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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.16 n.1 Temuco  1998

http://dx.doi.org/10.4067/S0716-98681998000100002 

STUDY OF THE MYENTERIC PLEXUS OF THE ILEUM OF RATS SUBJECTED TO PROTEIC UNDERNUTRITION

ESTUDIO DEL PLEXO MIENTERICO DEL ILEON DE RATAS SOMETIDAS A DESNUTRICION PROTEICA

* Marlos Meilus
** Maria Raquel Marçal Natali
** Marcílio Hubner de Miranda Neto

* Department pf Cell Biology and Genetics, state University of Maringá-PR. Brazil.
** Department of Morphophysiological Science, State University of Maringá-PR. Brazil.

SUMMARY: With the purpose of verifying the effects of proteic undernutrition on the myenteric plexus of the ileum, 20 rats of the Wistar strain whose dams were subjected to undernutrition during gestation and/or lactation were killed at 60 days of age. Whole-mount preparations of the ileum were stained with GIEMSA to allow visualization of the myenteric neurons and further analysis and quantification. Proteic undernutrition does not cause reduction on the number of myenteric neurons por cm2 on the ileum and medium neurons with intermediary basophily predominate in all groups.

KEY WORDS: 1. Ileum; 2. Myenteric plexus; 3. Myenteric neurons; 4. Proteic undernutrition.

INTRODUCTION

The major feature of the innervation of the gastrointestinal tract is the great development of the intramural system, which consists of a large number of neurons probably related to the complexity of the intestinal functions of the vertebrates, especially the regulation of the activity of propulsion.

Studies that evaluate the morphological, quantitative and morphometric aspects of the myenteric plexus (BARBOSA, 1978; SANTER & BAKER, 1988; GABELLA, 1987 and 1989) are various.

Nevertheless, the literature is scarce with regard to the neurons of the myenteric plexus under proteic deficit conditions; SHRADER & ZEMAN (1969); NATALI & MIRANDA-NETO (1996) deal with this issue. Many works (WINICK & ROSSO, 1969; WINICK, 1970) demonstrate that undernutrition causes alterations on the neurons of the Central Nervous System.

Taking into consideration that many motor, sensory and association neurons are found on the myenteric plexus as intrinsic reflex arcs which function either independently or with the CNS to mantain an adequate intestinal function, and that morphological or quantitative alterations of these neurons could occur in conditions of proteic undernutrition, we carried out this work with the purpose of investigating the effects of proteic undernutrition during gestation and lactation on the neurons of the myenteric plexus of the ileum of young rats.

MATERIAL AND METHOD

The ileum of 20 rats of the Wistar strain was used. The method used to induce the proteic undernutrition was the following: NUVILAB ration of normal proteic level (around 22%) was mixed with corn starch to decrease the proteic level to 8%.

This low-protein rations was supplemented with vitamin B complex and mineral salts; both rations were offered ad libitum.

According to the proteic level of the ration supplied to the dams, the groups were named NN: normal protein during gestation and lactation; DN: low protein during gestation and normal protein during lactation; ND: normal protein during gestation and low protein during lactation; DD: low protein during gestation and lactation.

After the period of breast feeding (21 days) the young rats of all groups received normal ration until the 60th day of age, when they were weighted, anesthetized with sulphuric ether inhalation, and killed.

To verify the location of the ganglia of the myenteric plexus segments of ileum were fixed in formol, placed in paraffin and stained with Hematoxilin-Eosin.

The morphology of 100 neurons per animal was analyzed using whole-mount preparations stained with GIEMSA (BARBOSA, 1978) with the help of an optical microscope Olympus CBB equiped with WF 10x lens, coupled to a micrometered disc and 40x objective with the purpose of measuring the largest transverse and longitudinal axes of the cell body for further comparison.

Data of the greatest axes of cell body, as well as cell shape, nucleus position and cytoplasmic basophily served as a basis for the classification of the neurons as small, medium and large (COOK & BURNSTOCK, 1976). The shapes of the nerve ganglia were also observed.

For neuronal quantification an optical microscope Olympus CBB equiped with WF 10x lens and 40x objective was used. The method chosen was counting by sampling. Each whole-mount preparation was divided into four quadrants of the same size. In each quadrant 10 fields were chosen at random, and all their neurons were counted. Half-neurons of a given quadrant were discarded, while those of another were counted. Considering the whole preparation of each animal, all neurons of 40 fields were counted, area of the field was calculated and the number of neurons per cm2 was established.

Statistic treatment employed the x2 test for analysis of frequency and basophily of the small, medium and large neurons of the ileum from the different groups. Student's test was used for data analysis of total length of the small intestine, weight of animal, largest axis of the cell body and number of neurons per cm2 in the ileum. The significance level on both tests was 5%.

RESULTS

Body weight and intestinal length

Means of body weight and length of the small intestine of animals from the different groups are presented on Table I.

Table I - Mean values of body weight and small intestine length (ID) of rats from the different nutritional groups.

Variables
NN
DN
ND
DD
Weight (g)
229,52
194,68
186,26
167,24
*
S
38,43
29,71
39,34
23,53
Length ID (cm2)
114,60
107,60
114,70
103,80
*
S
8,82
10,26
19,19
4,55

NN - normal ration during gestation and lactation
DN - low-protein ration during gestation and normal ration during lactation
ND - normal ration during gestation and low-protein ration during lactation
DD - low-protein ration during gestation and lactation
*
Differs significantly from control group.
Student's test p £ 0,05
t ³ 2,31

Table II Frequency of small, medium and large neurons that exhibited weak (Bf), intermediary (Bm) and intense (Bi) basophily, obtained by differencial counting of 100 neurons per group.

Groups
Small
Medium
Large
  Bf Bm Bi Bf Bm Bi
Bf
Bm

Bi

NN

07 10 - 19 26 24 - 06 08
DN 10 02 - 16 37 23 - 03 09
ND 15 15 - 08 24 32 - 02 04
DD 09 06 01 17 13 38 - 02 14

* When comparing groups in pairs the following significant differences were found: NN and DD (x2 = 13,05); DN and DD (x2 = 13,05); ND and
DD (x2 = 13,31).

pc = 5,99

Table III - Mean values of the number of neurons/cm2 on the ileum of rats from the different nutritional groups.

Variables
NN
DN
ND
DD

Number of neurons/cm2

           
on the ileum
63.525
74.691
*
63.467
94.630
*
S
5.807
6.314
 
7.738
6.803
 

* Differs significantly from control group.
Student's test p £ 0,05
t ³ 2,31

Table IV - Frequency of small, medium and large neurons on the ileum of rats from the different nutritional groups.

Variables
NN
DN*
ND*
DD*
Small neurons (£13,09µm)
17%
12%
39%
16%
Medium neurons (14,40-28,81µm)
69%
76%
64%
68%
Large neurons (³30,12µm)
14%
12%
06%
16%*

When comparing groups in pairs the following significant differences were found: NN and ND (x2 = 10,43); DN and ND (x2 =13,24); ND and DD (x2 = 11,91).
pc = 5,99


Fig. 1. Tranverse section of 6µm evidencing a ganglion of the myenteric plexus (g) between the circular (c) and longitudinal (l) muscle fibers. HEMATOXILIN-EOSIN, 722x.


Fig. 2. Myenteric ganglion of triangular shape. Whole-mount preparation stained with GIEMSA, 722x.

Location and shape of the myenteric ganglia

The ganglia of the myenteric plexus were found between the inner circular and the outer longitudinal muscle layers (Fig. 1). On these sites the muscle layers are separated to give room to the clusters of myenteric neurons. The ganglia of the myenteric plexus are generally positioned in roughly regular and parallel rows, and show various shapes and sizes. Ganglia had a stelete, triangular (Fig. 2) and round shapes; most of them were elongated (Fig. 3).

Morphology of the neuronal cell body

Neurons were found with diverse shapes and sizes within the different experimental groups. Based on features of morphology, staining and length of the cell body, neurons were distributed into three groups: small, medium and large (Fig. 3).

Largest length of cell body in small neurons ranged from 6,55µm to 13,09µm; round shape; nucleus generally in central position and occupying most of the cell body; almost always lacking nucleolus; only chromatin clusters on the nucleopasm.

Medium neurons had cell body with the largest axis ranging from 14,40µm to 28,81µm. Nuclei were large and located in different regions of the cytoplasm; generally eccentric. The cell body of these neurons was predominatly oval and elongated. Clusters of chromatin were observed in some nucleoplasms, while in others up to three well-defined nucleoli were seen.

Large neurons had cell body with the largest axis ranging from 30,12µm to 47,14µm. Most of them were elongated; oval shape; round neurons were also observed in smaller numbers. Nuclei were large and mostly eccentric. One to three nucleoli were observed.

Table II shows the relation of cytoplasmic basophily. Denominations of weak, intermediary and intense basophily are according to affinity for the stain.

Number of neurons per cm2 on the ileum

Table III presents the mean number of neurons per cm2 on the ileum for each group studied.

Frequency of small, medium and large neurons on the animals from the groups studied

Table IV gives data of frequency of neurons according to size. The analysis of these frequencies revealed statistically significant differences when all groups were compared (x2 = 15,28). When the groups were compared in pairs, the differences did not attain significance between groups NN and DN, NN and DD, DN and DD. Nevertheless, when groups NN and ND, DN and ND, ND and DD were compared the differences were significant.


Fig. 3. Myenteric ganglion of elongated shape, evidencing small (s), medium-size (m) and large (l) neurons. Whole-mount preparation stained with GIEMSA, 722x.

DISCUSSION

Statistical analyses of the mean values of body weight and intestinal length of the animals showed significant differences when groups NN and DD were compared.

After receiving normal diet from the 21st to 60th day of age the animals of group DD still had weight and intestinal length significantly smaller than those of other groups. These data are compatible with those found in young rats whose dams were subjected to proteic deficit also during gestation and lactation (PATHAK et al., 1981; YOUNG et al., 1987). In spite of the mean value of body weight not being significant at the level of 5%, a smaller weight is observed similar to group DD, demonstrating that a relatively long period is necessary for in group ND, complete recovery. It is possible that the differences on the intestinal lengths among the groups are not larger may be due to a fast recovery; this would problably occur because the gastrointestinal tract is the first tissue to get in contact with nutrients absorbing them to sustain its compensatory growth (HILL et al., 1968; SUGHIRARA, 1986).

Ganglia of the myenteric plexus in all groups studied lie between the inner circular and the outer longitudinal muscle layers (HAM & CORMACK, 1991). Their shape varied from round or pyramidal to stelate or elongated, with the predomination of the latter (HERNANDEZ, 1994; NATALI & MIRANDA-NETO, 1996; TORREJAIS et al., 1995). It was verified that undernutrition does not alter the location and disposition of the myenteric ganglia.

The observations made in this work revealed large differences on the length of the largest axis of the neuronal cell body, which varied from 6,55µm to 47,14µm. When studying the ileum of diabetic rats HERNANDEZ (1994), found neurons with the largest axis ranging from 8,48µm to 57,24µm, while COOK & BURNSTOCK (1976) found neurons with the largest axis of up to 45µm in the guinea-pig. NATALI & MIRANDA-NETO (1996) measured neurons varying from 3,92 to 28,8µm the duodenum, demonstrating that along the length of the intestinal tract morphometric variations occur on the neurons.

According to COOK & BURNSTOCK (1976), there are two distinct types of small neurons: the first type measures 9,00µm to 13,00µm in length and groups of rough endoplasmic reticulum and numerous ribosomes are found on the cytoplasm, which confer great basophily. The second type consists of small granular neurons, with the largest diameter of up to 12,00µm, scarce endoplasmic reticulum and many free ribosomes which confer the granular aspect. In our results we did not find small neurons of strong basophily, but we did find neurons of granular aspect. There may be considered as small neurons of intermediary basophily and from our point of view, are of the type mentioned above.

Neurons with greater synthetic activity possess larger amounts of rough endoplasmic reticulum and free polyribosomes (HAM & CORMACK, 1991). Once their structures have affinity for basic stains, they can account for the intense color observed on the cytoplasm of the medium-sized and large myenteric neurons. A synthetic activity greater than that of the small neurons may be suspected.

In relation to the medium and large neurons, SOUZA et al. (1988) reveal that the cell body of the myenteric neurons of the human esophagus stain intensely, and HERNANDEZ (1994) observed that 70,40% of the medium neurons on the ileum rats were strongly basophilic. Weak basophilic neurons were not found. In our research it was observed that strong basophilic neurons predominate among the medium and large neurons while among the small neurons the weak basophilic were most abundant.

With regard to the position of the nucleus, our observations are in accordance with the description of the autonomic neurons of the Peripheral Nervous System (HAM & CORMACK, 1991), of the myenteric neurons of the duodenum of rats (NATALI & MIRANDA-NETO, 1996) and of the ileal neurons of rats (HERNANDEZ, 1994). Like these authors we verified that most of the neurons of the myenteric plexus considered as medium and large have eccentric nucleus and thus the peripheral location of this organelle cannot be indicative of neuronal degeneration.

Analyzing the frequency of myenteric neurons, the predomination of medium neurons in all groups was registered.

When we analyze each group we observe that the animals receiving low-protein diet only during lactation show greater incidence of small neurons and smaller incidence of medium and large neurons. It is possible that group ND was the most affected by the proteic deficiency and even after restoring the normal diet the small neurons could not recover as efficiently as those from the other groups. Thus, the ability of storing proteic supplies on the cytoplasm was smaller on group ND, while the data of groups DN and DD were similar to the control group (NN).

It may be emphasized that the animals of group DN were able to recover until the 60th day of age; in spite of having 39 days of normal diet, group DD showed neurons similar to those of the control group.

NATALI & MIRANDA-NETO (1996) observed that the duodenum of rats subjected to undernutrition during gestation and lactation shows greater incidence of large neurons. They considered this an indicative that animals suffering prolonged undernutrition store protein on their neurons when normal diet is restored as a means of adaptation to a possible proteic deprivation.

The number of myenteric neurons varies among species (SOUZA et al., 1982; GABELLA, 1987) and age (GABELLA, 1989), between intestinal segments (BARBOSA, 1973), and as a function of experimental conditions to which the animal may be subjected (GABELLA, 1989; HERNANDEZ, 1994; NATALI & MIRANDA-NETO, 1996).

Through our results we verified that statistically significant differences exist in the frequency of neurons between all groups, except for group NN compared to group ND. In our opinion these differences have little or no physiopathological influence because, contrary to expectation, the group that suffered most with undernutrition, group DD, was the one with the greatest number of neurons (94.630/cm2); group DN (74.691/cm2) was the next.

It must be taken into account that it is exactly in these two groups that there was lesser intestinal growth, 103,80 cm and 107,60 cm, respectively. Furthermore, group DD also had the smallest mean body weight (187,24 grs.).

Results suggest that undernutrition in the periods studied does not cause decrease in the number of neurons per cm2 on the ileum; on the other hand, the greater neuronal density observed is due to the smaller body growth of the animals subject to undernutrition, such that a relation between intestinal length, body weight and number of neurons is notorious.

RESUMEN: Con el objetivo de verificar los efectos de la desnutrición proteica sobre el plexo mientérico del íleon, fueron utilizados 20 ratas del linaje Wistar, cuyas madres fueron desnutridas en los períodos de gestación y/o lactacia sometidas a sacrificio a los 60 días de edad. Se realizaron preparados de membrana del íleon teñidos con GIEMSA, para observación de las neuronas mientéricas y posteriores análisis y cuantificación. Verificamos que la desnutrición proteica no provoca redución en el número de neuronas mientéricas por cm2 de íleon, y que las neuronas medias con basofilia intermediaria predominan en todos los grupos.

PALABRAS CLAVE: 1. Ileon; 2. Plexo mientérico; 3. Neuronas mientéricas; 4. Desnutrición proteica.

REFERENCES

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Correspondence to:
Prof. Ms. Maria Raquel Marçal Natali
Department of Morphophisiological Sciences
State University of Maringá
Av. Colombo, 3690 CEP 87020-900
Maringá - Paraná
Brazil

Recibido : 20-10-1997
Aceptado : 24-02-1998

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