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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.19 n.3 Temuco dic. 2001

http://dx.doi.org/10.4067/S0716-98682001000300006 

MORPHOMETRIC ANALYSIS OF SEROMUCOUS ACINI AND GRANULAR
DUCTS OF SUBMANDIBULAR GLANDS FROM RATS (Rattus norvegicus
SUBMITTED TO EXPERIMENTAL CHRONIC ALCOHOLISM

ANÁLISIS MORFOLÓGICO DE LOS ACINOS SEROMUCOSOS Y DUCTOS GRANULOSOS DE

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

*Tirapelli, L. F.; **Tirapelli, C. R.; *Tirapelli, D. P. C.; ***Cassel. F. D.; ***Petroni, S. & ****Tamega, O. J.

*Department of Anatomy - Faculty of Medicine - UNIMAR - Marília (SP) - Brazil.
**Department of Pharmacology - Faculty of Medicine - USP - Ribeirão Preto (SP) - Brazil.
***Department of Biology and Chemistry - UNIJUI - Ijuí (RS) - Brazil.
****Department of Anatomy - Institute of Biology - UNESP - Botucatu (SP)- Brazil.

SUMMARY: It has been described that chronic ingestion of ethanol leads to morphological changes in different tissues. In the present work we carried out a morphometric analysis of seromucous acini and granular ducts of submandibular glands from rats submitted to experimental chronic alcoholism. Male Wistar rats received an alcoholic diet containing ethanol 6% (v/v). At 5, 10 and 15 months after the beginning of the treatment, samples of submandibular glands were collected and the area of seromucous acini and granular ducts were analysed. The area of seromucous acini cells and granular ducts cells were also measured. The results indicated that chronic ethanol consumption significantly reduces the area of seromucous acini cells and granular ducts cells. On the other hand, there was an enlargement in the area of the seromucous acini and granular ducts after chronic ethanol consumption. We concluded that the effects of alcohol accentuated as the period of treatment with alcohol increased.

KEY WORDS: 1. Chronic alcoholism; 2. Submandibular glands; 3. Seromucous acinus; 4. Granular duct; 5. Morphometric analysis; 6. Rats.

INTRODUCTION

Alcohol abuse is considered as one of the most worrying problems of Brazilian public health nowadays (Fortes & Cardo, 1991). Except on skin, alcohol easily diffuses through all kinds of biological membranes. This diffusion occurs mainly through the gastrointestinal tract (Geokas et al., 1981; Kalant, 1983; Clair, 1991). Once ethanol is ingested, it begins to be absorbed immediately by the mucous epithelium of the mouth (Batt, 1989 apud Rosas et al., 1997). However, the amount of ethanol absorbed through this pathway is practically insignificant, and for this reason, all the ingested alcohol can be considered to go straight to the stomach, where it is absorbed by simple diffusion through the epithelium of the stomach and intestines (Batt, 1989 and Watson, 1989 apud Rosas et al., 1997). Thus, when ethanol arrives at the stomach, it is absorbed in small proportions in the bloodstream through the gastric mucosae (approximately 20 to 30%), while the largest amount is channelled through the same gastric emptying toward the small intestine. Once ethanol is in the blood, it is distributed throughout all the body fluids, crossing freely over the totality of the biological membranes (Watson).

In this way, the chronic ingestion of ethanol may be related to morphological changes in a variety of tissues. Researches demonstrated that the excessive consumption of alcohol commits mainly the Central Nervous System (Clair) but it can also affect a variety of corporal structures, including several organs of the gastrointestinal tract (Mincis et al., 1973; Geokas et al. and Palmer, 1989). Alcohol abuse leads to functional and structural alterations on the gastrointestinal tract. These effects occur because the gastrointestinal tract is the first tissue that receives high amounts of ethanol (Fortes & Cardo).

The chronic consumption of ethanol may also result in alterations of 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). Alcoholism can also increase the size of salivary glands (Bhaskar, 1989).  Despite the fact that we can find a great number of studies describing the effects of chronic alcoholism on the submandibular glands, we were not able to find a systematic study of the temporal effects of alcoholism on the morphological structure of these glands.

Therefore, due to the importance of those glands in the oral biology, the aim of the present study was to verify the relation between the period of treatment with ethanol and the possible morphometric alterations in the area of seromucous acini and granular ducts from submandibular glands. Rats were maintained on a liquid diet containing ethanol 6%, with samples being collected at 5, 10 and 15 months after the beginning of  treatment.

MATERIAL AND METHOD

Twenty-seven four-month-old male Wistar rats, initially weighing 400g,  were housed in groups of three in a cage on a constant light-dark cycle.

The animals were randomly divided into three groups: control group (Group C), isocaloric group (Group I) and alcoholic group (Group A). Control rats received tap water ad libitum. Rats from the isocaloric group received a solution containing an isocaloric amount of sucrose (87.14 g/l) instead of ethanol. Rats from the alcoholic group were maintained on a liquid diet containing ethanol 6% (v/v). All animals had free access to Purina Lab Chow. Food and liquid consumption were recorded weekly.

For the completion of this experiment, a semi-voluntary alcoholism model was used with ethanol being the only liquid diet provided to group A. In order to avoid a considerable loss of animals, the alcoholic experimental group was submitted to a brief and gradual adaptation period. The animals received 2% ethanol in their drinking water in the first week, 4% in the second and 6% in the third week. At the end of the third week the experimental stage began.

At 5, 10 and 15 months after the beginning of the experiment, three rats of each group were intraperitoneally anaesthetised with Hypnol 3% (30 mg/kg) and weighed (Marte balance, model 1001). The animals were transferred to a wooden support and then laid on their backs (decubitus dorsal). The submandibular glands, located ventrally in the cervical area, were collected. After the collection and hystological routine, the glands were submitted to a morphometric analysis using a LEICA DMRB light microscope. Four slides were prepared for each animal (two slides for each gland). Three sections were randomly selected from each slide. Measurements of an area of five seromucous acini cells (total amount of 180 cells) and five granular ducts cells (total amount of 180 cells), as well as an area of thirty-six seromucous acini and thirty-six granular ducts, were performed in each section. The measurements were taken in different regions of the secretory epithelium from submandibular glands. The parameters of these measures were the inferior limit of the basal membrane and the apical portion of the secretory cells from submandibular glands. This procedure was performed using an image digitalizer coupled to a JVC TK C620 video camera, which captured the images directly from the slide. KS 300 (from Carl Zeiss) was used to analyse the images.

Statistical analysis of the data was obtained using a one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test. The level considered for significance was 0.05. Data are represented by means ± standard deviation (S.D.).

RESULTS

There were no statistically significant differences in food consumption in the alcoholic group (23.4 ± 3.03 g) when compared to the control (25.6 ± 1.33 g) or isocaloric groups (25.4 ± 3.06 g). The liquid consumption of the alcoholic group was 49.9 ± 4.7 ml, not different from the control (52.3 ± 3.16 ml) or the isocaloric (61.1 ±  7.1 ml) groups. At the end of the period of treatment, body weight did not vary among the three groups (alcoholic; 129.7 ± 60.66 g, control; 159.4 ± 30.06 g and isocaloric; 156.4 ± 48.83 g).

After different periods of treatment (5, 10 and 15 months), there was not macroscopic alteration of submandibular glands from rats of the alcoholic group when compared to the control and the isocaloric groups. However, the morphometric analysis showed an enlargement in the area of the seromucous acini cells (Table I) as well as in the whole seromucous acini from control and isocaloric rats (TableII). Similar results were found with granular ducts cells. An enlargement in the area of granular ducts cells from control and isocaloric rats was observed (Table III). This enlargement, however, was not seen in the whole granular ducts from control and isocaloric rats (Table IV).

Table I. Mean of the area of five seromucous acini cells from submandibular glands at different periods of treatment.


Groups 5 months 10 months 15 months
Control 1232.24 ± 35.70 1336.14 ± 40.17a 1396.95 ± 44.95b
Isocaloric 1236.26 ± 36.63 1348.66 ± 32.17 1403.28 ± 50.74
Alcoholic 1310.56 ± 42.72* 1081.28 ± 32.05*,c 881.80 ± 42.78*,d

Means ± S.D. are expressed in µm * Difference from respective control. a Difference from control 5 months. b Difference from control 5 and 10 months.
c Difference from alcoholic 5 months. d Difference from alcoholic 5 and 10 months.

In alcoholic-treated rats, there was a progressive reduction in the area of seromucous acini cells at 10 and 15 months but not at 5 months. This reduction was more accentuated as the period of treatment with alcohol progressed. So the reduction observed after 15 months was more pronounced than the ones at 5 and 10 months; the reduction after 10 months being more accentuated than the one after 5 months (Table I). Conversely, there was a progressive enlargement of the whole seromucous acinus in the alcoholic group, which was also time-dependent (Table II).

Table II. Mean of the area of the whole seromucous acini from submandibular glands at different periods of treatment.

Groups 5 months 10 months 15 months
Control 13007.19 ± 175.36 14956.42 ± 164.37a 15024.89 ± 95.70a
Isocaloric 13015.19 ± 173.33 14999.31 ± 87.59 15049.64 ± 124.99
Alcoholic 14183.06 ± 287.13* 18029.06 ± 124.82*,c 19975.69 ± 128.10*,d

Means ± S.D. are expressed in µm. * Difference from respective control. a Difference from control 5 months. c Difference from alcoholic 5 months.
d Difference from alcoholic 5 and 10 months.

Similar results were found with the granular ducts cells and the whole granular ducts in rats from the alcoholic group. A progressive reduction in the area of seromucous acinus cells was observed (Table III). The enlargement of the whole seromucous acinus after chronic alcoholism was seen only at 15 months, when compared to control groups. Nonetheless, an enlargement among the animals from alcoholic group was found at different periods of treatment, which was also time-dependent (Table IV).

Table III. Mean of the area of five granular duct cells from submandibular glands at different periods of treatment.

Groups 5 months 10 months 15 months
Control 1582.48 ± 309.53 1884.94 ± 479.54a 2112.84 415.54b
Isocaloric 1584.36 ± 308.20 1905.68 ± 426.88 2318.66 ± 475.94
Alcoholic 2469.38 ± 553.13* 1933.77 ± 434.27*,c 1625.84 ± 298.85*,d

Means ± S.D. are expressed in µm. * Difference from respective control. a Difference from control 5 months. b Difference from control 5 and 10 months.
c Difference from alcoholic 5 months. d Difference from alcoholic 5 and 10 months. months.

Table IV. Mean of the area of the whole granular duct from submandibular glands at different periods of treatment.

Groups 5 months 10 months 15 months
Control 18967.56 ± 3861.0 19590.78 ± 3270.8 20183.42 ± 4147.7
Isocaloric 18804.67 ± 3912.7 19946.22 ± 2080.6 20504.69 ± 289.31
Alcoholic 13123.14 ± 2088.4* 19350.36 ± 3223.3c 22552.97 ± 278.77*,d

Means ± S.D.are expressed in µm * Difference from respective control. c Difference from alcoholic 5 months. d Difference from alcoholic 5 and 10 months.

Figures 1 to 4 show photomicrographs of the submandibular gland from alcoholic rats. In these animals, some significant histological alterations were verified along the whole experiment, but mainly after 10 and 15 months of treatment. These alterations included an increase in conjunctive tissue among the lobes, demonstrated by the present spacing among the seromucous acinus and between those and the granular ducts (Fig. 1, 3 and 4). In the interlobular conjunctive tissue, a deposit of adipose tissue was found located among the collagen fiber with moderate frequency and in different concentrations (Fig. 1 and 2). This study was performed using a LEICA DMRB light microscope.


Figs. 1 and 2. Detail of the interlobular conjunctive tissue stroma. Please note the accumulation of adipose tissue (ta) among the collagen fibre. Trichromic of Masson, 400x; Trichromic of Mallory, 400x. Seromucous acinus (sm); artery (a); excretory duct (dx); granular duct (dg); space between the seromucous acinus and between these and the granular ducts (es); nervous axis (*); ganglion (ga); conjunctive tissue (tc). The areas of seromucous acinus and granular ducts were demarcated.


Figs. 3 and 4. Detail of the parenchyma glandular. Notice slight increase of space (es) between the seromucous acinus and between these and the granular ducts, besides the accumulation of the space (ei) among the cells of the granular duct. Trichromic of Mallory, 800x; Trichromic of Masson, 800x. Seromucous acinus (sm); granular duct (dg); striated duct (de). The areas of seromucous acini cells and granular ducts cells were demarcated.

DISCUSSION

The submandibular glands are tubule-acinar composed organs. The parenchyma of the submandibular glands consists of a secretory portion formed by seromucous acinus and serous tubules of the granular ducts. Septa of conjunctive tissue involves and supports the secretory portion and the intercalary, striated and excretory duct systems. The seromucous acini are formed by pyramidal cells with spherical nucleus of basal position. They rest on a basal lamina, and are separated by interposed myoepithelial cells.

Our results revealed that in the control group, a comparison among the different periods of treatment (5, 10 and 15 months) showed an enlargement in the area of seromucous acini cells and granular ducts cells. This enlargement was also observed in the whole seromucous acini, but not in granular ducts. Similar results were seen in the isocaloric group. These findings are in accordance with Ham (1967) and Testut & Latarjet (1968) who have supported the idea that the cells grow and penetrate into the subjacent connective tissue during their development. These cells would be responsible for the formation of secretory specialized structures.

The present findings indicated that chronic ethanol consumption significantly reduced the area of seromucous acini cells and granular duct cells. Furthermore, it was found that this reduction was more accentuated as the period of treatment with alcohol increased. The reduction observed after 15 months was more pronounced than the ones at 5 and 10 months; the reduction after 10 months being more accentuated than 5 months, further suggesting that the effect of alcohol is time-dependent.

We have also shown that chronic ethanol consumption significantly increased the area of seromucous acini and granular ducts. Our findings are in accordance with Borsanyi & Blanchard (1961). These authors examined biopsies of parotids from alcoholics, concluding that there is an increase in glandular acinus that suggests its hyper-function. However, our results indicate that the increasing in the area of seromucous acini and granular ducts was more accentuated as the period of treatment with alcohol increased, suggesting that the effect of alcohol is time-dependent.

As it has been discussed earlier, there was a natural enlargement in the area of seromucous acini and granular ducts in the control group. However, in the alcoholic group, the enlargement of these two structures was more accentuated. This fact suggests that ethanol consumption is responsible for the enlargement observed in the area of seromucous acini and granular ducts, further excluding the possibility that the enlargement observed in ethanol treated group is due to the natural growing of the cells observed in the control group.

Taken together our data look contradictory, since it was found that chronic ethanol consumption reduced the area of seromucous acini cells and granular ducts cells, but it is also responsible for an increasing in the area of seromucous acini and granular ducts. Nevertheless, our histological analysis showed an enhancement of the conjunctive tissue among the lobes. Moreover, a deposit of adipose tissue was also found, located among the collagen fibres in the interlobular conjunctive tissue. These alterations could explain, at least in part, the enlargement in the whole volume of the seromucous acinus and granular ducts from animals submitted to chronic alcoholism despite the reduction in the area of seromucous acini cells and granular ducts cells.

Adipose tissue deposition due to alcohol consumption has been previously described (Mandel & Baurmash). Therefore, an ignored disturbance in the lipidic metabolism gives place to fat deposit in the salivary glands that results in its increase. The hypertrophy of the acinar cells has also been referred to be the cause of oedema in salivary glands. It is possible that adipose infiltrations, as well as cellular hypertrophy, are manifestations of the same process (Mandel & Baurmash).

In submandibular glands of cirrhotic individuals, several authors (Maier et al., 1986, Scott et al.; Banderas et al. and Lamano Carvalho et al., 1993) have shown a proportional increase of adipose tissue and reduction in fibrovascular tissues, when compared to the control group. No reduction in the proportion of the acinar volume was found in cirrhotic cases. It is believed that the fibrous and adipose degenerative alterations by infiltration of adipose cells are related to ageing. Those alterations happened concomitantly with the reduction in the amount of glandular parenchyma, mainly of the acinus (Ten Cate, 1985). Scott, et al., suggested that a generalized disturbance in adipose metabolism could lead to an intraglandular adipose tissue deposition, as a consequence of alterations in the function of the liver due to hepatic cirrhosis. In some cases, there is a tendency to human salivary glands becoming more adipose with ageing, and the hepatic cirrhosis could be a factor that accelerates this process. In contrast to the increase of adiposity, a greasy degeneration of parenchymal elements of salivary glands was not detected (Scott et al.)

The way ethanol acts in salivary glands is still uncertain. However, some possibilities can be suggested:

1) The alcohol could alter the control of the autonomous nervous system, influencing and altering its receivers, since the glands depend on that enervation for its structural maintenance; 2) The alcohol could alter the secretion of the growth hormone, tyroxine, adrenocorticoids, testosterone and insulin, which are responsible for the structural maintenance of those glands; 3) the alcohol would act directly in the glandular cells (Lamano Carvalho et al.).

In summary, the present findings indicate that chronic ethanol consumption significantly reduces the area of seromucous acini cells and granular ducts cells. Conversely, there is an enlargement of seromucous acini and granular ducts after chronic ethanol consumption which could be related to an enhancement of the conjunctive tissue among the lobes and/or a deposit of adipose tissue located among the collagen fibers in the interlobular conjunctive tissue. The main finding of our study was that the effects of chronic ethanol consumption were more accentuated as the period of treatment with alcohol increased.

ACKNOWLEDGEMENTS: We thank Cláudia Maria Padovan for helpful english revision.

RESUMEN: Ha sido reconocido que la ingesta crónica de etanol causa alteraciones morfológicas en varios tejidos. En el presente trabajo fue realizado un análisis morfométrico de los acinos seromucosos y de los conductos granulosos de las glándulas submandibulares de ratones sometidos a alcoholismo crónico experimental. Ratones Wistar machos fueron sometidos a dieta alcohólica con etanol al 6% (v/v). A los 5, 10 y 15 meses tras el inicio del tratamiento, fueron recolectadas muestras de las glándulas submandibulares para analizar el área de los ácinos seromucosos y conductos granulosos. Los resultados indicaron que el consumo crónico de etanol reduce significativamente el área de las células de los acinos seromucosos y de las células de los conductos granulosos. Además, hubo un ensanchamiento en el área de  los  acinos  seromucosos  y  de  los conductos  granulosos  tras  el  consumo  crónico de etanol. Concluimos que los efectos del alcohol fueron más graves mientras mayor fue el periodo de tratamiento.

PALABRAS CLAVE: 1. Alcoholismo crónico; 2. Glándulas submandibulares; 3. Acinos seromucosos; 4. Conductos granulosos; 5. Análisis morfométrica; 6. Ratones.

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

Recibido : 09-07-2001
Aceptado: 28-08-2001

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