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International Journal of Morphology

On-line version ISSN 0717-9502

Int. J. Morphol. vol.21 no.3 Temuco  2003

http://dx.doi.org/10.4067/S0717-95022003000300010 

Int. J. Morphol., 21(3):245-250, 2003.

DENSITY AND MORPHOMETRY OF MYENTERIC NEURONS
OF THE ILEUM OF RATS SUBJECTED TO CHRONIC ALCOHOLISM

DENSIDAD Y MORFOMETRÍA DE LAS NEURONAS DEL ÍLEON DE RATAS
SOMETIDAS A ALCOHOLISMO CRÓNICO
Marli Aparecida dos Santos Pereira; Sonia Lucy Molinari;Fernando Carlos de Sousa;
Oriana Elaine André;Marcílio Hubner de Miranda Neto*


PEREIRA, M. A. S.; MOLINARI, S. L.; DE SOUSA, F. C.; ANDRÉ, O. E. & MIRANDA NETO, M. H. Density and morphometry of myenteric neurons of the ileum of rats subjected to chronic alcoholism. Int. J. Morphol., 21(3):245-250, 2003.

SUMMARY: This work verified the changes caused by ethanol ingestion on the myenteric neurons of the ileum of rats subjected to chronic alcoholism. Twenty rats (Rattus norvegicus) were divided in control (GC) and alcoholic (GA) groups during 210 days. The animals from group GA received sugar-cane brandy diluted to 30 GL and those from group GC received water. Both groups were fed with rodent chow ad libitum. The ileum was prepared as whole-mounts for neuronal staining according to Barbosa (1978) and Gabella (1979). The neurons were counted and the cell bodies were measured. The statistical analysis revealed a significant decrease in the number of large neurons in GA compared with GC, while there was a significant increase in the number of small neurons in GA. There was a smaller body growth in GA, thus causing a smaller spread of the nerve cells and a larger number of neurons evidenced by the techniques of Giemsa and NADH-diaphorase in the alcoholic animals than in the controls, and a decrease in the size of the cell bodies, evidenced by the increase in the proportion of small neurons and decrease in the proportion of large nerve cells.

KEY WORDS: 1. Alcoholism; 2. Ileum; 3. Myenteric neurons. 


INTRODUCTION

The myenteric nervous system has an important role in gastrointestinal motility as well as other functions. The quime is propelled along the small intestine by peristaltic waves, which are determined by the gastroenteric reflex triggered by gastric distention. The myenteric plexus is directly concerned with the physiologic mechanisms of this reflex in the small intestine. The plexus is formed by neurons that, according to the species, can be found isolated or clustered in ganglia linked by connectives composed of nerve fibers, and are dispersed between the two layers of the muscular tunica (Irwin, 1950; Burnstock, 1959; Santana et al., 1997; Gabella, 1971; Furness & Costa, 1887; Molinari et al., 1994; Natali & Miranda Neto, 1996).

The myenteric neurons vary widely in shape, structure and size, often having an oval or round nucleus eccentrically placed in the cell body and bearing one or two nucleoli (Furness & Costa; Santana et al.; Gabella, 1979). Several authors subjected this heterogeneous neuronal population to classification schemes, taking neuronal morphology Gabella1, 1971; Furness & Costa; Natali & Miranda Neto; Dogiel, 1989), neurotransmitter content ( Gabella, 1969; Bor-Seng-Shu et al., 1994) and cell body size (Natali & Miranda Neto) as parameters.

In addition to the phylogenetic studies of the myenteric neurons, information is found about them in animals subjected to experimental procedures such as aging ( Santer & Baker ) and desnutrition (Natali & Miranda Neto; Santana et al.) in an attempt to explain, based on quantitative and morphometric changes of the neurons, the clinical findings of idiopathic constipation or chronic diarrhea and malabsorption found in the elderly or in people having these pathologies.

It is well documented that ethanol impairs the function and morphology of the intestinal mucosa ( Baraona et al., 1962). It is known that alcoholics having hepatic cirrhosis develop major changes in the small intestinal motility that trigger gastrointestinal disturbances such as dispepsis, anorexia and nausea (Chesta et al.,1991; Chesta & Smok, 1993). Little is known about the effects of ethanol on the proteic turnover in the alimentary tract, especially in the smooth muscle fraction ( Preedy et al., 1993) , there being evidence that the functional disturbances can contribute to the gastrointestinal motility disorders as well. The reasons for the alterations in the motor function of the small intestine in alcoholics are not completely clarified, so that we proposed the investigation of the effect of ethanol on the morphology, density and cell body size of the myenteric neurons of the ileum, with the purpose of correlating possible anatomical alterations of the myenteric nervous system with the intestinal dysfunctions verified in chronic alcoholics.

MATERIAL AND METHOD

The ileum of 20 male rats (Rattus norvegicus), albinos variety and Wistar strain from the Central Biotery of the State University of Maringá was analyzed. The rats, aging 60 days, apparently healthy and having no physical impairments, were kept in individual cages at the animal house of the Department of Morphophysiological Sciences during 210 days under constant temperature and light-dark alternations of 12 hours. These animals were divided in two groups: the control group, of 10 animals, received ad libitum NUVILAB chow (recommended by the national Research Council & National Institute of Health ­ USA) with normal protein level (22%) and water. The alcoholic group, also having 10 animals, received ad libitum NUVILAB chow with normal protein level (22%) and sugar-cane brandy ("51" trademark, 39 GL, Indústrias Muller, Pirassununga, SP, Brasil), diluted to 30 GL (30% v/v). The model of alcoholism adopted was the "semi-voluntary", in which sugar-cane brandy was the only source of liquid available to the animal. After 210 days of treatment the animals of both groups were killed through inhalation of ethylic ether. The small intestine was removed and the ileum sectioned and subjected to the neuronal staining techniques.

Transverse segments spanning the whole intestinal circumference and opened at the mesenteric region were removed from five animals of each group and prepared as whole-mounts stained with the method of Giemsa ( Barbosa, 1978). Basically, this consists in appropriate fixation for 24 hours, microdissection under stereomicroscope for removal of the mucosa and submucosa; evidencing of theneurons with

Giemsa stain followed by dehydration and diaphanization and mounting of the whole-mounts between slide and coverslip with Permount synthetic resin. These whole-mounts were used for neuron counts and morphometric analyses of the nerve cell bodies.

The whole-mounts were divided in mesenteric and antimesenteric regions, in which nerve cell bodies were counted and measured. Counts were made by sampling, the neurons of 40 microscopic fields (8.96 mm2) of each region being counted in both experimental groups.

The sizes of the nerve cell bodies of 500 neurons of each region in both groups were measured. The major longitudinal and transverse axes of the cell bodies were measured with an Olympus CCB microscope equipped with 10X lens coupled to a micrometer disc and 40X objective. The data obtained were grouped and the calculated means were used to name the myenteric neurons as small, medium and large (Cook & Burnstock, 1976; Natali & Miranda-Neto). Medium neurons were those whose sum of the axes yielded values between the valid intervals of the mean. Neurons whose sum of the axes yielded values smaller than the mean minus the standard deviation, were regarded as small. Large neurons were those whose sum of the axes was larger than the mean plus the standard deviation.

Five animals of each group were used for counts of NADH-diaphorase positive neurons, evidenced through whole-mounts subjected to the technique of the NADH-diaphorase of Gabella (1969), having Nitro Blue Tetrazolium (NBT) as the final electron acceptor.

In the statistical analysis the Chi-squared test was employed to compare the incidence of small, medium and large neurons found in the groups. Student's t test was applied to the means (number of neurons, solid and liquid ingestion, body weight). The significance level adopted was 5%.

RESULTS

At the end of the experimental period, the animals of the control group had a mean body weight of 511.0 ± 41.74 g and the alcoholic group, 422.4 ± 42.5 g. The alcoholic rats showed a mean body weight significantly smaller when compared to the control rats (t=3.33; vc=1.86).

The myenteric nervous system in both groups, control and alcoholic, is formed by neurons clustered in ganglia, which are elongated and oriented circularly in the intestine. In the ileal wall, the ganglia are observed between the longitudinal and circular layers of the muscular tunica and are surrounded by collagen fibers. These features are observed regardless of the method employed.

The whole-mounts stained with Giemsa (Fig.1A) showed ganglia composed of densely packed neurons, while on those subjected to the NADH-diaphorase technique ( fig. 1B) the ganglia present neuronal cell bodies which are less clustered, leaving empty spaces in the ganglion, in both experimental groups.


Fig. 1. Whole-mount of the mesenteric region of the ileum of alcoholic rats. Bars calibrations: 10mm
A. Myenteric neurons stained with o method of Giemsa.
B. Myenteric neurons NADH-diaphorase positive

Te table I presents the results of the quantitative analysis of the whole-mounts in the control and alcoholic group, stained with both techniques. The density of neuronal cell bodies in the myenteric plexus varied according to the region of the intestinal circumfe-rence. A larger neuronal density was observed in the mesenteric region in both groups and with both staining techni-ques.

Table I. Incidence of neurons in the ileum, in an area of 8.96 mm2, in the mesenteric (RM) and antimesenteric (RA) regions of the control and the alcoholic groups, with both techniques.


  Giemsa NADH-diaforase
Regions Control Alcoholic Control Alcoholic
RM 2032,6 a 2411,8 a 1246 a 1724 a
RA 1441,2 b 1855,4 b 975,2 b 1576 a

Means followed by the same letter in the same column do not differ at the level of 5%
(Technique of Giemsa, comparison between the mesenteric and antimesenteric regions in the control group (t=7.09, vc=1.86) and in the alcoholic group (t=4.55, vc=1.86)
(Technique of the NADH-diaphorase, comparison between the mesenteric and antimesenteric regions in the control group (t=5.7, vc=1.86) and in the alcoholic group (t=1.77, vc=1.86)

The analysis of the mean neuronal density between both groups is in Table II. The alcoholic rats had a significantly higher neuronal density when compared to the controls.

Table II. Indicence of neurons in the ileum, in an area of 8.96 mm2, in the mesenteric (RM) and antimesenteric (RA) regions, in the two experimental groups, with both techniques.


Groups
Giemsa
NADH-d
  RM RM RA RA
Control 2032,6 a 1441,2 a 1246 a 975,2 a
Alcoholic 2411,8 b 1855,4 b 1724 b 1576 b

Means followed by the same letter in the same column do not differ at the level of 5%
(Technique of Giemsa. Comparison of the mesenteric regions between the groups t=4.8, vc=1.86)
(Technique of Giemsa. Comparison of the antimesenteric regions between the groups t=3.2, vc=1.86)
(Technique of the NADH-diaphorase. Comparison of the mesenteric regions between the groups t=8.03, vc=1.86)
(Technique of the NADH-diaphorase. Comparison of the antimesenteric regions between the groups t=7.95, vc=1.86)

Through the measurement of the major longitudinal and transverse axes of the cell bodies it was observed that neuronal size varied from 22.5 to 57.5 mm, so that small neurons were those measuring from 22.5 to 30 mm, medium neurons where those between 32.5 and 42.5 mm and large neurons where those ranging from 45 to 57.5 mm. With these measures as a basis, the ileum of the control rats had 90 small neurons, 322 medium neurons and 78 large neurons. In the alcoholic group 176 small, 287 medium and 37 large neurons were observed (Fig. 2).


Fig. 2. Number of small, medium and large neurons in the ileum of control and alcoholic rats.

The Chi-squared test at the significance level of 5% indicated that there existed a relationship between the experimental condition and the amounts of small, medium and large neurons in the ileum (c2=45.6; vc=5.99).

DISCUSSION

The arrangement of the ganglia relative to the intestinal circumference, that is, circularly, was observed in both experimental groups and with both techniques, agreeing with the findings in the guinea-pig ileum with methylene blue (Irwin), in the ileum of rats with NADH-d (Santer & Baker), in the small intestine of guinea-pigs with cuprolinic blue ( Karaosmanoglu et al.,1996), and in the ileum of mice with Giemsa (Bor-Seng-Shu et al.). The location of the ganglia between the longitudinal and circular layers of the muscular tunica, and the collagen fibers around them as well, in both experimental groups, are similar to the findings described in the literature (Fiorini et al., 1999; Miranda- Neto et al., 2001).

In the ileum, the farther the counts are from the mesenteric attachment, the smaller is the neuronal density, so that the antimesenteric region has the smallest neuronal density. Differences in the neuronal density in the myenteric plexus in distinct regions of the circumference of a given intestinal segment were also found in the large intestine of guinea-pigs (Irwin; Gabella, 1990), in the chicken colon (Ali, et al., 1979) and in the middle portion of the small intestine of rats (Santer, 1994).

Through the technique of Giemsa we found the following neuronal means in areas of 8.96 mm2: 2032.6 and 1441.2 in the control rats; 2411.8 and 1855.4 in the alcoholic rats in the mesenteric and antimesenteric regions, respectively. As this technique is supposed to stain all the neurons because of the affinity of methylene blue by polyribosomes and all the neurons, regardless of physiologic features, have this organelle, we would then have the total number of neurons.

The mean of NADH-diaphorase positive neurons in the control rats was 1246 in the mesenteric region and 975.2 in the antimesenteric region and in the alcoholic rats it was 1724 and 1576, respectively. The smaller proportion of NADH-d positive neurons in relation to the technique of Giemsa was also found by others (Sant'Ana et al.).

The NADH-d technique evidenced wide gaps between the cell bodies of the ganglia forming the myenteric plexus. Heinicke et al. (1987) argued that such gaps might represent small unstained neurons.

In both experimental groups the myenteric nervous system is composed of small, medium and large neurons. Differences in the neuronal cell body size was also verified in other investigations and intestinal segments of different species (Natali et al.; Cook & Burnstock; Fiorini et al.; Miranda-Neto et al., 1999).

In the ileum of the control and alcoholic rats 18% and 26.2% of the neurons, respectively, were small. Medium neurons accounted for 66.4% in the controls and 62.2% in the alcoholics. Large neurons represented 15.6% of the total neuronal population in the control group and 11.6% in the alcoholic group. These differences found in the percentages of small, medium and large neurons in the myenteric neuronal population are probably related to the ingestion of alcohol. The rats subjected to alcoholism had a final body weight of 422.4 ± 42.5 g, while those of the control group weighted 511.0 ± 41.74 g (t=3.33; vc=1.86), so that the former had a reduced body growth. The calories from the alcohol lead to a reduced ingestion of food, and desnutrition ensues (Weinberg & Vogl, 1998; Pereira et al., 2003). In turn, desnutrition, especially concerning protein, impairs synthetic processes, and could explain the reduced neuronal cell body size as well as the smaller dispersal of the neurons, as the neuronal density observed in the alcoholic rats was greater than that found in the control rats. This "alcoholic desnutrition" can also explain why our morphologic and morphometric data of the ileal myenteric neurons are similar to those in the ileum of rats subjected to hypoproteic diets (Sant'Ana et al.; Fiorini et al.; Miranda-Neto et al.). Our morphometric data corroborate those of Bogliolo (1981), who states that, because of the reduced availability of nutrients or of necessary stimuli, the cell adapts its metabolism, which may diminish the structural turnover and lead to decreased cell volume.

The present study allows the conclusion that the experimental chronic alcoholism lasting for 210 days induced a significant decrease in the final body weights of the rats. This smaller body growth was reflected in the lesser dispersal of the nerve cells with greater neuronal densities evidenced with the techniques of Giemsa and NADH-diaphorase in the alcoholic animals; and in the reducedsizes of the neuronal cell bodies evidenced by the increased proportions of small neurons and decreased proportions of large neurons in the alcoholic rats. 


PEREIRA, M. A. S.; MOLINARI, S. L.; DE SOUSA, F. C.; ANDRÉ, O. E. & MIRANDA NETO, M. H. Densidad y morfometría de las neuronas del íleon de ratas sometidas a alcoholismo crónico. Int. J. Morphol., 21(3):245-250, 2003.

RESUMEN: Verificamos los cambios causados por la ingestión de etanol en las neuronas mioentéricas del íleon en ratas sometidas a alcoholismo crónico. 20 ratas (Ratus novergicus) fueran divididas en grupo control (GC) y grupo alcohólico (GA). Durante 210 días los animales del GA recibieron aguardiente de caña diluida a 30° y los animales del GC recibieron agua. El íleon fue sometido a la elaboración de preparados totales para marcación neuronal de acuerdo con Barbosa (1978) y Gabella (1969). Las neuronas fueran contadas y medidos los cuerpos celulares. En ambas técnicas, las ratas alcoholizadas presentaron una mayor densidad neuronal. El análisis estadístico, reveló una redución significativa del número de neuronas grandes en el GA cuando fue comparado con el GC; en el GA hubo un aumento significativo en el número de neuronas pequeñas. Ocurrió un menor crecimiento en las ratas GA, causando una menor dispersión de las células nerviosas y un amplio número de neuronas evidenciado a través de las técnicas de Giemsa y NADH-diaforase tanto en los animales del GA como del GC y reducción en el tamaño de los cuerpos celulares, comprobado por el aumento de la proporción de neuronas pequeñas.

PALABRAS CLAVE: 1. Alcoholismo; 2. Ileon; 3. Neurona mientérica.


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Correspondence to:
Profa. Dra. Marli Aparecida dos Santos Pereira
Universidade Estadual de Maringá
Av. Colombo, 5790 ­ Bloco H 79 DCM
Maringá ­ Pr. 87020-900
BRASIL

e-mail: madspereira@uem.br

Received : 30-06-2003
Accepted: 07-08-2003 


Department of Morphophysiological Sciences, State University of Maringá, Pr. Brasil.

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