investigated the mechanisms of 131I damage in submandibular glands (SMGs) and evaluated the cytoprotective effect of phenylephrine (PE) against mitochondrial radiation damage. METHODS AND MATERIALS: Rats were randomly divided into 4 groups: control, PE alone, 131I alone, and 131I with PE pretreatment. The mitochondrial structure of SMGs was observed under transmission electron microscopy. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cytochrome c, cleaved-caspase 3, SIRT1, NAMPT, and PGC-1α protein levels were determined with Western blot and immunohistochemistry. Levels of mitochondrial membrane potential, nicotinamide adenine dinucleotide (NAD), and adenosine triphosphate (ATP) were measured with relevant kits. RESULTS: After exposing rat SMGs to 131I, the mitochondrial membrane structures were destroyed, the mitochondrial membrane potential decreased, the release of cytochrome c increased, and cleaved-caspase 3 and cell apoptosis were activated. Moreover, the expression of SIRT1, NAMPT, and PGC-1α was downregulated, and the levels of NAD and ATP decreased. In contrast, PE alleviated the 131I-induced mitochondrial damages and upregulated the expression of SIRT1/NAMPT/PGC-1α and the levels of NAD and ATP. CONCLUSIONS: These findings demonstrate that 131I impairs the salivary glands via the downregulation of SIRT1/NAMPT/PGC-1α signal pathways, which disturbs mitochondrial homeostasis. PE alleviated the 131I damage in SMGs at the mitochondrial level, suggesting that PE could be used as a potential radioprotector for patients with differentiated thyroid cancer with radiation sialadenitis.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α, ß, γ in the three major salivary glands in situ of mice and their response to ß-adrenoceptor stimulation.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which is composed of three isozymes (α, ß and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)-triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice in situ. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno-light microscopy under non-stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kß was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a ß-adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno-light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post-exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.

Expression patterns of tight junction proteins in porcine major salivary glands: a comparison study with human and murine glands.

Tight junction (TJ) proteins play a dynamic role in paracellular fluid transport in salivary gland epithelia. Most TJ studies are carried out in mice and rats. However, the morphology of rodent salivary glands differs from that of human glands. This study aimed to compare the histological features and the expression pattern of TJ proteins in porcine salivary glands with those of human and mouse. The results showed that porcine parotid glands were pure serous glands. Submandibular glands (SMGs) were serous acinar cell-predominated mixed glands, whereas sublingual glands were mucous acinar cell-predominated. Human SMGs were mixed glands containing fewer mucous cells than porcine SMGs, whereas the acinar cells of murine SMGs are seromucous. The histological features of the duct system in the porcine and human SMGs were similar and included intercalated, striated and excretory ducts, but the murine SMG contained a specific structure, the granular convoluted tubule. TJ proteins, including claudin-1 to claudin-12, occludin and zonula occludin-1 (ZO-1), were detected in the porcine major salivary glands and human SMGs by RT-PCR; however, claudin-6, claudin-9 and claudin-11 were not detected in the murine SMG. As shown by immunofluorescence, claudin-1, claudin-3, claudin-4, occludin and ZO-1 were distributed in both acinar and ductal cells in the porcine and human SMGs, whereas claudin-1 and claudin-3 were mainly present in acinar cells, and claudin-4 was mainly distributed in ductal cells in the murine SMG. In addition, 3D images showed that the TJ proteins arranged in a honeycomb-like structure on the luminal surface of the ducts, whereas their arrangements in acini were irregular in porcine SMGs. In summary, the expression pattern of TJ proteins in salivary glands is similar between human and miniature pig, which may be a candidate animal for studies on salivary gland TJ function.

Planimetric correlation between the submandibular glands and the pancreas: a postmortem ductographic study.

The salivary glands and pancreas have comparable anatomic and antigenic properties and can share common pathogenetic mechanisms involving toxic or autoimmune processes. The aim of this study is to assess the correlation in size between the normal submandibular glands and the pancreas. The study was based on human autopsy specimens of the pancreas, neck and oral base from 22 adults, both sexes (mean age, 57.9 years). The pancreatic and submandibular ducts were injected with a contrast medium, and the area of the salivary and pancreatic glandular ductograms was measured with the aid of software for quantification of visual information. Samples of tissue from the salivary glands and the pancreas were studied by means of light microscopy. A high correlation was found between the planimetric size of the pancreas and the submandibular glands (correlation coefficient 0.497 and 0.699 for the right and the left gland, respectively). This ratio was close to 5:1. There were no significant differences in size for the left vs. right submandibular gland (p = 0.39). The ductograms were significantly larger in size in males than in females (p < 0.001). This study has proven a positive correlation in planimetric size between the normal submandibular glands and pancreas, a result that is expected to have possible clinical implications in the long-term follow-up of patients with chronic pancreatitis.

Acinar cell response to liquid diet during rats' growth period differs in submandibular and sublingual glands from that in parotid glands.

Continuously feeding a liquid diet to growing rodents strongly inhibits parotid gland growth, due to suppressed growth of acinar cells. This study investigated whether a liquid diet had a similar effect on submandibular and sublingual glands of growing rats. Rats were weaned on day 21 after birth and then fed a pellet diet in the control group and a liquid diet in the experimental group for 0, 1, 2, 4, and 8 weeks. Their submandibular and sublingual glands were excised, weighed, and examined histologically, immunohistochemically (using antibodies to 5'-bromo-2-deoxyuridine and cleaved caspase 3), and ultrastructurally. The submandibular glands did not significantly differ between the control and experimental groups at all tested points. Only at Week 8, acinar cell area and 5'-bromo-2-deoxyuridine-labeling index of acinar cells in sublingual glands were significantly lower in the experimental group than in the control group. These results show that a liquid diet during rats' growth period had no effect on acinar cells in their submandibular glands, and only a slight effect on acinar cells in their sublingual glands of growing rats, in contrast to the marked effect of a liquid diet on parotid glands.

Similar synapse elimination motifs at successive relays in the same efferent pathway during development in mice.

In many parts of the nervous system, signals pass across multiple synaptic relays on their way to a destination, but little is known about how these relays form and the function they serve. To get some insight into this question we ask how the connectivity patterns are organized at two successive synaptic relays in a simple, cholinergic efferent pathway. We found that the organization at successive relays in the parasympathetic nervous system strongly resemble each other despite the different embryological origin and physiological properties of the pre- and postsynaptic cells. Additionally, we found a similar developmental synaptic pruning and elaboration strategy is used at both sites to generate their adult organizations. The striking parallels in adult innervation and developmental mechanisms at the relays argue that a general strategy is in operation. We discuss why from a functional standpoint this structural organization may amplify central signals while at the same time maintaining positional targeting.

Three-dimensional cultures of mouse submandibular and parotid glands: a comparative study.

Freshly isolated salivary cells can be plated on an extracellular matrix, such as growth factor-reduced Matrigel (GFR-MG), to induce the formation of three-dimensional (3D) structures. Cells grown on GFR-MG are able to form round structures with hollow lumina, capable of sustaining amylase expression. In contrast, cells grown on plastic do not exhibit these features. Our recent studies have used mouse parotid gland (PG) cells, grown on different extracellular matrices, as a model for acinar formation. However, PG cells were not able to respond to the secretory agonist carbachol beyond 5 days and did not sustain polarity over time, regardless of the substratum. An alternative option relies in the use of mouse submandibular glands (SMG), which are more anatomically accessible and yield a larger number of cells. We compared SMG and PG cell clusters (partially dissociated glands) for their ability to form hollow round structures, sustain amylase and maintain secretory function when grown on GFR-MG. The results were as follows: (a) SMG cell clusters formed more organized and larger structures than PG cell clusters; (b) both SMG and PG cell clusters maintained α-amylase expression over time; (c) SMG cell clusters maintained agonist-induced secretory responses over time; and (d) SMG cell clusters maintained secretory granules and cell-cell junctions. These results indicate that mouse SMG cell clusters are more amenable for the development of a bioengineered salivary gland than PG cell clusters, as they form more organized and functional structures. Copyright © 2014 John Wiley & Sons, Ltd.

Morphology of the intralobular duct of the submandibular gland in rats in case of experimental diabetes mellitus.

The objective of study is to study the peculiarities of morphological changes in different subdivisions of the intralobular duct of the submandibular gland (SMG) in rats in case of experimental diabetes mellitus (DM). The study included sexually mature male Wistar rats. Experimental DM was induced by streptozotocin. Electron microscopic study of subdivisions of the intralobular duct of the SMG was carried out on the 14th, 28th, 42nd, 56th, and 70th days of the experiment. In early stages of experimental DM the intercalated ducts are characterized by a relatively unchanged structure, and in late stages vacuolization of the cytoplasm of their epithelial cells is observed. Since the 14th day vacuolization of mitochondria is observed in epithelial cells of the granular ducts being the most pronounced on the 28th day and not apparent over the subsequent periods. The degree of filling with granules reduces till 56th day, however, it increases sligthly on the last day of the experiment. On the 28th-70th days vacuolization of the cytoplasm is observed in epithelial cells of the striated ducts. In addition, on the 14th day the mitochondrial matrix of these cells condenses; over the next periods it becomes enlightened and mitochondrial cristae are clearly visualized and disorganized. CONCLUSION: In the intralobular duct of the SMG in experimental DM dystrophic changes of different intensity occur in the granular and striated ducts on the 14th day and in the intercalated ducts only since the 42nd day of the experiment.

[STUDYING OF MORPHOLOGICAL PECULIARITIES OF SUBMANDIBULAR SALIVARY GLANDS AFTER GASTRIC RESECTION IN EXPERIMENT].

The impact of gastric resection on the submandibular salivary gland (SSG) state, using histological and histochemical methods of investigation in experiment, was studied up. A relative mass of a SSG after gastric resection conduction have had reduced, and the accompanying changes in stroma were revealed with the gland's secretion enhancement. Essential dystrophic changes in the SSG parenchyma and stroma after gastric resection conduction may cause a pronounced disorders of their function.

Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose.

FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini remained unaffected even after increased oxidation. Staining with the subunit was also reduced significantly by adding galactose to the incubation medium. Binding of CTB to cell surfaces apparently requires intact sialic groups on most, but not all, cell surfaces. Oxidation of the sialic acid residues may influence the structure of the sialylated GM1 molecules on the cell surface in different ways. It is possible that both the sialic acid residue and the terminal galactose are oxidized. Alternatively, the sialic acid may be resistant to acid hydrolysis in gangliosides in which the sialic acid is attached to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human milk contains high levels of sialic acid glycoconjugates that may provide defense mechanisms.

Morphometric Study of Diabetes Related Alterations in Human Parotid Gland and Comparison with Submandibular Gland.

Type 2 diabetes mellitus represents one of the principal diseases that afflict the world population and is often associated with malfunction of salivary glands and consequent oral diseases. We recently described significant ultrastructural alterations in the human submandibular gland in diabetic patients without evident oral pathologies. Herein, an analogs morphometrical investigation was focused on the parotid gland in order to evaluate if one of the two glands is more affected by diabetes. Parotid fragments from diabetic and nondiabetic patients were fixed, dehydrated, and processed for light and electron microscopy. Serous cells were randomly photographed and the density and size of several structures involved in the secretory process were examined by morphometry. Scanning electron microscopy images revealed significant changes in the number of apically docked granules and vesicles, suggesting that the last steps in exocytosis are somehow altered in diabetic cells. Other variables analyzed by light and transmission electron microscopy such as the size of acini and secretory granules did not show significant changes, but comparison with previous data obtained with submandibular gland cells demonstrated that the two glands are affected differently.

Diabetes causes morphological changes in human submandibular gland: a morphometric study.

BACKGROUND: Dataon structural alterations in human diabetic salivary glands are scanty and conflicting. The goal of this study is based on the evaluation of the morphological changes in submandibular glands of subjects with well-controlled diabetes and without evident salivary malfunctions. METHODS: Submandibular gland pieces from diabetic and non-diabetic patients were fixed, dehydrated, and processed to obtain sections for light and electron microscopy. Randomly selected micrographs were statistically analyzed to reveal variations in serous acini. RESULTS: Morphometrical evaluation allowed us to reveal significant changes such as enlargement of acinar and granule size, reduction of mitochondrial size, increased density of microbuds and protrusions along luminal membranes. CONCLUSIONS: The results indicate that diabetes affects submandibular gland structure even when glandular function appears unaltered and suggest that morphological changes reflect functional changes chiefly regarding the secretory activity.

Biocompatibility of tungsten disulfide inorganic nanotubes and fullerene-like nanoparticles with salivary gland cells.

Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.

Immunohistochemical and ultrastructural investigation of acinar cells in submandibular and sublingual glands of rats fed a liquid diet.

In atrophic parotid glands induced by liquid diet, acinar cell apoptosis is increased while proliferative activity is reduced. This study aimed to clarify how liquid diet affects submandibular and sublingual glands, including acinar cell apoptosis and proliferation. Seven-week-old male Wistar rats were fed either a liquid (experimental group) or pellet diet (control group) from 3 to 21 days, respectively. Submandibular and sublingual glands were weighed and examined histologically, ultrastructurally, and immunohistochemically using antibodies to cleaved caspase-3 (Casp-3) and 5-bromo-2'-deoxyuridine (BrdU). Weights of submandibular and sublingual gland from the experimental group were not significantly different from controls at any time point. Histological and ultrastructural characteristics of experimental acinar cells in both glands were normal. Acinar cells in control and experimental submandibular glands were positively stained with periodic acid Schiff (PAS) and weakly stained by alcian blue (AB). In control and experimental sublingual glands, mucous acinar cells were PAS-positive and strongly AB-positive. Although Casp-3- and BrdU-positive acinar cells were identified in both glands in the experimental group, their labeling indices were not significantly different from controls. In conclusion, liquid diet in rats does not induce atrophic alterations to acinar cells, including apoptosis and proliferative activity in submandibular and sublingual glands.