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International Journal of Morphology

versão On-line ISSN 0717-9502

Int. J. Morphol. v.21 n.4 Temuco  2003 

Int. J. Morphol., 21(4):273-277, 2003.



*Maria Raquel Marçal Natali; *Marcílio Hubner de Miranda-Neto & **Antônio Marcos Orsi

NATALI, M. M. R.; MIRANDA-NETO, H. M. & ORSI, M. A. Morphometry and quantification of the myenteric neurons of the duodenum of adult rats fed with hypoproteic chow. Int. J. Morphol., 21(4):273-277, 2003.

SUMMARY: Morphometric and quantitative analyses were carried out on the myenteric neurons of the duodenum, to study possible alterations resulting from a supply of low-protein level chow to adult Wistar rats. The animals were divided in two groups: nourished (N) fed with ration containing 22% protein for 210 days and the undernourished (D) fed with ration containing 8% protein from the 90th day of age on and during the next 120 days. Whole-mounts were stainedby the method of Giemsa and the histochemical reaction of NADH-diaphorase enzyme, to estimate the neuronal population, which revealed a greater neuronal density in undernourished rats, regardless of the technique employed. The morphometric studies of the cell body profiles of 500 neurons of each group indicated a reduction on the cell body size and an increase on the proportion of small neurons on the rats subjected to the hypoproteic chow.

KEY WORDS: 1. Proteic deficiency; 2.Duodenum; 3.GIEMSA; 4.Myenteric neurons; 5.NADH-diaphorase; 6. Rats.


The wall of the digestory tube presents extensive plexi of nerve fibers and neuronal cell bodies responsible for the modulation of the rithmic gastrointestinal peristaltic activity, among other functions, even in the absence of control from the central nervous system. These plexi compose the Enteric Nervous System (ENS). One of the most developed ganglionated plexi of the intestinal wall is the myenteric plexus (or Auerbach's), located between the inner circular layer and the outer longitudinal layer of the smooth muscle tunica. The myenteric plexus has morphologic and quantitative features that change along the digestory tract both in man and in animals (Gabella, 1987; Furness & Costa, 1987).

Morphometric and quantitative assessments of the neurons of the myenteric plexus of the small intestine in rats subjected to diets with protein restriction during gestation and lactation revealed morphometric changes in the cell body and greater neuronal density in the undernourished animals (Torrejais et al., 1995; Natali & Miranda-Neto, 1996; Meilus et al., 1998; Brandão et al., 2003).

Among the several manifestations of neuronal plasticity there are the variations occurring on the peripheral neurons that accompany the changes in the organs they innervate, this plasticity not being restricted to the initial periods of body growth, but persisting in the completely differenciated nervous tissue (Gabella, 1987, 1990). Thus, the innervated organs could have the potential to promote the neuronal growth or the reduction of the nerve cell volume to some desired level, depending on the prevailing conditions.

This work had the aim of evaluating the effects of a prolonged protein restriction on the neurons of the myenteric plexus of the duodenum of adult rats, considering that malnutrition could lead to alterations in the cellular metabolism reflecting upon the morphometric and quantitative aspects of these neurons.


Twenty male albino rats (Rattus norvegicus) Wistar1 strain with 90 days of age were used, weighting on average 296 gr and were kept in individual cages, at constant temperature and light-dark cycles of 12-12 hr, for 120 days. The rats were divided in two groups:Nourished group (N): composed of 10 animals fed for 210 days with standard rodent chow ­ NUVILAB (recommendend by the National Research Council & National Health Institute, USA), with 22% proteic level. Undernourished group (D): composed of 10 rats that from the 90th day of age, received ration with 8% protein (Natali et al., 2000) for 120 days. Chow and water were offered ad libitum. This chow was supplemented with hydrosoluble vitamins of complex B and saline mixture (American Institute of Nutrition, 1977; Natali & Miranda- Neto, 1996; Natali et al.).

Five animals of each group had their blood collected for dosage of total protein, albumin and globulins. These parameters allowed the confirmation of the model of malnutrition used and have been already described (Natali et al., 2000).

Morphologic and quantitative study of the myenteric plexus. The duodenum was entirely removed and the excess of mesenteric fat was dissected out. Five segments of each experimental group were washed in 0.9% saline solution, and filled and immersed in fixative solution of Giemsa for 48 horas. Then, the segments were microdissected under stereomicroscope; the mucosa and submucosa were removed and the smooth muscle and serosa were preserved. This material was then stained with staining solution of Giemsa (methylene blue) in Sorensen phosphate buffer (pH 7.0) (Barbosa, 1978).

The other five segments of each group were washed and filled with Krebs solution(pH 7.3), washed twice with the same solution (10 minutes each) and immersed for five minutes in 0.3% Triton X-100 dissolved in Krebs solution. They were next washed twice with the same solution (10 minutes each) and incubated for 45 minutes, for evidenciation of the NADH-diaphorase enzyme, according to Gabella (1969).

Neurons counts were carried out in an Olympus BX 40 microscope under 40X objective. In each whole-mount preparation, either those stained by Giemsa or those subjected to the NADH-diaphorase technique, it was counted all neurons seen in 80 microscopic fields, 40 fields on the mesenteric region and 40 on the intermediate region. Half-seen neurons were counted on alternate fields. Total area of the 80 fields was 13.88mm2.

 In each one of the Giemsa-stained whole-mounts the areas of 100 neuronal cell bodies were measured, yielding 1.000 measured neurons. This analysis was made using the Computerized Image Analyzing System Optimas 4.10.

Statistical Treatment. For the analysis of the number of neurons, Variance Analysis was employed, followed by Tukey's test t, working with the square root of the mean number of neurons. For the analysis and classification of the cell profiles (areas), the test t of Student was carried out. The significance level adopted was of 5%.


Quantification of the myenteric neurons in the duodenum. The number of neurons, counted on the Giemsa-stained whole-mounts, revealed for the nourished group (N) a mean of 3.020 neurons (21.757 n/cm2), and for the undernourished group (D) a mean of 3.516,4 neurons (25.334 n/cm2).

The NADH-diaphorase technique revealed for the nourished group a mean of 786.6 neurons (5.667 n/ cm2) and for the group of undernourished animals a mean of 1.477,8 neurons (10.646 n/cm2).

The number of neurons found in the duodenum of the undernourished animals was greater than that observed in the nourished group, despite the technique employed. Nevertheless, the NADH-diaphorase technique stained a smaller population of neurons in both groups.

The statistical tests showed a significant difference both between the groups and the techniques used.

The square root of the mean number of myenteric neurons in the duodenum of rats, according to the technique used was with Giemsa 57.08a and with NADH 33.02b.

The square root of the mean number of myenteric neurons, according to the nutritional groups was: Nourished 41.41a and undernourished 48.70b

Determination of the cellular profile. The area of the cell body of 1.000 neurons of the myenteric plexus, 500 from each group, was estimated. Through these measures, expressed as µm2, it was observed that there is a large variation on the size of the areas of these cell bodies, neurons with profiles ranging from 25.44µm2 to 387.83µm2being found.

From the values obtained in the nourished rats, the neurons were classified as small, medium and large, according to the size of the area of their cell bodies.

The neurons with areas smaller than the mean minus its standard deviation were considered small, those above the mean plus its standard deviation were considered large, and those intermediate to these values were considered medium neurons. The mean value of the neurons from the nourished group was 147.86µm2 and the standard deviation was 61.83.

In this way, small neurons were those having cellular areas 86.03µm2, large neurons were those with cellular areas 209.69µm2, and medium neurons had their areas between 86.04µm2 and 209.68µm2.

The arithmetic mean, obtained using the 500 neurons of the undernourished group, was 130.13µm2 ± 54.10, which, when compared to the mean of the nourished group, was statistically smaller (p < 0,05).

Table I presents the absolute frequency of the neurons of the duodenal myenteric plexus, classified according the area of their cell bodies.

Table I. Absolute frequency of neurons of the myenteric plexus classified according to the area of the cell bodies in the nourished (N) and undernourished group (D).

Variable Small Medium Large Total

Nourished 77a(1) 343 a 80  a 500
Undernourished  112 b 338 a 50  b 500

1Frequencies followed by the same letter do not differ for the group, at the significance level of 5%.


Quantitative aspects of the myenteric neurons. In this investigation, two staining methods were employed to the studies of the neuronal population of the myenteric plexus: the method of Giemsa (Barbosa) and the histochemical method based on the activity of the NADH-diaphorase enzyme (Gabella, 1969) in whole-mounts.

The neuronal density we found in the control rats is similar to that observed by Bor-Seng-Shu et al., 1994, in the duodenum of adult mice stained with Giemsa, whose neuronal density was 20,212 neurons/cm2. In the small intestine of guinea-pigs, whole-mounts stained with Giemsa revealed a neuronal density of 15,600 neurons/cm2 (Liberti et al., 1994), and using cuprolinic blue as a stain a neuronal density of 15,929 neurons/cm2 was obtained (Karaosmanoglu, 1996).

We believe that the differences found among these authors stem from the fact that these densities are not restricted tospecific segments, but instead refer to the whole small intestine; variations in the number of myenteric neurons along the segments are commonplace in the literature.

The number of myenteric neurons is also subject to variations according to the species and the age of the animal. In the comparative evaluation of differences in the number of myenteric neurons in animals of different ages and species, (Gabella, 1989; Santer, 1994) employed different staining methods; a decrease was observed in the neuronal density in the older animals.

When analyzing the neuronal density in the undernourished animals, we evidenced that malnutrition, in this experimental condition, did not cause reduction in the neuronal density, as opposed to what is observed in experimental models using aging or diabetes (Buttow et al., 1997; Zanoni et al., 1997).

The greater neuronal density verified in the undernourished animals (25,334 neurons/cm2), is related to the smaller body growth of these animals and to the reduced intestinal wall and smooth muscle tunica, which lead to a smaller spread of the nerve cells and then a greater concentration per area (Natali & Miranda Neto; Torrejais et al.; Meilus et al.; Brandão et al.).

The concerned literature shows that the number of myenteric neurons stained with NADH-diaphorase is smaller than that found with techniques using cuprolinic blue stain (Heinicke et al., 1987; Karaosmanoglu et al., 1996) or Giemsa (Bor-Seng-Shu et al.; Liberti et al.; Torrejais et al.; Natali & Miranda Neto, 1996), this was observed in this work as well.

We believe that this difference is due to the fact that methylene blue and similar stains reveal the affinity of these stains for acidic cellular structures, such as the rough endoplasmic reticulum and free ribosomes (Nissl's corpuscules). These are cytoplasmic organelles abundant in the neuronal cell body.

On the other hand, the NADH-diaphorase technique evidences the action of dehydrogenases in the energy production pathways of the neurons. Once the intensity of this stain is given by the rate of enzymatic activity, revealing intensely stained cells, it is supposed that small cells, with low enzymatic activity, could remain weakly stained or unstained.

We consider that the Giemsa stain provides better estimates of the total neuronal population, and that the NADH-diaphorase technique would indicate the number of NADH- diaphorase positive neurons, which represent only a neuronal sub-population.

The NADH-diaphorase sub-population, obtained in the whole-mounts of duodenum of rats from the nourished group, was 786.6 neurons, in an area of 13.88 mm2, corresponding to 5.667 neurons/cm2. Thus, the results obtained for the nourished group were similar to those observed in the ileum, 4.872 neurons/cm2 (Heinicke et al.), and in the jejunum and ileum of rats, respectively 5.477 neurons/cm2 and 8.169 neurons/cm2 (Santer). On the other hand, for the group of undernourished rats a mean of 1.477,8 NADH-diaphorase positive neurons was obtained, corresponding to a neuronal density of 10.646 neurons/ cm2.

Analysis of the neuronal cell profile. The Giemsa-stained whole-mounts showed that there are neurons of different sizes in the myenteric plexus of the duodenal wall. This wide range of sizes resulted in a classification of the neurons as small, medium and large, based on the area of the neuronal cell body, this scheme being often seen in the literature (Fehér& Vajda, 1972; Natali & Miranda-Neto, 1996; Meilus et al., 1998).

The mean value of the area of the cell body was 147.86µm2, obtained from the measurement of 500 neurons of rats from the nourished group. A mean value of 104.14µm2 for the duodenum and mean values of 120.95µm2 for the jejunum and 126.86µm2 for the ileum in mice were obtained with this same technique (Bor-Seng-Shu et al.), although in a different species.

In adult guinea-pigs, a mean neuronal area of 195.40µm2 in the duodenum, and 185.58µm2 and 147.0µm2 in the jejunum and ileum, respectively, were measured (Liberti et al.). These authors used the same staining method, that is, the Giemsa stain.

We consider that the variations that reveal larger values for the neuronal sizes may be due to the technique used; as previously discussed, small neurons probably remained unstained. This possibility would explain our results with Giemsa stain, in which the smallest neuron had an area of 25.44µm2, and the largest 387.83 µm2. Clearly, the small neurons were not stained.

The comparative analysis of the mean area of the cell bodies from the nourished group (147.86 µm2) demonstrated a

significant difference relative to the mean from the undernourished group, which was 130.13 µm2, thus showing the effect of protein restriction on the neuronal size.

Decreases of the neuronal cell body were also observed in the ileum of rats aging 60 days (Torrejais et al.; Meilus et al.) and in the ascending colon of adult rats (Sant` ana et al., 1997 ) which were subjected to protein restriction (8% protein in the chow).

The relation between the predominance of a given neuronal population, the size of the cell body and the development period of the animal, was analyzed in the myenteric plexus of adult and newborn rats (Gabella, 1971). The author concludes that, during growth, there is an increase in the neuronal size, and that the prevailing population in the newborn rats is of small neurons. However, in adult animals, although the small neurons were present, larger neurons made up the major component of the ganglionic population.

Our results agree with these, the population of small neurons being less representative. There was also agreement with the results obtained for animals of the same age, subjected to the proteic restriction. In these, the population of small neurons increased relative to the larger ones which, according to the proposal of the author referred, would be explained by the absence of growth of the animal.

Cellular hypotrophy, including in neurons, can be considered as a basic response mechanism in instances where the cells are under agression, such as decrease of nutrient supply or of necessary stimuli for their functioning. As a result the cells get adapted, with decreased metabolism, leading to a decrease in the turnover of their own structures and then to a reduction in their volume (Bogliolo, 1981).

The considerations made here allow us to state that the greater proportion of small neurons observed in the undernourished group results from the short supply of proteins in the chow delivered to the rats, halting their growth. In the specific case of the myenteric neurons, the hypertrophic growth would be commited, and this would be one of the reasons why a larger number of small neurons would be mantained.

NATALI, M. M. R.; MIRANDA-NETO, H. M. & ORSI, M. A. Morfometría y cuanificación de las neuronas mioentéricas del duodeno en ratones adultos alimentados con ración hipoproteica. Int. J. Morphol., 21(4):273-277, 2003.

RESUMEN: En el plexo mioentérico del duodeno de ratones Wistar albinos adultos, se realizaron análisis morfológicos y cuantitativos para estudiar posibles alteraciones en el transcurso de su alimentación con raciones de bajo contenido de proteínas. Los animales fueron divididos en dos grupos: normoalimentados (N) cuya ración contenía 22% de proteínas y fueron alimentados con ella por 210 días, y el grupo desnutrido (D) alimentado con ración conteniendo 8% de proteínas, a partir de los 90 días de edad y durante los 120 días subsiguientes. Los micropreparados de membrana fueron teñidos con el método GIEMSA, y a través de la reacción histoquímica de la enzima NADH-diaphorasa se estimó la población neuronal, que reveló mayor densidad neuronal en los animales desnutridos, independientemente de la técnica empleada. Los estudios morfométricos de los perfiles de los cuerpos celulares de 500 neuronas de cada grupo, indicaron reducción en el tamaño del cuerpo celular y aumento de la proporción de neuronas pequeñas en los ratones alimentados con ración hipoproteica.

PALABRAS CLAVE: 1. Desnutrición proteica; 2. Duodeno; 3. GIEMSA; 4. NADH-diaphorasa; 5. Neuronas mioentéricas; 6. Ratones.


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Correspondence to:
Prof. Dra. Maria Raquel Marçal Natali
Laboratory of Enteric Neurons
Department of Morphophysiological Sciences
State University of Maringá
Paraná, BRASIL

Fone: 55-44-261-4706
Fax : 55-44-261-4340

E-mail :

Received : 18-08-2003
Accepted : 22-09-2003

* Laboratory of Enteric Neurons, Department of Morphophysiological Sciences, State University of Maringá, PR, Brazil

** Department of Anatomy, Institute of Biosciences, UNESP, Botucatu, São Paulo, Brazil.

1Approved by the Ethical Committee on Animal Experimentation (CEEA) of the Biosciences Institute/UNESP- Botucatu, SP, Brazil.

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