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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/S0716-98682002000100003 

NADH-DIAPHORASE POSITIVE MYENTERIC NEURONS OF THE
AGLANDULAR REGION OF THE STOMACH OF RATS (Rattus norvegicus)
SUBJECTED TO DESNUTRITION

 NURONAS MIOENTÉRICAS NADH-DIAFORASA POSITIVAS DE LA REGIÓN NO GLANDULAR
DEL ESTÓMAGO DE RATONES (Rattus norvegicus) SOMETIDOS A DESNUTRICIÓN

*Sonia Lucy Molinari; ** Carlos Alexandre Fernandes; *** Larissa Renata de Oliveira;
*** Débora de Mello Gonçales Sant'Ana & * Marcílio Hubner de Miranda Neto

* Department of Morphophysiological Science, State University of Maringa, State of Parana, Brazil.
** Science Biological Students, State University of Maringa, State of Parana, Brazil.
*** Department of Morphophysiological Science, University Paranaense, Umuarama, Parana, Brazil.

SUMMARY: The stomach carried out movements characterized by potent peristaltic slow waves which serve mixture, mechanical digestion and slow emptying through the pylorus. The control of this movement is made by hormone action and neural activity, especially the intrinsic innervation by the neurons of the myenteric plexus. Once the condition of desnutrition can cause morphological alterations even upon the neurons this work was proposed with the purpose of verifying the effects of protein desnutrition lasting 120 days on the quantitative aspects of the NADH-diaphorase positive neurons. It was used 10 adult rats aging 90 days, which were divided in two groups, control and disnurtured. The rats of the disnurtured group (n=5) were subjected to a diet of 8% protein level, and those of the control group (n=5) received chow with normal protein level (22%). After 120 days, the aglandular stomach was subjetec to the histoenzymologic technique of the NADH-diaphorase for neuronal evidenciation. The aglandular regions was divided in a region near the major gastric curvature and a region anterior to the limitant ridge. According to the sampling method of counting, the observed neurons in 40 microscopic fields (6.64 mm2) of each region in all the animals of the two groups were counted using light microscope with 40X objective. We verified that the neurons can be isolated or grouped into ganglia. Next to the limitant ridge it was found a mean of 570.8 neurons in the control group and 718.4 in the disnurtured group. On the other hand, next to the major gastric curvature, we observed 25.8 neurons in the control group and 52.6 neurons in the disnurtured group. The low-protein diet resulted in less body growth with a weight decrease of about 30.33% when compared to the control animals, as well as a decrease in the area of the stomach of 20.13%. We concluded that the neurons are not uniformly distributed in the stomach wall and that the disnurtured animals showed less neuronal dispersion, thus exhibiting a greater neuronal density per mm2.

KEY WORDS: 1. Desnutrition; 2. Stomach; 3. Myenteric neuron.

INTRODUCTION

To carry out its functions, the stomach's motility has features of potent slow peristaltic waves which serve for mixture, mechanical digestion and slow emptying through the pylorus. The control of this movements is made through hormone action and neural activity, a major constituent of which is the intrinsic innervation represented by the neurons of the myenteric plexus.

The myenteric plexus is defined as being formed by a network of ganglia, disposed in regular rows and interspersed with the nerve fibers, which extends from the esophagus to the anal canal (Sternini, 1988) between the circular and longitudinal layer of the muscle tunica (Irwin, 1931; Gabella, 1971). The intrinsic components of the enteric nervous system, especially of the myenteric plexus, play an important role in the control of the intestinal motility and secretion and their study reveals valuable information on the many gastrointestinal pathologies, especially those concerned with nutritional alterations.

As most of the biomolecular reactions require proteins, it is expected that each cell and each tissue can be affected when subjected to conditions of protein desnutrition (Deo, 1978). It has been described that the cellular response to desnutrition is time-dependent, that is, is linked to the period in which it begins and the duration of the period of food restriction (Winick & Noble, 1966; Firmansyah et al., 1989).

Studies on the alterations caused by protein desnutrition on the morphology of the enteric nervous system are of great value, once it is composed of a tissue of low turnover index. Changes on the number, shape of function of its cells may lead to drastic alterations in the individual, because the lack of normal delivery of aminoacids can disrupt processes of the normal cellular metabolism. In the myenteric plexus few are the work made; in this way, experimental research relating desnutrition and its effects on the myenteric plexus contribute to the knowledge of the pathophysiology of illnesses related to gastric motility.

In face of the data on the literature, the purpose of this work was to verify the effects of protein desnutrition on the morphology and density of the myenteric neurons in the aglandular stomach of rats.

MATERIAL AND METHOD

The stomach of 10 laboratory rats, Rattus norvegicus of the Wistar strain, coming from the Central Biotery of the State University of Maringá, were used. The animals were mantained and killed according to the rules of ethics and conduct in animal experimentation.

The adult rats aging 90 days (+- 280 g) were kept in individual cages, at constant temperature and light-dark cycles of 12 hours, and were divided in two grous: a) control group, made up of five animals receiving Nuvilab chow with normal protein level (22%) for 120 days (chow recommended by the National Council & National Institue of Health _ USA) and water, both ad libitum. b) disnurtured group, made up of five animals that, from the age of 90 days were fed for 120 days with Nuvilab chow with protein level reduced to 8%, through the addition of corn starch and supplemented with vitamins and minerals (Natali et al., 1995) and water, both ad libitum.

After 120 days, the rats were killed through inhalation of ethylic ether.

After removal, the stomachs' profiles were taken on a cardboard and the area was calculated in a computerized image analyzer (ImagePro Plus).

For the analysis of arrangement and shape of the myenteric ganglia, as well as the counting of the NADH-diaphorase positive neurons, it was employed the histochemical technique for evidenciation of nerve cells through the activity of the NADH-diaphorase enzyme, using Nitro Blue Tetrazolium as artificial electron acceptor (Gabella, 1969). The aglandular region of the stomach was sectioned at the limitant ridge and under stereomicroscope the mucosa and some muscle fibers were dissected out. Next, it was dehydrated, diaphanized and mounted in slide and coverslip with Permount sinthetic resin.

The aglandular region of the stomach was divided into two segments: next to the major gastric curvature and anterior to the limitant ridge (Fig. 1). According to the method of counting by sampling, the neurons observed in 40 microscopic fields (6.64 mm2) under 40X objective in each segment were counted in each animal of both groups.


Fig. 1. Schematic representation of the regions analyzed of the aglandular stomach of the rat. (a) major gastric curvature and (b) region anterior to the limitant ridge.

The mean, standard deviation and variation coefficient of the number of neurons found in each segment of both groups were calculated. Student's t test was employed to compared the differences of the means between the control and disnurtured groups. The significance level adopted was 5%. Photographic documentation was obtained in photomicroscope BIMAX-50 with photographic equipment PM10-AK.

RESULTS

Table I. Mean and standard deviation of the number of neurons in an area of 6.64 mm2 in the regions of the major gastric curvature (A) and anterior to the limitant ridge (B) of the aglandular stomach of rats from the control and disnurtured groups.

GROUPS  

REGIONS

 
  A   B
Control 25.8±4.59*     570.8 ±120.31*
Disnurtured 52.6± 6.43**     718.4  ±109.81**

t: 8.99; vc: 1.86 *
t: 6.86; vc:1.86 **

The observations made in the whole-mounts of the aglandular region in both groups show that the myenteric plexus is organized in ganglia, although some isolated neurons are also found. Different sized neurons are observed, and they are generally oval with espherical and eccentric nucleus. The distribution, arrangemente and density of the ganglia is not homogeneous in the segments studied (Figs. 2a and 2b).

00
Fig. 2. Whole-mount of the aglandular region of the rat stomach, showing the arrangement of the myenteric plexus and the neuronal density next to the major gastric curvature (a) and anterior to the limitant ridge (b). NADH. 60X.

In the region of the major gastric curvature the ganglia are small, with few and sparse neurons (Fig 2a). In the region anterior to the limitant ridge, the ganglia have irregular shapes, are not far apart from each other and are formed by many neurons (Fig. 2b). This arrangement shows that the density of the neurons varied according to the region under observation.

The quantitative data in the differente regions of the aglandular stomach of the rats from the control and disnurtured groups are summarized in Table 1. The use of Student's t test showed significant differences between the regions studied, both in the control (t=8.99; critical value=1.8.) and the disnurtured group (t=6.97; critical value=1.86).

The experimental diet the rats were fed with caused changes of body weight. The rats of the control group ended with 362.98±27.70 g, while those receiving low-protein diet obtained 252.88±21.20 g body weight (t=7.8; critical value =1.86).

DISCUSSION

The myenteric plexus in the wall of the aglandular stomach of rats is distributed in a non-uniform manner, regardless of the group considered. It is observed that there is a decrease in the number of cell bodies, with larger neuronal densities in the region anterior to the limitant ridge and smaller densities in the region next to the major gastric curvature.

We verified, in both groups, that the ganglia observed next to the limitant ridge are large, while those next to the major gastric curvature are smaller are more dispersed. This findings are similar to those described by Fregonesi et al., (1998) in the body of the stomach of rats, where a larger neuronal density was verified in the lesser gastric curvature as compared to the major gastric curvature. The region anterior to the limitant ridge corresponds to the findings of Fregonesi et al., as far as the features of the ganglia found in the lesser gastric curvature are concerned.

The neurons, of varied size and mostly oval, showed spherical nucleus, generally eccentric, and were, in this respect, similar in both groups and in both regions studied. Similar data were found in other segments of the gastrointestinal tract of the rat, such as in the body of the stomach (Fregonesi et al.,), duodenum (Natali et al., 1995), jejunum (Miranda-Neto et al., 1999), ileum (Fiorini et al., 1999), cecum (Zanoni et al., 1997), and colon (Romano et al., 1996 and Sant'Ana et al., 1997).

As for the neuronal densitu, we verified that the region anterior to the limitant ridge shows larger numbers of neurons per mm2, when compared with the region next to the major gastric curvature. The difference in neuronal density can be related to the thickness of the muscle tunica. Saffrey & Burnstock, 1994) mentioned that the number of neurons in the myenteric plexus is larger in areas where the layer of smoth muscle are thicker; this fact was observed here in the different regions of the aglandular stomach. In the region next to the limitant ridge it was found a mean of 570.8 and 718.4 neurons in an area of 6.64 mm2 in the control and disnurtured groups, respectively. In the region of the major gastric curvature 140.8 and 325.8 neurons/6.64 mm2 were found, respectively. The observatin of greater neuronal density in the disnurtured group (25.8%) can be explained by the fact that during 120 days these animals received low-protein diet, leading to a 30.33% loss of body weight relative to the controls, as well as a decrease in the area of the stomach profile of about 20.13%. Probably these factors influenced the neuronal distribution, making neurons more densely packed and leading to the larger amount of neurons per area in the disnurtured animals. Similar data were obtained in the rat ileum with diet containg a protein level of 8% (Fiorini et al.). The change of body weight in animals receiving protein-deficient diets was evidenced by Takano (1964), Maffei et al. (1980), Natali et al. (1995), Natali & Miranda-Neto (1996), Sant'Ana et al. and Natali et al. (2000).

This study verified that the number of neurons of the myenteric plexus are heterogeneously distributed in the wall of the aglandular stomach, with greater neuronal density in the region anterior to the limitant ridge. The low-protein diet led to a decrease of both body weight and stomach profile, thus causing a greater packing of neurons per mm2.

RESUMEN: El estómago realiza movimientos caracterizados por potentes ondas peristálticas lentas, útiles en la mezcla del bolo alimenticio, la digestión mecánica y su lenta liberación por el píloro. El control de este movimiento se realiza por acción hormonal y por actividad nerviosa, destacando la inervación intríseca que representan las neuronas del plexo mioentérico. Considerando que un estado de desnutrición puede provocar alteraciones morfológicas, incluso neuronales, se ha propuesto este  trabajo, con el objetivo de verificar los efectos provocados por la desnutrición proteínica durante un período de 120 días, en el aspecto cuantitativo de las neuronas NADH-diaforasa positivas. Para este estudio se utilizaron 10 ratones adultos, los cuales, a los 90 días de vida fueron divididos en dos grupos; control y desnutridos. A los ratones del grupo desnutridos (n=5) se les alimentó con una dieta proteínica de, aproximadamente, 8% y a los ratones del grupo control (n=5) con dieta de valor proteínico normal, 22%. Transcurridos 120 días, al estómago aglandular se le hizo un ensayo de histoenzimología del NADH-diaforasa, para verificar características neuronales. La región glandular fue dividida en dos áreas: una, próxima al lóbulo gástrico mayor y la otra, anterior al repliegue límite. En el recuento por muestreo, las neuronas observadas en 40 campos microscópicos (6,64 mm2) de cada área, de las muestas de los ratones de los grupos control y desnutridos, fueron contadas con un microscopio de 40x. Constatamos entonces que las neuronas pueden estar aisladas o agrupadas en sus ganglios. Cerca del repliegue límite encontramos un promedio de 570,8 neuronas en el grupo control y 718,4 en el grupo desnutridos. Por otra parte, cerca del lóbulo gástrico mayor, encontramos 25,8 neuronas en el grupo control y 52,6 en el grupo desnutridos. La dieta hipoproteínica implicó un menor crecimiento físico, con una descompensación de alredededor de 30,33%, al compararlo con ratones del grupo control, como también hubo una reducción de la superficie del perfil estomacal, de aproximadamente 20,13%. Concluimos que, las neuronas no están distribuidas uniformemente en la pared estomacal y que en los ratones desnutridos presentaron una menor dispersión, presentando por lo tanto una mayor densidad neuronal por mm2.

PALABRAS CLAVE: 1. Desnutrición; 2. Estómago; 3. Neurona, mientérico.

REFERENCES

Deo, M.G. Cell biology of protein-calorie malnutrition. Wld. Ver. Nutr. Diet., 32:49-95, 1978.           [ Links ]

Fiorini, A.; Molinari, S. L.; Natali, M. R. M.; Miranda-Neto, M. H. Quantitative morphological analysis of the myenteric neurons of the ileum in rats under experimental desnutrition. Acta Scientiarum, 21(2):409-13, 1999.           [ Links ]

Firmansyah, A.; Suwandito, L.; Penn, D. & Lebenthal, E. Biochemical and morphological changes in the digestive tract of rats after prenatal and postnatal malnutrition. Am. J. Clin. Nutri.,50:261-8, 1989.         [ Links ]

Fregonesi, C. E. P. T.; Miranda-Neto, M. H.; Molinari, S.L.  Estudo morfológico e quantitativo dos neurônios mientérico do corpo do estômago de Rattus norvegicus. Acta Scientiarum, 20(2):221-4, 1998.           [ Links ]

Gabella, G. Neuron size and number in the myenteric plexus of the newborn and adult rat. J. Anat., 109:81-95, 1971.           [ Links ]

Gabella, G. Detection of nerve cells by a histochemical technique. Experientia, 23(52):218-9, 1969.           [ Links ]

Irwin, D. A. The anatomy of Auerbach's plexus. Am. J. Anat., 49:141-66, 1931.           [ Links ]

Maffei, H.V. L.; Rodrigues, M.A.M.; Camargo, J. L.V. & CAMPANA, A. Intraepithelial lymfocytes in the jejunal mucosa of  malnourished rats. Gut, 21: 32-36, 1980.           [ Links ]

Miranda-Neto, M. H.; Molinari, S.L.; Stabille, S.R. & Sant'Ana, D.M.G. Morphologic and quantitative study of the myenteric neurons of the jejunum of  malnourished rats (Rattus norvegicus). Arq. Neuropsiquiatr., 57(2-B):387-91, 1999.           [ Links ]

Natali, M.R.M.; Miranda-Neto, M, H.; Balhs, A.S. & Watanabe, I.S. Effects of maternal protein malnutrition n the duodenal mucous layer of rats. Braz. J. Morphol Sci., 12: 71-6, 1995.           [ Links ]

Natali, M.R.M. & Miranda-Neto, M.H. Effects of maternal proteic undernutrition on the neurons of the myenteric plexus of duodenum of rats. Arq. Neuropsiquiatr., 54(2):273-9, 1996.           [ Links ]

Natali, M.R.M.; Miranda-Neto, M.H. & Orsi, A.M. Effects of hypoproteic diet supply  on adult wistar rats (Rattus norvegicus). Acta Scientiarum, 22(2):567-71, 2000.         [ Links ]

Romano,E.B.; Miranda-Neto,M.H. & Cardoso, R.C.S. Preliminary investigation about the effects of strptozotocin-induced chronic diabets on the nerve cell number and size of myenteric ganglia in  rat colon. Rev. Chil. Anat., 14:139-45, 1996.         [ Links ]

Saffrey, M. J. & Bunstock, G. Grouth factors and the developments and plasticity of the enteric nervous system. J. Aut. Nervous System., 49:183-96, 1994.           [ Links ]

Sant'Ana, D.M.G.; Miranda-Neto, M. H.; Souza, R.R. & Molinari, S.L. Morphological and quantitative study of the myenteric plexus of the ascending colon rats subjected to proteic desnutrition. Arq. Neuropsiquiatr., 55(4):687-95, 1997.           [ Links ]

Sternini, C. Structural and chemical organization of the myenteric plexus. Ann. Rev. Physiol., 50:81-93, 1988.           [ Links ]

Takano, J. Intestinal changes in protein-deficient rats. Exp. Mol. Pathol., 3:224-31, 1964.         [ Links ]

Winick, M. & Noble, A. Cellular response in rats during malnutrition at varion ages. J. Nutrition, 89:300-6, 1966.           [ Links ]

Zanoni, J. N.; Miranda-Neto, M.H; Bazotte, R.B. & Suza, R.R. Morphological and quantitative analysis of the neurons of myenteric plexus of the cecum of streptozotocin-induced diabetics rats. Arq. Neuropsiquiatr., 55(4):696-702, 1997.         [ Links ]

Correspondence to:
Prof. Dra. Sonia Lucy Molinari
Universidade Estadual de Maringá
Departamento de Ciências Morfofisiológicas
Av. Colombo, 5790 - Bloco H-79 Sala 03
87020-900 Maringá Pr.
BRAZIL

Recibido : 18-10-2001
Aceptado: 02-11-2001

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