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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.20 n.1 Temuco  2002

http://dx.doi.org/10.4067/S0716-98682002000100001 

ULTRASTRUCTURAL ALTERATIONS IN SUBMANDIBULAR GLANDS OF RATS
(Rattus norvegicus) SUBMITTED TO EXPERIMENTAL CHRONIC ALCOHOLISM

ALTERACIONES ULTRAESTRUCTURALES DE LAS GLÁNDULAS SUBMANDIBULARES DE
RATONES (Rattus norvegicus) SOMETIDOS A ALCOHOLISMO CRÓNICO EXPERIMENTAL

*Tirapelli, L. F.; *Tirapelli, D. P. C. & **Tamega, O. J.

* Faculty of Medicine, Marília University, UNIMAR, Marília, SP, Brasil.
**
Universidade Estadual Paulista, UNESP, Botucatu, Brasil.

SUMMARY : The objective of this study was to analyze the submandibular glands of rats (Rattus norvegicus) submitted to experimental chronic alcoholism at electronic transmission microscopy. The experiment was programmed for a 15-month period, and in every five months, three animals from each group were sacrificed. Twenty-seven male rats, with average weight of 300g, were used and divided into three experimental groups with the following characteristics: 1) alcoholic: received ethanol, daily, at 6% for 15 months; 2) isocaloric: received diluted sucrose at a concentration of 87.14 g/L, corresponding energetically to the caloric content of the alcoholic group; and 3) control: received tap water "ad libitum" as liquid diet. The animals were fed with the same amount of Purina ration as a conventional weekly solid diet. It was observed several ultrastructural alterations in the alcoholic animals mainly in seromucous cells: dilation and enlargement of Golgi complex cisterns; the presence of digestive vacuoles, a large number of electronlucent vesicles and an increase in intercellular space, an increase between basolateral interdigitation cell and an enlargement of connective tissue. It was observed at some cells of the intercalary ducts at a lower part of lateral membranes, only after 15-month treatment, the presence of wide and clear vesicles with electronlucent material located among cell secretion granules of granular ducts. No alterations were observed in the animals of the control and isocaloric groups as well as the other parts of glandular parenchyma of animals of the alcoholic group.

KEY WORDS: 1. Ultrastructural alteration; 2. Ethanol; 3. Submandibular gland; 4. Rats.

INTRODUCTION

In rodents, the submandibular salivary glands present histologic differences when compared to other mammals, including men, probably related to its different alimentary habits. In the rats are tubule-acinar composed organs, but with its parenchyma constituted exclusively by seromucous acinus, having characteristics of the two cellular kinds. Another important difference is that, in rodents, this gland is characterized for exhibiting, interposed to the intercalary and striated ducts, a tubular segment highly developed and formed by serous cells, the granular duct or tubule (De Assis et al., 1994). The increase of the production of alcoholic drinks, accompanied by its great consumption, has been accentuating the medical and psychosocial problems related to abusive use and alcoholic dependence (De Lima, 1997).

Researches demonstrate that the excessive consumption of alcohol commits mainly the Central Nervous System (Clair, 1991) and it can also affect several corporal structures, among them several organs of the gastrointestinal treat (Mincis et al., 1973; Geokas et al., 1981 and Palmer, 1989), including the salivary glands, that are of clinical importance (Borsanyi, 1962; Mandel & Baurmash, 1971; Scott et al., 1988; Sasahara et al., 1990 and Banderas et al., 1992).                        

Therefore, due to the importance of those glands in the oral biology, this study aimed to verify, in electronic microscopy of transmission, the possible morphologic alterations in the submandibular gland of rats submitted to experimental chronic alcoholism, subsidizing possible correlations of those pathological alterations with the ones observed in human beings.

MATERIAL AND METHOD

Twenty-seven male rats were used (Rattus norvegicus), lineage Wistar, with at the age of two months and weighing approximately 300 g. The animals, obtained from Biotério Central of UNESP - Campus Botucatu, were divided into three groups: Alcoholic (A), Isocaloric (Ci) and Control (C). Experimental group A: daily received absolute ethyl alcohol diluted to 6% in water; Experimental group Ci: received sucrose diluted at a concentration of 87.14 g/L, corresponding energetically to the caloric content of the alcohol of group A; and Group C: received tap water "ad libitum" as liquid diet. The animals of the three groups were fed with the same weekly amount of conventional solid diet (ration for rats of the trademark Purina). Before the beginning of the experiment, the animals of group A were submitted to a brief period of gradual adaptation to the consumption of alcohol, that consisted of the supply of absolute ethyl alcohol diluted in water with concentration gradually altered in weekly growing doses of 2%, 4% and 6%, and the experimental phase started after the third week of treatment. After a 5, 10 and 15-month period from the beginning of the experiment, three rats of each group were intraperitoneally anesthetized with Hypnol 3% (30 mg/kg), weighed and fixed by using the perfusion method with glutaraldehyde 2.5%, after previous wash with physiologic solution. Samples of the submandibular glands were collected, processed by the routine for the electronic microscopy of transmission and analyzed through a microscope PHILIPS IN 301.

RESULTS

The parenchyma of the submandibular glands consists of a secretory portion formed by seromucous acinus and serous tubules of the granular ducts (Fig.1). Septa of conjunctive tissue involve and support the secretory portion and the intercalary (Fig. 1), striated and excretory duct system. The seromucous acinus are formed by pyramidal cells with spherical nucleus of basal position. They rest on a basal lamina, and are separated by interposed myoepithelial cells (Fig. 3). Its basolateral walls presented interdigitations (Fig. 3), while the more apical portion of its lateral walls presented desmosomes (Fig. 4) and, the lower portion, intercellular canaliculi (Fig. 3). Microvillosities were described in its apical wall (Fig. 4). It was noticed, in the cytoplasm, the presence of  RER (Figs. 3 and 4); mitochondria (Fig. 3); Golgi complex (Fig. 3) and occasional lysosomes (Fig. 3). The secretion granules presented a kind of spherical shape, different dimensions and they occupied mainly its apical area (Figs. 3 and 4). The intercalary ducts are between the seromucous acinus and the granular ducts (Fig. 1). The granular ducts are constituted by barrel shape cells, cytoplasm replete with great electron-dense granules and basal nucleus, the same happening with other cytoplasmatic constituents (Figs. 1 and 2). Finally the striated ducts were formed by high collunaria cells and the excretory ducts presented high cells. The other glandular portion, the stroma, was constituted by weak conjunctive tissue with great amount of collagen fibers and that sustained blood vessels and nervous fibers (Figs. 1 and 5).


1. General aspect of the gland. Observe part of a granular duct (dg) and a seromucous acinus (sm), in continuity to an intercalary duct (di). Sanguineous capillary (ca); nucleus (nu); conjunctive tissue (tc). 3600x.


2. Granular duct in transversal cut. Secretion granules (gr); lumen (l); nucleus (nu). 3600x.


3. Seromucous cells showing, in its base, part of a myoepithelial cell (mi). Intercellular canaliculi (ca); Golgi complex (g); secretion granules (gr); basolateral interdigitations (i); intercellular junction (j); basal lamina (lb); contractile myofilaments (mr); mitochondrion (m); nucleus (nu); endoplasmatic wrinkled reticule (re); conjunctive tissue (tc). 10000x.


4. Apical portion of a seromucous cell. Desmosomes (d); secretion granules (gr); intercellular junction (j); microvillosities (mv); endoplasmatic wrinkled reticule (re). 13000x.


5. Arteriola and amielinic nervous fiber in the conjunctive tissue (tc). Notice the nucleus of the schwann cell (sw). Axon (ax); fibroblast (f); lumen of the arteriola (l); endothelium nucleus (en); muscular fiber nucleus (fm) 16500x. Electromicrographies of the submandibular gland of alcoholic rats, with 5 and 10  months of treatment.  

In the animals of groups C and Ci, there were no differences verified in the  ultrastructure of several glandular constituents, in all periods of the research (Fig. 1).  Gradual ultrastructural alterations could be observed in the animals of group A, mainly in its acinar seromucous portion. After five months of treatment with ethanol, in the cytoplasm of the acinar seromucous cells, dilation could be observed in the Golgi complex, mainly in the perinuclear area, with a great number of extensive and voluminous vesicles of different dimensions (Figs. 6, 7 and 8), and the presence of concentric membranes (Fig. 6) similar to myelinic figures. Furthermore, some cells presented granules with different dimensions and shapes from the normal pattern, in other words, big or small and of circular contour (Fig. 7), while others did not present defined shape (Fig. 8). Its aspect was, most of the times, clear and they left the impression of coalition of several granules, resembling to vesicles containing electron-lucid material (Fig. 8).

Electromicrographies of the submandibular gland of control and isocaloric rats, with 5 months.


6 and 8 : Seromucous cells, presenting dilation of the Golgi complex cisterns (g); presence of membranes in a disorganized way and with different electron-densities inside some secretion granules (mb); granules without defined form (*), and presence of concentric membranes similar to myelinic figures (mc). Myoepithelial cell (mi); secretion granule (gr); intercellular junction (j); basal lamina (lb); lumen (l); mitochondrion (m); nucleus (nu); conjunctive tissue (tc). 10000x.


7 : Vesicles with electron-lucid content (ve); dilation of the Golgi complex cisterns (g), secretion granules with circular format ( ), and the presence of concentric membranes inside some secretion granules (mb) in the cytoplasm and a seromucous cell. Mitochondrion (m); nucleus (nu). 10000x.

The alterations described so far were observed in the animals of group A, in all the periods of the experiment. However, other alterations could be verified in those animals, after 10 and 15 months of treatment with ethanol. After ten months of treatment, it was noticed a dilation of the space among basolateral interdigitations of the acinar seromucous cells (Fig. 11), besides an increase of its intercellular spaces basally to the unitive complex (Fig. 10, 11 and 12). Among seromucous acinus, a small edema could be observed in the space occupied by the conjunctive tissue (Fig. 10). It was also observed in the cytoplasm, the presence of some secondary lysosomes (Fig. 12) and of possible digestive vacuoles (Fig. 11). In the cells of some of the intercalary ducts, an increase of the intercellular space could be verified in the most basal portion of its lateral membranes (Fig. 9).


9 : Cells of the intercalary duct where it is observed, in its base, extensive intercellular space (ei). Intercellular junction (j); lumen (l); nucleus (nu); nucleus of the myoepithelial cell (mi). 13000x.


10 : General aspect of a seromucous acinus. Notice the presence of vesicles with electron-lucid content (ve), occupying great part of the cytoplasm; and an increase of the intercellular space (ei). Myoepithelial cell (mi); nucleus (nu); conjunctive tissue (tc). 3600x.


11 : Apical portion of a seromucous acinus, where a dilation of the intercellular space (ei) is observed, and between the basolateral interdigitations (i), of the Golgi complex cisterns (g), besides the presence of a great vesicle containing electron-lucid material (ve), of wide fused clear granules (* ), and of a digestive vacuole (vd). Desmosomes (d); secretion granules (gr); lumen (l). 13000x.


12 : Seromucous cell. Cytoplasm presenting vesicles of electron-lucid content (ve), dilation of the Golgi complex cisterns (g), and the presence of secondary lysosomes (arrow head) and of a great number of blended clear granules (*). Notice dilations in the intercellular space (ei). 10000x.

In the animals of group A after 15 months of treatment, it could also be verified in some cells of the granular duct, the presence of wide and clear vesicles, containing electron-lucid material, located among its secretion granules (Fig. 13),  besides a slight increase in the intercellular space and in the space between the basolateral interdigitations of those cells (Fig. 14). In the other constituents of the glandular parenchyma, myoepithelial cells, striated and excretory ducts, no ultrastructural alteration was observed along the whole period of the experiment.


13:  Cell of the granular duct. Notice the presence of great vesicles of electron-lucid content (ve) in the base of the cell. Secretion granule (gr). 5000x.


14 : Spaced basolateral interdigitations (i) between two cells of the granular duct. Basal lamina (lb); mitochondrion (m); endoplasmatic wrinkled reticule (re). Spaced intercellular increased (ei). 16500x.

DISCUSSION

The larger salivary glands can have their size increased through a variety of metabolic states, such as starvation, protean deficiency, pregnancy, hepatic diseases and alcoholism (Bhaskar, 1989). Our studies demonstrated several ultrastructural alterations in the submandibular glands of the alcoholized animals: dilation of the Golgi complex cisterns; presence of concentric membranes similar to the myelinic figures; dilation of the intercellular canaliculi, of the intercellular spaces and of the basolateral interdigitations of the seromucous acinar cells, and the presence of secondary lysosomes and possible digestive vacuoles, after 15 months of experiment. Those alterations were also verified in the parotid glands of rats alcoholized by Sasahara et al. (1990), that also observed an increase of the number of secretory granules after three months of ethanol consumption. In our study, morphometric studies were not accomplished, but differentiated aspects were verified in some secretion granules of alcoholic animals: coalition of granules, indefinite shape and clear aspect, resembling to vesicles containing electron-lucid material.

From the described and observed morphologic alterations, we can suggest a functional alteration of the seromucous cells and, consequently of the submandibular gland, in chronic alcoholic individuals. Histologic alterations in the granular ducts of those alcoholized rats glands, were described by Soubhia (1989), such as: an increase in its number, volume and cytoplasmatic content. Tirapelli et al. (2001) also observed some histologic alterations principally the deposit of adipose tissue located among the collagen fibers. In our studies, some cells of this duct presented wide and clear cytoplasmatic vesicles. A slight increase of the intercellular space and the space between the basolateral interdigitations, could also be verified.

Also in cells of some intercalary ducts of alcoholized animals, our results showed an increase of the intercellular space and intercellular canaliculi, alterations not verified in the compiled works. Our results showed that myoepithelial cells, the striated and excretory ducts did not present morphologic alterations during the whole experiment, possibly due to the low ethanol concentration (6%). However, Maier et al. (1986) and Sasahara et al. observed an atrophy and a fat accumulation and the presence of intumesced mitochondria in the cells of the striated duct, in salivary glands of rats chronically alcoholized.

Electromicrographies of the submandibular gland of alcoholic rats, with 10 and 15 months of treatment.  

In the stroma, the occasional presence of wide clear spaces similar to vacuoles can be observed, distributed among its collagen fibers, also observed by Abelson et al. (1976). In submandibular glands of cirrhotic individuals, Maier et al., Scott et al., Banderas et al. and Lamano Carvalho et al. (1993) showed a proportional increase of adipose tissue and reduction in the fibrovascular tissues, when compared to the one of the Control group. It is believed that the fibrous and adipose degenerative alterations, by infiltration of adipose cells are related to aging. Those alterations happen concomitantly with the reduction in the amount of glandular parenchyma, mainly of the acinus (Ten Cate, 1985). Scott et al. suggest the intraglandular adipose tissue as a result of a generalized disturbance in the adipose metabolism, consequent of alterations in the function of the liver due to hepatic cirrhosis. In some cases, there is a tendency of human salivary glands becoming more adipose with aging, and the hepatic cirrhosis could be a factor that would accelerate that answer. In contrast to the increase of adiposity, a greasy degeneration of the parenchymal elements of the salivary glands was not detected (Scott et al.). The way ethanol acts in the salivary glands is still uncertain. Here are some possibilities: 1) The alcohol would alter the control of the autonomous nervous system, influencing and altering its receivers, since the glands depend on that innervation for its structural maintenance; (2) The alcohol would alter the secretion of the hormones of growth, tyroxine, adrenocorticoids, testosterone and insulin, responsible for the structural maintenance of those glands;  3) the alcohol would act directly in the glandular cells (Lamano Carvalho et al.).      

For the results obtained in that work, we also suggested that one or more factors previously described can be considered responsible for alterations described in submandibular glands of rats chronically alcoholized.

RESUMEN: El objetivo de este estudio fue analizar las glándulas submandibulares de ratones (Rattus norvegicus) sometidos al alcoholismo crónico experimental, utilizando el microscopio electrónico de transmisión. El experimento fue programado para un período de 15 meses. Cada 5 meses, 3 animales de cada grupo fueron sacrificados. Utilizamos 27 ratones (machos) adultos, con peso medio de 400g los cuales fueron divididos en tres grupos experimentales: 1) Alcohólico, el cual recibió etanol diluido al 6% por 15 meses; 2) Isocalórico que recibió sacarosa diluida en concentración de 87,14 g/L, correspondiendo energéticamente al contenido calórico del grupo alcohólico, y 3) Control, el cual recibió agua de grifo "ad libitum"como dieta líquida. Los animales de todos los grupos fueron alimentados con una dieta sólida establecida semanalmente, que consistió en idéntica ración de Purina.

Observamos varias alteraciones ultraestructurales, principalmente, en las células seromucosas: dilatación y ensanchamiento de las cisternas del complejo de Golgi, presencia de vacuolas digestivas, un gran número de vesículas electrodensas, aumento en el espacio   intercelular,  aumento  entre  las  interdigitalizaciones  basolaterales de  las células y ensanchamiento del tejido conjuntivo. Además, observamos en algunas células de los ductos interpuestos en la posición más baja de sus membranas laterales, tras 15 meses de tratamiento, la presencia de vesículas claras y llenas de material electrodenso localizados entre los gránulos de secreción de las células de los ductos granulares. No observamos ninguna alteración en los animales los grupos control e isocalóricos, así como en otras partes del parénquima glandular de los animales del grupo alcoholizado.

PALABRAS CLAVE: 1. Glándulas submandibulares; 2. Alteración ultraestructural; 3. Etanol; 4. Ratón.

REFERENCES  

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Tirapelli, L.F.; Tirapelli, D.P.C.; Tirapelli, D. P. C.; Cassel, F. D.; Petroni, S. & Tamega, O. J. Morphometric analysis of seromucous acini and granular ducts of submandibular glands from rats (Rattus norvegicus) submitted to experimental chronic alcoholism. Rev. Chil. Anat., 19(3):263-9, 2001.         [ Links ]

Dirección para correspondencia:
Prof. Dr. Luís Fernnado Tirapelli
Rua Larindo Fontana, 175 Apto. 61
CEP 17519-390
Marília SP
BRASIL

Recibido : 09-07-2001
Aceptado: 25-10-2001