Diabetes-related ultrastructural and immunohistochemical changes in human salivary gland parenchyma and a study on a native scaffold obtained from salivary gland stroma

Systemic pathologies such as diabetes and Sjögren’s syndrome, medications, and radiation therapy often affect salivary gland morphology and functionality, giving rise to changes in quantity and composition of saliva and to numerous oral complications that severely compromise the patient’s life quality. The aim of this thesis, articulated into two sections, was to evaluate: 1) if salivary gland morphology and functionality are affected by type 2 diabetes mellitus even when there are not evident signs of oral injuries; 2) the possibility to use a native scaffold derived from human salivary glands as a substrate in which salivary cells can be host to restore damaged salivary glands. The first part of the investigations was carried out with surgical samples of salivary glands obtained from subjects, half diabetics and half non-diabetics, all without evident oral diseases. The samples were processed for light and electron microscopy and random images were subjected to morphometrical evaluation. The calculations revealed diabetes-related alterations such as acinar swelling and remarkable changes in serous cells, where secretory granules appeared enlarged but reduced in their total number. On the other hand, an increased number of granules anchored to the apical membrane was found, as well as an altered number of apical vesicles and microvilli (both involved in the mechanisms of membrane recycle). Taken together these findings suggest the occurrence of difficulties in the last step of exocytosis, and demonstrate that the structures involved in the secretory process are altered by diabetes per se. Other samples were analyzed by means of immunogold staining method to reveal the ultrastructural localization of melatonin and its receptors MT1 and MT2, until now never reported. Both melatonin and receptors were reactive in the secretory granules, in cytoplasmic vesicles and on cell surfaces, suggesting that their interaction could allow melatonin storage within specific cell compartments. Melatonin staining when performed on diabetic major salivary glands highlighted changes in the labeling intensity related with the diabetic condition. 4 The second part is focused on the characterization of a native salivary gland scaffold in view of its use in gland reconstruction. As well as diabetes, radiation therapy, Sjögren’s syndrome, and several medications also give rise to salivary gland degeneration and xerostomia, but the therapies commonly used are not satisfactory so far. An emerging alternative approach to ameliorate the life quality of xerostomic patients is the regeneration of salivary parenchyma. A “Native Human Submandibular Gland Scaffold” (nHSMGS) was isolated from healthy submandibular glands, and was analyzed by light and electron microscopy and by histochemistry. Morphological examinations showed a fiber arrangement similar to that of the intact glands, while histochemistry revealed the presence of collagen type I, III, and IV. Then, a human salivary gland cell line and human fibroblasts were seeded and cultured on the scaffold in order to verify its reliability as autologous substrate for cells expansion. Results showed that a high cell percentage was proliferating after 4 days of culture, and that most cells were still alive after 8 days. All these data encourage the use of the nHSMGS as autologous scaffold in which expand salivary cells for the restoring of damaged salivary glands

A morphometric study of human submandibular gland in type 2 diabetic status

Diabetes Mellitus Type 2 represents one of the principal diseases that afflict the world population. It is well documented that diabetes affects both morphology and function of several organs. In diabetic rats significant structural changes have been demonstrated in salivary glands, such as accumulation of secretory material and lipid droplets within secretory cells, parenchymal degeneration and its replacement with fibrous connective tissue (1). With regard to human salivary glands, the data are scanty and conflicting. Our work, carried out by light and electron microscopy, is based on the evaluation of the morphological changes which occur in human submandibular glands of diabetic with respect to non diabetic patients. Surgical fragments of glandular tissue were fixed, dehydrated, and processed for light and electron microscopy. Randomly chosen images were analyzed with Image Pro Plus software to record the dimension of acini, serous cells, secretory granules and other variables. Data were analyzed by Student’s t-test and Mann Whitney test. In diabetic glands statistically significant morphological changes were observed, such as enlargement of serous acini and increase of secretory granules area. These results suggest that the secretory activity of human submandibular gland is severely affected by the diabetic status. Obviously these data need to be confirmed with further measurements in order to explain better how diabetes affects human salivary glands

Dynamics of the melatonin MT1 receptor in the rat parotid gland upon melatonin administration

Our recent ultrastructural study of human parotid glands revealed that the melatonin receptors, MT1 and MT2, are localised in the plasma cell membranes of acinar and ductal cells but also, and intriguingly, predominantly in acinar secretory granules, giving rise to the working hypothesis that secretory granules are a part of a transcytotic transport system for melatonin. To put this hypothesis to the test in rat parotid glands, anaesthetised animals were exposed to a high melatonin dose (3 mg/kg per hour), infused intravenously over two hours and aiming to stimulate a glandular melatonin-receptor-dependent intracellular transport system, if any. Thirty minutes later, the right parotids were removed. Pre-stimulation, left parotid gland tissue was removed to serve as (untreated) controls. Gland tissues were processed for the gold post-embedding technique and for western blot analysis. In untreated glands, on transmission electron microscope images, melatonin receptors displayed a distribution pattern similar to that in human parotids, i.e. here, too, the receptors were principally associated with the acinar secretory granules. In melatonin- treated glands, the number of granules associated with the MT1 receptor was twice that in untreated glands, despite the same total granule number in the two glands. Moreover, the density of gold particles showing MT1-receptor immunoreactivity associated with granules in melatonin-treated glands was 2.5 times that in untreated glands. The number of MT1 receptors associated with the granule membrane was about three times higher in melatonin-treated glands than in untreated glands, while the number of MT1 receptors inside the granules was about twice that in untreated glands. The immunoblotting of membrane-enriched samples showed that the MT1-receptor expression was about three times that of untreated glands. When it came to the MT2 receptor, no changes were observed. Melatonin itself thus exerts dynamic effects on its MT1 receptor, which may reflect an adaptive receptor-linked carrier system for melatonin, delivering - upon gland stimulation - melatonin to the saliva by exocytosis

Luigi Castaldi (Pistoia 1890-Firenze 1945) and his outstanding contribution to the history of Florentine wax modelling

Luigi Castaldi a pupil of Giulio Chiarugi and promoter, in 1929, with Nello Beccari and Emerico Luna, of the Società Italiana di Anatomia was, from 1927 to 1943, Director of the Anatomy Institute of the University of Cagliari. His publications, encompassing various fields of anatomy, neuroanatomy, auxology, applied anatomy, biology, and experimental morphology were awarded with several prestigious national and international prizes. Similar acknowledgements were granted to his many publications on the History of Anatomy and Medicine. Among them, there are the biographies of Filippo Civinini (1805-1844), Filippo Pacini (1812-1883), Atto Tigri (1813-1875) and the oration given the 29th of September 1935 for the translation of the remains of these illustrious anatomists from Pistoia to the church of S. Maria delle Grazie, adjacent to the Ospedale del Ceppo, the seat of the old Medical School of Pistoia. Nowadays, the most well known of his historical studies, regarded as a true classic, is the masterly essay: “Francesco Antonio Boi primo cattedratico di anatomia a Cagliari e le cere fiorentine di Clemente Susini”. The book, published by L Olschki posthumously in 1947 through the good offices of his friends, and still present in the catalogue of the original publisher, is scrupulously supported by documents found in the archives of Cagliari and Florence. Besides illustrating the figure of FA Boi the Sardinian Anatomist who, by order of Carlo Felice of Savoy, commissioned the wax models for the University of Cagliari to Clemente Susini, Castaldi gives a vivid and unprecedented description of La Specola Museum, and of the personalities responsible for its establishment. Moreover, though highly praising the outstanding scientific achievements of Felice Fontana, for the first time, he ascribes to Clemente Susini the wax modelling, formerly known as the work of Fontana

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 201

Ultrastructural evidence of a secretory role for melatonin in the human parotid gland

In vivo animal studies show that pentagastrin, cholecystokinin and melatonin cause the secretion and synthesis of salivary proteins. Melatonin occurs in large amounts in the gut and is released into the blood on food intake. In vitro experiments suggest that pentagastrin exerts secretory activity in human salivary glands, as judged by ultrastructural changes, reflecting secretion, and an actual protein output. Currently, it is hypothesised that melatonin induces secretory exocytotic events in the human parotid gland. Human parotid tissues were exposed to a high single concentration of melatonin in vitro, processed for high resolution scanning electron microscopy and then assessed morphometrically with the emphasis on the membrane of the intercellular canaliculi, a site of protein secretion. Compared with controls and in terms of density, the melatonin-exposed parotid tissues displayed increases in protrusions (signalling anchored granules) and microbuds (signalling membrane recycling and/or vesicle secretion) and decreases in microvilli (signalling cytoskeletal re-arrangement related to exocytosis), phenomena abolished or very largely reduced by the melatonin receptor blocker, luzindole. In conclusion, acinar serous cells of parotid tissue displayed in vitro exocytotic activity to melatonin, signalling protein secretion. Whether, under physiological conditions, melatonin influences the secretion of human parotid glands remains to be explored, however

A morphometric study of human submandibular gland in type 2 diabetic status

Diabetes Mellitus Type 2 represents one of the principal diseases that afflict the world population. It is well documented that diabetes affects both morphology and function of several organs. In diabetic rats significant structural changes have been demonstrated in salivary glands, such as accumulation of secretory material and lipid droplets within secretory cells, parenchymal degeneration and its replacement with fibrous connective tissue (1). With regard to human salivary glands, the data are scanty and conflicting. Our work, carried out by light and electron microscopy, is based on the evaluation of the morphological changes which occur in human submandibular glands of diabetic with respect to non diabetic patients. Surgical fragments of glandular tissue were fixed, dehydrated, and processed for light and electron microscopy. Randomly chosen images were analyzed with Image Pro Plus software to record the dimension of acini, serous cells, secretory granules and other variables. Data were analyzed by Student’s t-test and Mann Whitney test. In diabetic glands statistically significant morphological changes were observed, such as enlargement of serous acini and increase of secretory granules area. These results suggest that the secretory activity of human submandibular gland is severely affected by the diabetic status. Obviously these data need to be confirmed with further measurements in order to explain better how diabetes affects human salivary glands. Maria Alberta Lilliu gratefully acknowledges Sardinia Regional Government for the financial support of her PhD scholarship (P.O.R. Sardegna F.S.E. Operational Programme of the Autonomous Region of Sardinia, European Social Fund 2007-2013 - Axis IV Human Resources, Objective l.3, Line of Activity l.3.1.). Michela Isola gratefully acknowledges Sardinia Regional Government for the financial support (P.O.R. Sardegna F.S.E. Operational Programme of the Autonomous Region of Sardinia, European Social Fund 2007-2013 - Axis IV Human Resources, Objective l.3, Line of Activity l.3.1 “Avviso di chiamata per il finanziamento di Assegni di Ricerca”)

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