Maria Alberta Lilliu

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.

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. Anat Rec, 298:1911–1918, 2015.

The antipsychotic amisulpride: ultrastructural evidence of its secretory activity in salivary glands.

OBJECTIVE Amisulpride is reported to inhibit clozapine-induced sialorrhea. Preclinically, clozapine evokes muscarinic-M1-type-mediated secretion that, however, amisulpride does not reduce. Instead, amisulpride, without causing any overt secretion per se, enhances both nerve- and autonomimetic-evoked salivation by unknown mechanism(s). Hypothesizing that amisulpride prepares the gland for secretion, we looked for ultrastructural events indicating secretory activity in intercellular canaliculi of serous/seromucous cells, that is, density increase in protrusions (reflecting anchored granules) and in microbuds (reflecting recycling membranes and/or vesicle secretion) and decrease in microvilli (reflecting the cytoskeletal re-arrangement related to exocytosis). MATERIAL AND METHODS Rat parotid and submandibular glands were exposed to amisulpride in vivo or in vitro. Glands were processed for transmission electron and scanning electron microscopy and then morphometrically assessed. RESULTS Cells were packed with secretory granules. The density of protrusions increased in both glands, whereas significant and parallel changes in microvilli and microbuds occurred only in parotid glands, and in vitro. CONCLUSIONS Amisulpride induced ultrastructural signs of secretory activity but to varying extent; in submandibular glands, in contrast to parotid glands, changes were not brought beyond the granular anchoring stage. Amisulpride may provide an overall readiness for secretion that will result in augmented responses to agonists, a phenomenon of potential interest in dry-mouth treatment.

Subcellular distribution of melatonin receptors in human parotid glands

The hormone melatonin influences oral health through a variety of actions, such as anti‐inflammatory, anti‐oxidant, immunomodulatory and antitumour. Many of these melatonin functions are mediated by a family of membrane receptors expressed in the oral epithelium and salivary glands. Using immunoblotting and immunohistochemistry, recent studies have shown that the melatonin membrane receptors, MT1 and MT2, are present in rat and human salivary glands. To date, no investigation has dealt with the ultrastructural distribution of the melatonin receptors. This was the aim of the present study, using the immunogold method applied to the human parotid gland. Reactivity to MT1 and, with less intensity, to MT2 appeared in the secretory granules of acinar cells and in the cytoplasmic vesicles of both acinar and ductal cells. Plasma membranes were also stained, albeit slightly. The peculiar intracytoplasmic distribution of these receptors may indicate that there is an uptake/transport system for melatonin from the circulation into the saliva.

Melatonin localization in human salivary glands

BACKGROUND Circulating melatonin is believed to reach body fluids by crossing passively the cell membranes, but alternative ways for melatonin transport also are hypothesized. This investigation was carried out to furnish ultrastructural evidences for melatonin transport by salivary gland cells in order to indicate plausible routes by which circulating melatonin can reach saliva. METHODS Bioptic samples of parotid, submandibular and labial glands were processed for the electron microscopy and treated to demonstrate melatonin reactivity by the immunogold staining method. RESULTS AND CONCLUSIONS The preferential sites of melatonin reactivity were the granules and vesicles of serous cells. Our results suggested that the acinar cells are able to store melatonin and that the hormone can be released into saliva through granule and vesicle exocytosis. The quantitative evaluation of labelling showed that the parotid gland is the most involved in the release of melatonin in saliva.

Animal models are reliably mimicking human diseases? A morphological study that compares animal with human NAFLD

Non‐alcoholic fatty liver disease (NAFLD) is a clinical‐pathological syndrome that includes a wide spectrum of morphological alterations. In research, animal models are crucial in evaluating not only the pathogenesis of NAFLD and its progression, but also the therapeutic effects of various agents. Investigations on the ultrastructural features of NAFLD in humans are not copious, due to the difficulty to obtain human samples and to the long time of NAFLD to evolve. Translational comparative studies on the reliability of animal models in representing the histopathologic picture as seen in humans are missing. To overcome this lack of investigations, we compared the ultrastructural NAFLD features of an animal model versus human. Sprague‐Dawley rats were fed with a high fat diet (HFD) for 1–4 weeks, while control rats were fed with a standard diet. Human specimens were collected from patients with diagnosed fatty liver disease, undergoing liver biopsies or surgery. Rat and human samples were examined by light microscopy and by transmission and high resolution scanning electron microscopy. The present work demonstrated that NAFLD in animal model and in human, share overlapping ultrastructural features. In conclusion, animal HFD represent an appropriate tool in studying the pathogenesis of NAFLD. Microsc. Res. Tech. 77:790–796, 2014.

Diabetic Status Influences the Storage of Melatonin in Human Salivary Glands: MELATONIN IN DIABETIC SALIVARY GLANDS

Recently we reported on the detailed localization of melatonin (and its receptors) in human salivary glands, revealing that serous cells are able to store and secrete melatonin into saliva. Since we found that type 2 diabetic patients display reduced melatonin content in saliva, our next step was to examine the presence of melatonin in salivary glands removed from type 2 diabetic subjects. The resulting data were compared with those previously obtained by identical procedures in non‐diabetics, to establish if the diabetic status may affect melatonin distribution. Bioptic samples of diabetic parotid and submandibular glands were fixed, dehydrated, embedded in Epon Resin and processed to demonstrate melatonin reactivity by the immunogold staining method. The labeling density (expressed as the number of gold particles per μm2/granule) and the percentage of melatonin‐positive granules were assessed in diabetic samples. These values were compared with those in non‐diabetic samples and differences were evaluated. In parotid and submandibular diabetic glands the reactivity for melatonin was specifically associated with secretory granules and small vesicles in serous cells. Melatonin reactivity was higher in parotid than in submandibular glands. Our data were in line with those obtained in our previous study on non‐diabetic glands. Diabetic salivary glands showed a higher labeling density and a lower number of melatonin‐positive granules compared to non‐diabetic glands. Taken together, these data might explain the decreased salivary melatonin content and the associated oral problems observed in diabetics. Anat Rec, 301:711–716, 2018.

Diabetes induces changes in salivary gland melatonin reactivity

The fine localization of melatonin and its receptors in the human salivary glands were reported in our previous works revealing, by transmission electron microscopy (TEM), that serous cells are able to store melatonin and to secrete it by regulated pathways (1, 2). Moreover, changing in morphology during secretion was observed after melatonin treatment by high resolution scanning electron microscopy (3). As in saliva of patients suffering from type 2 diabetes melatonin was reduced, we focused our study on salivary glands removed from diabetic subjects, in order to add diabetic data to our survey on melatonin and salivary glands. Aim of this investigation was to establish if diabetic status may affect subcellular melatonin distribution and traffic. Bioptic samples of parotid and submandibular glands, removed from diabetic patients, were fixed, dehydrated, embedded in Epon Resin and processed to search for melatonin reactivity by the immunogold staining method. The labelling density (expressed as number of gold particles per μm2/granule) and the percentage of melatonin-positive granules were estimated in diabetic samples. The resulting values were compared with those of non-diabetic ones and the differences were statistically evaluated. In diabetic samples the pattern of melatonin staining was unchanged with respect to non-diabetic ones, as the gold particles were specifically localized within secretory granules and vesicles of serous cells. The quantitative evaluation of gold particles showed that the labeling density changed in parotid diabetic samples with respect to those measured in non-diabetics, as the percentage of melatonin positive granules showed a tendency to decrease in the diabetic status in both glands.This work was supported by grant from RAS, L7/2007, Progetti di Ricerca Fondamentale o di Base, Bando 2012

Melatonin localization in serous cells of human salivary glands

The presence of melatonin in human saliva and its beneficial effects in the oral cavity have been extensively demonstrated, but little is known about the route by which it enters saliva. Although it is commonly believed that blood melatonin crosses the salivary cell membranes and reaches saliva by passive diffusion, its intracellular route remains unexplored. This study was carried out to provide ultrastructural evidence for the melatonin presence in human salivary glands and to suggest a plausible mechanism for its secretion. Surgical samples of parotid, submandibular and labial glands removed from 15 consenting patients were routinely processed for electron microscopy. Thin sections were treated to demonstrate melatonin localization by the immunogold postembedding method. Melatonin positivity was found in secretory granules of serous cells, testifying the capacity of salivary gland cells to store the hormone within cytoplasmic compartments and to release it into the oral cavity in response to a variety of stimuli. Moreover, immunoreactivity was quantified by counting the gold particles per surface unit, showing the strongest labeling in parotid gland, which probably is the most involved in melatonin release.

Subcellular distribution of melatonin receptors in rat parotid salivary glands: effect of melatonin intravenous infusion.

The ultrastructural localization of melatonin receptors Mt1 and Mt2 in human salivary glands has previously been reported (Isola el al, 2013). In the acinar cells, the intragranular distribution of these receptors suggested to us that they may serve as a transcytotic system for melatonin. In an attempt to verify our working-hypothesis we currently exposed anaesthetized rats to the intravenous infusion of melatonin for 2 hrs. After another 30 min, the parotid gland on the right side was removed to be compared with prestimulation removed pieces of the left parotid gland serving as control specimens. Parotid gland tissues from 5 rats were cut into small pieces and fixed with a mixture of paraformaldehyde and glutaraldehyde. They were dehydrated, and embedded in Epon Resin. The ultrathin sections were collected on nickel grids and incubated overnight at 4oC with an antibody specific for Mt1 and Mt2, respectively. The grids were then incubated for 1 hr with the appropriate secondary antibody conjugated to gold particles and, finally examined using electron transmission microscopy. In all sections, the immunoreactivity to Mt2 and, with less intensity, to Mt1 was principally expressed in secretory granules of acinar cells. Occasionally, plasma membranes were also stained, although slightly. Statistical analysis showed that, in response to melatonin, the immunogold particles reactive for Mt1, but not those for Mt2, increased. To conclude, in line with our hypothesis, melatonin stimulates an acinar carrier system, which seems predominantly to involve the Mt1 receptor, in order to delivery this hormone into saliva.