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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.19 n.2 Temuco ago. 2001

http://dx.doi.org/10.4067/S0716-98682001000200009 

ULTRASTRUCTURAL ALTERATIONS OF THE PAROTID GLANDS OF RATS
(Rattus norvegicus) SUBMITTED TO EXPERIMENTAL CHRONIC ALCOHOLISM

ALTERACIONES ULTRAESTRUCTURALES DE LAS GLÁNDULAS PARÓTIDAS
DE RATONES (Rattus norvegicus) SOMETIDOS AL ALCOHOLISMO CRÓNICO EXPERIMENTAL

*Tirapelli, L. F.; **Tirapelli, D. P. C. & *Schimming, B.C.

***Professor of Anatomy - School Medicine - Universidade de Marília - UNIMAR - Marília SP - Brasil
**  Graduating of Biomedice - Universidade de Marília - UNIMAR - Marília SP - Brasil

SUMMARY: Adult male rats (lineage Wistar) were alcoholized with ethanol diluted to 6%, after a brief adaptation period with weekly growing doses of ethanol to 2%, 4% and 6%. After a 10 and 15-month period of experiment, 4 animals of each group _ control (C) and alcoholized (A) _ were intraperitoneally anesthetized with 3% Hypnol and fixed by perfusion  with 2,5% glutaraldehyde.  Samples of parotid glands were collected and examined in an Transmission Electron Microscope. Gradual ultrastructural alterations were observed in those animals during the whole experiment: dilation of the cisterns of the Golgi complex, presence of digestive vacuoles and a great amount of empty vesicles in the entire cytoplasm, an increase and accumulation of secondary lysosomes, besides an enlargement of the intercellular space, between basolateral interdigitations of the cells, as well as in the conjunctive tissue. The described morphologic alterations are due, probably, to disturbances in the hydroelectrolitic homeostasis in the saliva production, therefore contributing to serious morpho-functional disturbances of those glands in alcoholic individuals. No alteration could be observed in the animals of the control group.

KEY WORDS: 1. Parotid gland; 2. Alcoholism; 3. Rats; 4. Ultrastructural alterations.

INTRODUCTION

  Due to its great importance in the oral physiology, the parotid glands are widely studied mainly in human beings. According to Lima (1997)  the ethanol is one of the oldest and most popular drugs in history. In Brazil, as well as in other countries, the great increase of consumption of alcoholic drinks takes a significant portion of the population (15 to 20%), in other words, about 30 million people. The social, clinical and physiological consequences of its excessive use are well documented. Larato (1972) reported that the chronic ingestion of ethanol produces a variety of systemic modifications including hepatic cirrhosis, hypoproteinemia, ascites, macrocytic anemia, neurological alterations and vitamin deficiencies. In the gastrointestinal tract, Mincis et al. (1973) and Palmer (1989) cited that the ethanol can affect several organs as esophagus, stomach, small intestine, pancreas and liver. Important clinical alterations were observed in the salivary glands after chronic consumption of ethanol. For instance, an increase of the parotid gland was found in 12% of the chronic alcoholic individuals, and in 50% of the patients with alcoholic hepatitis and/or alcoholic cirrhosis, and reported by some authors (Borsanyi, 1962; Mandel & Baurmash, 1971; Martin & Pangborn, 1971; Schuckit, 1979 and Bode, 1980).

  Morphologic modifications could be verified in the parotid glands of rats submitted to chronic consumption of ethanol by Sasahara et al. (1990) and Banderas (1992).  However, there are several very known studies reporting the influence of ethanol in the parotid glands of rats submitted to long periods of treatment.

 The aim of this study was to demostrate the possible ultrastructural alterations in the parenchyma and stroma of the parotid glands of rats after the use of ethanol for a long period (15 months).

MATERIAL AND METHOD

 Sixteen adult male rats (Rattus norvegicus), lineage Wistar, weighing approximately 250 g and 3 months old used. The animals were obtained from Central Biotério - UNESP - Campus Botucatu. Two experimental groups divided into 2 groups of 8: control and alcoholic groups. Control group (C): received water "ad libitum" as liquid diet. Alcoholic group (A): received ethanol to 6% as liquid diet for 15 months.

            The animals of both groups received the same amount of Purina ration as solid diet.

            Before the beginning of the experiment, the animals of Group A were submitted to a brief adaptation period with ethanol supply, in weekly growing doses of 2%, 4% and 6%. At the end of the third week, the experimental phase started. After a 10 and 15-month period of experiment, 4 animals of each group were intraperitoneally anesthetized with 3% Hypnol and fixed by the perfusion, method with 2,5% glutaraldehyde by blood vein, through the left ventricle, after previous wash with physiologic solution. Samples of the parotid glands were collected, submitted to the routine for Transmission Electron Microscope, examined and photo-documented in a microscope PHILIPS EM 301.

RESULTS

            The parenchyma of the parotid gland shows a secretory portion constituted by organized serous cells in lobes involved by fine septa of fibrous conective tissue, and for an excretory portion formed by ducts that transports the secretion. Interposed to the parenchyma, a stroma of conective tissue supports these structures. The serous acinus (Fig. 1A) are formed by pyramidal cells (Fig. 1C) whose apical portions define the acinar lumen. Among the microvilli and the thin basal lamina, myoepithelial cells can be found with long cytoplasmatic extensions (Fig. 1D).  In the cytoplasm, a circular basal nucleus (Figs. 1A, 1B and 1D) is involved by long parallel cisterns of RER and scarce amount of Golgi complex (Figs. 1B and 1D). The mitochondria are distributed by the whole cytoplasm (Figs. 1B, 1C and 1D). The secretion granules show circular format, different sizes and they are quite electron-dense (Figs. 1A, 1B and 1C). Intercalary ducts connect the cells of the serous acinus to the striated ducts. formed by pyramidal cells with a basal  nucleus (Figs. 2A, 2B and 2C). The striated ducts present specializations in the basal surface of its cells. (Fig. 2C). The stroma is constituted by a great amount of fibrous conjunctive tissue rich in collagen fibers, and it supports blood vessels and nerves (Figs. 1A, 2D and 2E). In controls no morphological alteration could be observed during the experiment period.

In the animals of group A, during the whole treatment period, alterations could be observed in the stroma but mainly in the secretory portion of the glandular parenchyma. Therefore, in the alcoholic animals after 10 months of treatment with ethanol, it was observed, in the cytoplasm, an increase in the volume of the cisterns of the Golgi complex, mainly in the perinuclear area, showing a great number of expanded vesicles, of different dimensions and presenting voluminous aspect (Fig. 3A). In some areas the presence of concentric membranes was observed (Fig. 3B) similar to myelin figures. Prominent empty vesicles, of irregular form and size, were found in the whole cytoplasm, presenting membranes of circular format or not, and sometimes concentric (Fig. 3C). The occasional presence of secondary lysosomes (Fig. 3D) distributed among the secretion granules, were also observed in the cytoplasm of the secretory serous cells. The intercellular space and the space between basolateral interdigitations (Figs. 3A, 3D and 3E) were widely enlarged, therefore, allowing the individualization of the acinar cells.

  The alterations previously mentioned could also be observed in the alcoholized animals, after 15 months of treatment. Some of them were more accentuated, such as: the presence of  empty vesicles (Fig. 4A) with varied volume and distributed in the whole cytoplasm, as well as the increase of the intercellular space (Fig. 4A). It could also be observed, in the cytoplasm of the acinar serous cells, the occasional presence of digestive vacuoles (Fig. 4D).

 It was observed, in the tubular portion of the glandular parenchyma, an increase of the intercellular space and in the space between basolateral interdigitations of the cells of the intercalary duct, as well as the occasional formation of secondary lysosomes (Fig. 4C).

  The conjunctive stroma was widely edematous (Figs. 4A and 4B) mainly in the 15-month-treatment animals, causing an individualization of the collagen fibers and an empty aspect.

 

Fig. 1. Electromicrography of parotid gland of animals of control group.

1A. Serous acini: connective tissue (ct); lumen (l); nucleus (nu); rough endoplasmic reticulum (re); secretion granule (sg). 5.000x.
1B. Details of basal portion of serous cell: desmosome (d); Golgi complex (g); intercellular space (is); mitochondria (m); nucleus (nu); rough endoplasmic reticulum (re); secretion granule (sg). 13.000x.
1C. Serous cell: basal lamina (bl); connective tissue (ct); desmosome (d); endothelium (en); Golgi complex (g); intercellular space (is); lumen (l); microvilli (mi); mitochondria (m); nucleus (nu); rough endoplasmic reticulum (re); secretion granule (sg). 10.000x.
1D. Myoepitheliall cell: basal lamina (bl); citoplasmatic prolongation (cp); connective tissue (ct); Gogi complex (g); mitochondria (m); nucleus (nu); rough endoplasmic reticulum (re); serous cell (sc). 16.500x.

Fig. 2. 2A. Intercalated duct: connective tissue (ct); lumen (l); nucleus (nu); serous cell (sc). 2.750x.

2B. Detail of intercalated duct cell: basal lamina (bl); desmosome (d); nerve fibers (nf); Golgi complex (g); intercellular space (is); mitochondria (m); nucleus (nu); rough endoplasmatic reticulum (re). 13.000x.
2C. Basal portion of  striated duct cell: basal lamina (bl); basal membrane pleat (bmp); collagen fiber (cf); mitochondria (m); nucleus (nu). 16.500x.
2D. Striated duct: blood vessel (bv); connective tissue (ct); lumen (l); nucleus (nu). 2750x.
2E. Connective tissue: collagen fibers (cf); fibroblasts (f); nerve fibers (nf); Schwann cell's nucleus (*). 8.000x.

 

DISCUSSION

The data showed severe alterations in the parotid glands of rats treated chronically with ethanol. In agreement with Abelson et al. (1976), a slight increase in the parotid glands of 60 to 80% in patients with alcoholic cirrhosis is usually caused by the fat accumulation mainly in the glandular stroma. In this phase, Scott et al. (1988) reported that the parotid glands show a reduction in the acinar volume, substituting it for adipose tissue. Those authors also suggest that this accumulation of adipose tissue happens due to disturbances in the adipose metabolism, as a consequence of hepatic dysfunction in cases of hepatic cirrhosis. The results described  by Abelson et al. are also in agreement with the works of  Mayers et al. (1986; 1988) and Banderas. Carvalho et al. (1993), studying the effects of ethanol in salivary glands of rats, did not observe statistically significant alterations in the relative volume of the acinus and of other ducts. However, the medium and relative volumes of the nuclei of acinar cells were significantly reduced in the glands of the treated animals. On the other hand, the relative volume of the cytoplasm of the parotid acinar cells of alcoholized animals was significantly larger. Thus, confirming the decrease in the nucleus-cytoplasm relation of this group. A significant reduction in the medium cellular volume was also shown, without any alteration in the amount of acinar cells. Such decrease in the cellular volume of alcoholized animals seems to be mainly due to an atrophy of the cells of the glandular acinus (Carvalho et al.).

In this work, some ultrastructural alterations in the parotid glands of alcoholized animals, could be observed mainly in the secretory acinar area of the gland. The intercellular space of the acinar cells showed a great enlargement, with the presence of cells joined to each other just for the components of the unitive complex. This could also be observed by Sasahara et al. in studies on the effect of ethanol in the parotid glands of mice. Therefore, it is suggested that enzymatic alterations and alterations in the membranes, induced by ethanol, can cause fluid circulation disturbances among the membranes, leading to the accumulation of such fluids in extracellular spaces.

Other pathological aspects detected in our studies happened in the conjunctive tissue, that seemed to be partially destroyed by the presence of great spaces among its components. This could be seen as a direct action of ethanol or a fluid accumulation. Mayer (1986; 1988); Scott et al. and Banderas suggested also, the accumulation of adipose tissue, thus, contributing to a disturbance of the active production of saliva by the parotid gland, but were not observed in our studies. In the alcoholized animals additional alterations could also be verified in relation the control animals: enlargement in the volume of Golgi cisterns; presence of concentric membranes similar to myelinic figures, presence of empty vesicles of different sizes, increase in the number of secondary lysosomes, alterations also observed by Sasahara et al. and the appearing of digestive vacuoles in the whole cytoplasm, due to possible fluid accumulation between basolateral interdigitations of the acinar cells.

The presence of empty vesicles in the whole cytoplasm can be explained by the great penetration of fluids in the cytoplasm of the acinar cells, result of the fluid accumulation in the intercellular space and in the conjunctive tissue. Regarding the presence of a great number of secondary lysosomes and digestive vacuoles, it is suggested that this happens due to disturbances in the secretion of the saliva products and/or due to the "death" of some cytoplasmatic organelles due to the direct action of ethanol. The increase of intercellular spaces observed in the cells of the intercalary duct were also described by Sasahara et al. Alterations, as the decrease in the number of secretion granules and dilation of RER found in the acinar cells due to alterations in the cytoplasm of the striated duct, as described by Sasahara et al., were not observed in our studies. Therefore, the observed morphologic alterations after a long period (15 months) are probably, due to disturbances in the hydroelectrolitic homeostasis in the saliva production, thus contributing to serious morpho-functional disturbances of such glands, in alcoholic individuals.

 

Fig. 3. Eletromicrography of parotid gland of animals of alcoholic group with 10 months treatment.
3A. Cytoplasm of two serous acini cells, showing widely expanded cisterns of Golgi (g). Increase of intercellular space (is);  lumen (l); nucleus (nu); rough endoplasmatic reticulum (re); secretion granule (sg). 13.000x.
3B. Detail evidencing the presence of concentric membranes (cm) similar to myelin figures in cytoplasm of an acini cell. Secretion granule (sg). 36.000x.
3C. Concentric membranes (cm) inside a empty vesicles. Mitochondria (m); nucleus
(nu); rough endoplasmatic reticulum (re); secretion granule (sg). 16.500x.
3D. Serous acini cell. Note: Concentric membranes (cm) similar to myelin figures; dilation of cisterns of Golgi (g); presence of empty vesicles and of secondary lisosomes (*), besides an increase of intercellular space (is) and between  basolateral interdigitation (i).
Connective tissue (ct); lumen (l); secretion granule (sg). 13.000x.
3E. Presence of empty vesicles (ec) and also an increase in intercellular space (is) and between the basolateral intergitations (i). Secretion granule (sg). 10.000x.

Fig. 4. Eletromicrography of parotid gland of animals of the alcoholic group with 15 months of treatment.

4A. Serous acini. Note the presence of wide empty vesicles (ve) in the cytoplasm of the cells and also an increase of intercellular space (is), between basolateral interdigitations (i), and in the space occupied by connective tissue (ct).Lumen (l); nucleus (nu); secretion granule (sg). 5.000x.
4B. Wide space occupied by connective stroma (ct), individualizing some serous acini (sa) of glandular parenchyma. Capillary (c). 2.750x.
4C. Note an increase of intercellular space (is) and between basolateral interdigitations (i) in the cells of an intercalated duct.
Lumen (l); myoepithelial cell (mi); nucleus (nu); secondary lisosomes (*). 10.000x.
4D. Presence of digestive vacuole (dv) in cytoplasm of a serous acini cell. Mitochondria (m); nucleus (nu); rough endoplasmatic reticulum (re); secretion granule (sg). 21.000x.

RESUMEN: Ratones machos adultos (linaje Wistar) fueron alcoholizados con etanol diluido al 6%, tras un corto periodo de adecuación con dosis crecientes semanales de etanol al 2%, 4% y 6%. Después de 10 a 15 meses de iniciado el ensayo, 4 animales de cada grupo - control y alcoholizado - fueron anestesiados intraperitonealmente com Hypnol al 3% y fijados por perfusión con glutaraldeido al 2,5%. Muestras de glándulas parótidas fueron extraidas y examinadas en un Microscopio Electrónico de Transmisión. En las glándulas de estos animales, durante todo el experimento, se observaron alteraciones ultraestructurales crecientes: dilatación de las cisternas del complejo de Golgi, presencia de vacuolas digestivas y una gran cantidad de vesículas vacías en todo el citoplasma, aumento y acúmulo de lisosomas secundarios, observándose, además, un ensanchamiento de los espacios intercelulares, entre las interdigitalizaciones basolaterales de las células, así como en el tejido conjuntivo. Las alteraciones morfológicas descritas se deben, probablemente, a las perturbaciones en la homeostasis hidro-electrolítica en la producción de la saliva, contribuyendo de esta manera, a una seria alteración morfofuncional de la glándula parótida en los individuos alcoholizados. Ninguna alteración pudo ser observada en los animales del grupo de control.

PALABRAS CLAVE: 1. Glándula parótida; 2. Alcoholismo; 3. Ratones; 4. Alteraciones ultraestructurales.

Dirección para correspondencia:

Prof. Dr. Luís Fernando Tirapelli 
Rua Laurindo Fontana, 175 Apto 61 
Portal do Sol - CEP  17519-390 
Marília - SP
BRASIL

Recibido : 03-05-2001
Aceptado: 05-06-2001

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