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

Coastline and climatic changes in the Punic period (V-III century B.C.): archaeological and paleobotanical indicators (South Sardinia – West Mediterranean Sea)

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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

Mitochondrial metabolism and morphology in a rat model of NAFLD

NAFLD (non-alcoholic fatty liver disease) is a condition, characterized by fatty liver, that, if untreated, can progress to NASH (non-alcoholic steatohepatitis) and, finally, to cirrhosis. Mitochondrial dysfunction is a crucial element for the progression of NAFLD to NASH. Our objective was studying both morphological and physiological mitochondrial alterations in a rat model of NAFLD and NASH. Sprague Dawley rats were fed either by a standard diet (35% of energy from fat) or by a high fat diet (HFD) (71% of energy from fat). Diets were given ad libitum for a maximum of four weeks and then rats were killed by decapitation after 1, 2, 3, and 4 weeks. A portion of liver samples was processed for light and transmission electron microscopy (TEM) for steatosis assessment, a part for high resolution scanning electron microscopy (HRSEM) to evaluate 3D mitochondria morphology, the remaining part for oxidative phosphorylation experiments (OXPHOS). Measurements of OXPHOS were performed on freshly isolated mitochondria in a Clark-type oxygen electrode. Several substrates were added to mitochondria to test the efficiency of electron transport chain complexes (ETC) and to test the efficiency of several enzymes involved in fatty acid oxidation (FAO). At three and four weeks of HFD, signs of hepatic steatosis were evident (as an increased amount of intracellular lipid droplets) on histological sections and TEM images. OXPHOS measurements indicated that, compared to controls, in the first week, HFD rats had an increased oxygen consumption with each substrate, in the second, third and fourth week OXPHOS efficiency had a tendency to decline with substrates inherent to glycolitic metabolism, but it was higher with substrates stimulating FAO. There were not notable differences in Respiratory Control Ratio and ADP/O ratio between HFD and controls, proving that the integrity of mitochondria was preserved and coupled to phosphorylation. HRSEM observations indicated that variations on inner mitochondrial morphology in HFD rats, appeared in the third and fourth week, when mitochondrial cristae were less, and cristal shape was often lamellar rather than tubular. Concluding, following HFD mitochondria exhibited first an increase in oxidative metabolism (with normal ultrastructural morphology) and later an alteration in the oxidative phosphorylation apparatus associated to mitochondrial cristae injury. These preliminary results suggest that in order to develop a consistent mitochondrial impairment associated to NASH is necessary to extend HFD treatment for longer. This study was supported by a grant by RAS (Regione Autonoma della Sardegna)

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”)

Cardiovascular remodelling in female diabetic rats

Diabetic cardiomyopathy involves both cardiac and large vessels alterations in their biochemical and biomechanical properties. Part of these dysfunctions is due to ROS overproduction and advanced glycated end-products (AGEs) synthesis caused by high blood glucose concentrations (1). Epidemiological studies usually ignore sexgender outcomes of diabetes that has higher cardiovascular risk in women than in men (2). The aim of the present study was to assess the effects of diabetes on aorta, portal vein and myocardium morphology in females Wistar rats. Diabetes was induced by a single dose of streptozotocin 65 mg/kg, and, after 4 and half months, we evaluated the cardiovascular remodelling by light and transmission electron microscopy (TEM). Paraformaldehyde fixed samples of aorta and portal vein were stained with Masson Trichrome method (for collagen fibers), Weigert’s stain (for elastic fibers), Hematoxylin and Eosin (for nuclei), and underwent to morphometric analysis. TEM samples were prepared accordingly to common protocols. Morphometric analysis performed on diabetic aortas showed a reduction of tunica media thickness, but the internal diameter width or the lumen cross-area was unchanged compared to controls. The number of smooth muscle cells increased in tunica media of diabetic aortas. The main change observed in diabetic portal veins was a reduction of the area occupied by elastic fibers in tunica adventitia. TEM observations of papillary muscles did not reveal any changes in the sarcomere lengths across the two experimental groups. These results display slight differences on what was reported in male rats (3) and account for a different development of diabetes in female subjects

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

Nonalcoholic fatty liver disease (NAFLD) affects up to 20% of western population and, when untreated, it can progress from simple fatty liver or steatosis to a more severe condition, such as NASH (non alcoholic steatohepatitis) and cirrhosis (1). NAFLD is a clinical-pathological syndrome that include a wide spectrum of morphological alteration but such studies on humans are not copious, as human samples are difficult to obtain because of ethical limitations. Experimental models are crucial to study steatosis’ progression, not only for elucidating the pathogenesis of NAFLD but also in examining therapeutic effects of various agents. Animal models may be developed on genetic or nutritional basis, or a combination of both. It is important to select the best model fitted to the aim of the study. But the question that arises is: can the animal model reflect hepatic histopathology and pathophysiology of human NAFLD? This question is always neglected as well as the evaluation of ultrastructural features of NAFLD. In order to overcome this lack of investigations we compared ultrastructural features of NAFLD in an animal model and in human samples of NAFLD patients. NAFLD animal model was obtained using Sprague Dawley rats fed by a high fat diet (HFD) (71% of energy from fat), while control rats were fed by a standard diet (35% of energy from fat). Diets were given ad libitum and rats were killed after 1, 2, 3, and 4 weeks. Human specimens were obtained from patients with fatty liver disease undergoing to liver biopsies. Normal liver was taken from patients undergoing surgery for other pathologies. Hepatic steatosis and normality of the liver were assessed by parallel examinations at light microscopy, transmission and high resolution scanning electron microscopy. Light microscopy results showed that different degrees of NAFLD observed in human samples corresponded to similar morphological changes in treated rats. Ultrastructural examination revealed that in the HFD model the histopathology closely reflected that of human NAFLD, although the first did not replicate the full spectrum of the disease in humans. In summary, we showed that, at least morphologically, HFD model overlays to human NAFLD. This could point out for reliability in evaluating other pathological features in animal models. Moreover, animal HFD mimics human nutritional dysregulation that may induce the same biochemical and molecular modifications observed in human patients and might represent a more appropriate tool for studying the pathogenesis of NAFLD over genetic models (2)

Immunolocalization of AQP5 in human parotid salivary glands

Background. Aquaporins (AQPs) are channel protein essential in the transport of water across the biological cell membranes. Thirteen different AQPs have been identified in different tissues of mammals. In rat parotid, the AQP-5 is principally localized in the luminal membrane of the acinar serous cells (1). In mouse parotid a weak but distinct labeling has also observed in lateral, facing the neighboring cells, and basal plasma membranes (2). The AQP seems to be directly involved in transepithelial pathway for osmotic water flow, and in salivary fluid secretion. To date, in human only a report (3) testifies the presence of AQP-5 in luminal surface of secretory cells of parotid glands. So, in order to extend the knowledge regard to aquaporins, our intention was to define the sublocalization of AQP-5 in human parotid glands by immunogold post embedding method. Methods. Surgical samples of human parotid glands were cut into small fragments, fixed in a mixture of paraformaldehyde and glutaraldehyde and embedded in Epon resin without previous osmication. Ultrathin sections were incubated overnight at 4°C with primary antibody (1:25) against human AQP-5. Labeled sections were examined by transmission electron microscope (TEM). Results. The AQP-5 labeled was observed to the apical membrane of the serous cells. Gold particles were, occasionaly, found in the surface of intercellular canaliculi. Few small vesicles exhibited reactivity for AQP-5. Conclusions. Our preliminary studies show that the apical membrane of serous cells of human parotid glands is a specific site of AQP-5. Because the water-rich fluid is secreted in the luminal membrane where aqp5 was present, it is reasonable to speculate that aqp5 plays an important role in the secretion in the human parotid salivary gland. The reactivity of labeled vesicles could mean that in serous cells of human parotid gland occurs a translocation of aqp5 from the intracytoplasmatic compartment to the apical membrane (4). This study is supported by a grant by Regione autonoma della Sardegna. 1. Nielsen et al., Am J Physiol Cell Physiol, 1997. 2. Larsen et all., J Mol Hist, 2001. 3. Gresz et al., Am J Physiol Gastrointest Liver Physiol, 2001. 4. Hishikawa et all., biochemical and biophysical research communications, 1998