Revista chilena de anatomía
versão impressa ISSN 0716-9868
Rev. chil. anat. v.20 n.2 Temuco 2002
Rev. Chil. Anat., 20(2):125-130, 2002.
MORPHOQUANTITATIVE STUDY OF AGING OF THE GUINEA PIG'S
MYENTERIC PLEXUS COLLUM
DEL CERDO DE GUINEA
ALVES, N. & LIBERTI, E. A. Morphoquantitative study of aging of the guinea pig's myenteric plexum collum.Rev. Chil. Anat., 20(2):125-130, 2002.
SUMMARY: Applying the NADH technique in the myenteric plexus' collum of young and aged guinea pigs, we have studied the morphology of ganglions and neurons, the number and arrangment of neurons and the profile area of neuronal bodies. Ganglions preferentialy elongated or without a defined morphology were observed in five collum segments (proximal, intermediate proximal, medial, intermediate distal and distal). There were no important differences in this aspect from one segment to another, whether in the same age group or between the two studied groups. It has also been observed the presence of relatively small ganglions containing a reduced number of neurons and big ganglions containing a high number of these cells. The neurons were more compacted in the ganglions of the individuals from the younger group, while the majority of ganglions from the individuals of the aged group were more equally divided. Neurons mainly roundish and oval were found in both groups, neurons with the cytoplasmic contour greatly altered could be observed in various ganglions of aged animals. Concerning the area of cellular profile, the majority of neurons of both groups were found in the interval between 101 and 300 µm2. Based on the determination of the fragment's area of each collum division, the density of neurons for cm2 was estimated, as well as the total neuronal density, in which the individuals of the aged group was aproximately 46% smaller than that of the individuals from the younger group.
KEY WORDS: 1. Anatomy; 2. Myenteric plexus; 3. Guinea pigs.
The decrease of a certain cellular population during the individual's life can have important functional consequences. A significant loss of vital cells would take individual to death for a natural phenomenon. That is especially valid for the neuronal cellular population, wheather for its functional meaning or because when they are destroyed, these nerve cells can never be restored. It is known that there is a celular loss in several areas of the organism during life.
Remak's studies (1840) concerning the ganglial cells of the alimentary canal, as well as the first description of the submucous plexus (Meissner, 1857) and myenteric plexus (Auerbach, 1864), in the wall of the alimentary canal mark the beginning of the knowledge on enteric nervous system.
Since then, several works , in different animals and in humans, have been accomplished seeking to study their several aspects (Dogiel, 1895; Ramon Y Cajal, 1911; Schabadasch, 1930; Murat, 1933; Massueli, 1942, and others). Later, several works were developed in order to understand the structure of this nervous system area. Continually, the authors went on increasing a great number of relatively important details concerning the enteric plexus constitution (Andrew, 1961; Gabella, 1966; 1971, 1972, 1976, 1979; Rother & Leutert, 1974; Furness & Costa, 1987; Hamilton, 1985; Santer & Baker, 1988).
The knowledge of the enteric plexus is fundamental to understand several physiologic mechanisms of the alimentary canal, among them there's the coordineted moviments. These are intimaly related to the digestive tract innervations.
In senior patients, intestinal constipation is a commum finding, and although its etiology remains ignored, a decrease in the motor function of the collum has been suggested as a possible cause of that condition (Mc Dougal et. al, 1984; Devroede, 1989). In the esophagus and in the collum, the motion dysfuntions observed with the aging process were related to the alterations of the flat musculature of the wall (Hamilton).
Santer & Baker, noticed an important reduction in the number of neurons in the myenteric plexus, particulary in the collum of senior mice.
Once the motility of the collum is mainly controlled by myenteric plexus neurons, and taking into consideration the significant reduction of these neurons during the aging process in several areas of the alimentary canal of different animals, including the esophagus, stomach and human's small intestines (Gabella, 1971 and Santer & Backer), it is of great interest to sattle down the number of myenteric plexus' neurons from the regions of the collum in senior individuals, in order to observe the effects of aging in that part of the alimentary canal.
Considering that aging can determine alterations in the myenteric plexus, such as the reduction of the number of neurons, we tried in this study to:
-determine the number of neurons in the myenteirc plexus in several collum areas of young and senior guinea pigs;
-establish the size of the existing neurons in those groups, in order to verify if some neuronal group is more affected with the aging process.
We believe that these observations can contribute to a better understanding about the motor alterations observed in the collum during the aging process.
MATERIAL AND METHOD
Eigth male guinea pigs (Cavia porcelus) were used. They belong to the Technical Management Support of Teaching and Reasearch from USP College of Medicine. The animals were divided in two groups according to age. Thus, the younger group had 4 animals between 6-12 months (G I), and the senior group had 4 animals with ages above 30 months (G II). The animals were sacrified by ether overdose and had their anterior abdominal wall open for the removal of the large intestine.
One of the extremities of the organ was joinned, and an amount of KREBS solution (*) (Gabella, 1987) injected in the viscus in order to obtain a light distenction of the walls. After that, the collum had its other extremity tied, and its total length was obtained with a measuring tape. Next, five segments of 3 cm were removed and identified as: proximal, intermedium proximal, medium, intermedium distal and distal. Those segments were then submitted to the Nicotinamide Adenine Dinucleotide Technique (NADH) (Gabella, 1969) to detach the ganglial neurons, it happened as follows:
- immersion in a coloration solution (0,5 mg/ml of Nitro-Blue Tetrazolium) (SIGMA) in distilled water; 25 parts of lid phosphate of sodium (0,1 M pH 7,3) and 50 parts of water; 0,5 mg/ml of NADH in a reduced form (SIGMA); Pre-incubation for 1 to 3 minutes (solution of 0,3% Triton X 100) to facilitate penetration; Wash in Krebs solution; Immersion for 60 minutes in a coloration solution.
After the coloration, the pieces were opened in its longitudinal axis and kept like this among two glass sheets in order to have their areas traced in a especial paper.
Then it was fixed in the solution of 10% of phormalina for a period of 1 to 7 days. After that, they had its area traced again in order to calculate the retaction index. Soon afterwards, the mucous membrane and the submucosa layers were dissected under surgical microscope in order to stand out the myenteric plexus.
The neuronal density as well as the area of 50 neurons per segment were obtained with the aid of the computerized imaging for morphometric studies.
Study of the ganglions and neurons' morphology and cellular profile area of the neurons.
For this study the mixture of membrane segments obtained as already described was used.
Using a camera coupled to a light microscope1 that increases 500 times, we draw in withe paper using black pencil, the profiles of 50 neurons for each segment of the intestine in both groups. Our pourpose was to steem , with the help of computer imaging2 for morphologic studies, the profile's areas of celullar bodies and neurons.
The data were tabulated and the results expressed in frequency distribution's historiograms according to the size of the neurons. The medium values were also calculated for both groups.
Estimative of neurons' density.
The membrane mixtures obtained as described previously were used for the counting of neurons.
It was arbitrarily considered as ganglion all grouping of three or more neurons. Isolated or coupled neurons were considered as belonging to the closest ganglion. Fifty fields for each segment of the animals' intestine was aleatory chosen.
The counting of the number of neurons was done with the help of a 600x increase light microscope.
With the results obtained in those countings, and knowing that the area of each membrane and the medium lenth of the large intestine of both studied groups, the number of neurons per cm2 was calculated.
The quantitative data obtained for the younger and senior groups were statistically compared, and a variance analysis was accomplished with two factors: area and age.
The differences among the averages were analyzed inside of the same group through multiple comparisons (Tukey method) and among the two age groups for the comparison of two not parametric samples (Mamn-Whitney Method) (Zar, 1984).
The probability levels smaller or equall to 5% were considered significant.
Morphology of ganglions and neurons: qualitative aspects
Particulary in relation to the myenteric plexus of the collum of the guinea pig, it was verified that their meshes assume a polygonal aspect.
In the membrane mixture, ganglions with varied forms can be observed in the five areas of the collum, having no important differences in this aspect of segment in relation to others, whether in the same age group or between the two studied groups. Thus, we could notice since ganglions relatively small containing a reduced number of neurons, to big ganglions with a higher number of those cells. The ganglions were prolonged without a defined morphology, as some respected the polygonal aspect of the plexus meshes (Fig. 1)
Fig. 1. Ganglions of the collum's myenteric plexus of guinea pig, in a prolonged way (A) and in a complex way (B) according to the polygonal aspect of the animal (NADH 342 X, B 342 X)
When the disposition of the neurons were observed inside the ganglions, it was verified that these cells were found more compacted in the younger group ganglions (GI), (Fig. 2A). In the individuals of the senior group (GII), (Fig. 2B), in most ganglions, the neuronal agroupments presented a smaller number of cells, demonstrating wide aspects among themselves. In several ganglions of GII, a considerable neuronal reduction was observed (Fig. 2B). In relation to the size and form of the neurons in five areas of the collum of both groups, it was found small, big and medium neurons, with varied forms, prevailing those with round or oval aspect. In the animals of the senior group (GII), neurons in degeneration with citoplasmatic outline quite altered were very evident (Fig. 2B).
Fig. 2. Ganglions of the collum's myenteric plexus of young guinea pig (A), and senior (B). Observe the little amount of neurons in the senior animals, as well as the presence of muscli of sattelite cells (arrows) (NADH, A 420 X, B 420 X)
Density of neurons. The neuron's density/cm2 was determined for five segments of each case, in two age groups analized, and after the obtaintion of the total area of the collum of each animal, it was estimated the neuronal density for both groups.
The avarage and deviations of the neural densities and the number of neurons for both groups can be found in Tables I, II, and in Illustration 5. We could observe that the neuronal density (N/cm2) was on average 53.397,5± 15.300,5 for the younger group, and 18.905,0 ± 2697,5 for the senior group.
The total neuronal density varied from 5.768.070 to 8.652.618 in GI, with an average of 6.983.880 ± 1.412.650 and from 3.133.260 to 3.446.120 with an average of 3.280.730 ± 156.806 in GII. We concluded that the total number of neurons was larger in GI.
Dimensions of the area of the cellular's body image. The dimentions of the area of the myenteric plexus cellular's body image of the neurons in two dimensional projection of all areas of the collum do notcompose a homogenius population. So, it was verified that in GI, the dimentions varied from 100 to 800 µm2 and in GII, from 100 to 500 µm2.
The analysis of the distribution historiograms of the dimentions of the collum's cellular body area's image of both groups demonstrated that the largest frequency for GI was observed in the interval from 101 to 300 µm2, and the same interval goes to senior group (GII).
It can also be noticed that there is no significant differences among several areas of the collum in both groups and among the aged groups.
Statistical analysis. Neuronal density.
The statistical analysis for the neuronal density allowed us to verify that there is not any differences in the interactions age x area (p = 0.925 ) and among the areas in the two groups. Only the total neuronal density of the collum's myenteric plexus presented differences between the two groups (p = 0.035). Therefore, the total neuronal density estimated for the collum of young individuals islarger than the one of the senior individuals.
Dimensions of the area of the cellular body's image. The statistical analysis demonstrated that there is not any differences in the dimensions in the area of the image's myenteric plexus cellular bodies among the collums of the young (P = 0,958), and senior (P = 0,438) and in the interaction age X area (P = 0,864).
The membrane mixtures, submited to several coloration techniques have been used in this study of the nervous plexus, mainly in certain animal species, where the thickness of the organ's walls allows us a good visualization of the constituint elements of the plexus (Gabella, 1972, 1979, 1987). This technique offers better results than the techniques that use histological cuts, since they follow certain principles, such as avoiding retractions or distention of the tissue, and taking care in the dissection for the removal of muscular layers.
Besides, the mixtures make the study of relatively wide areas, if the directly quantification is done.
As for the evidenciation of neurons method applied in this study, developed by Gabella (1969), it was efficient because it colors by selecting the neurons of the plexus, without evidencing the other cells present in the alimentary canal. Besides, there was not any evidence that the neurons of the plexus were not coloured, although there was some variation in the intensity of the coloration.
Nervous cells and satellite cells that involve the pericary are predominant elements in the myenteric plexus ganglions of the human's collum, similar to the verified in other species and also in the guinea pigs (Gabella, 1979; Furness & Costa).
In this present study, it was not observed any gradient type for the neuronal density in the guinea pig's collum, once it was not verified significant differences among the interaction age X area in the two groups.
We found significant differences among the young and senior groups in what it refers to the total neural density, in other words, an important decrease in the number of neurons in the individuals of the senior group (around 46%) in relation to the individuals of the younger group.
Neurons predominantly round and oval were found in both groups. In several ganglions of the senior animals, it was observed neurons with a cytoplasm outline quite altered. Regarding the area of the celular profile, most of the neurons of both groups are in the interval from 101 at 300 µm2.
Differences related to age or region as described in other parts of the human's alimentary canal were not observed in the collum. We can then conclude that aging determinetes a decrease in the number of neurons, but it does not determinate morphologic modifications in the collum. At least, we couldn't observe it when using this methodology.
According to the results obtained and to the propositions established in this work, and also in agreement with the methodology used, it is important to conclude that:
1. The membrane mixtures of the guinea pig's collum colored with NADH are appropriate for morphoquantitative studies of the myenteric plexus in young and senior animals.
2. In all areas of the collum , the ganglions of the myenteric plexus had varied forms, there weren't differences in this aspect among young and senior individual.
3. In ganglions of the young individuals collum , the neurons were grouped in a more compacted way, different from the verified in seniors, where the neurons were more distributed, suggesting a decrease in the number of cells in this group.
4. The average density of neurons (neurons/cm2) for the young group was of 53.397,5 ± 15.300,5. For the senior group, it was of 18.905,0 ± 2.697,5.
5. There weren't differences stattistically significant in the neuronal density among the several parts of the individuals' collum of the same age group.
6.The total neuronal density for the individual's collum of the younger group was of 6.983.880 ± 1.412.650, and of 3.280.730 ± 156.806 for the individuals collum's of the senior group, demonstrating a reduction of 53% in density in the senior group;
7. The area's dimentions of the cellular body's image of the neurons from the the plexus demonstrated a heterogenious population, that varied from 100 µm2 to 800 µm2 for individuals for the younger group, and of 100 µm2 to 500 µm2 fot the individuals of the senior group.
8. The average of the area of the cellular body's image was of 245,98 in GI and 205,49 in GII. There was not any estatisticaly significant variation between the two groups.
RESUMEN: AplicandolatécnicadelNADH en el plexo mientéricode cerdos jóvenesyadultos,estudiamos la morfología de los ganglios y neuronas, el número y disposición de las neuronasy el áreade contornodelos cuerpos neuronales.
Encincosegmentosobservamos ganglios preferentementeelongadoso sinuna morfologíadefinida. No habíandiferencias importantes entre uno y otrosegmento, enel mismo grupo etario o entre los dos gruposestudiados. Se observó también la presenciade gangliosrelativamente pequeñosconteniendoun reducidonúmerosde neuronasy grandes gangliosconun alto número deesas células. Las neuronasestaban máscompactadasen los gangliosde los individuosdel grupo más joven,mientras que la mayoríade los gangliosen los individuosdel grupoadultofuerondivididos más equilibradamente. Neuronas redondeadasu ovales se encontraron en ambos grupos; y en varios ganglios de animales adultos se observaron neuronas con el contorno citoplasmático ampliamente alterado.Con relación al área de contornocelular,la mayoría de las neuronasde ambos grupos, fueron encontrados en un intervalo entre 101 y 300 µm2. Basadosen el áreadel fragmentode cadadivisión, se estimó la densidadde las neuronas por cm2, comotambiénla densidad neuronaltotal, la cualfue,aproximadamente, 46% menor en los individuosdel grupo adulto,con respectoa losjóvenes.
PALABRAS CLAVE: 1. Anatomía; 2. Plexo mientérico; 3. Cerdo de guinea.
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Prof. Dr. Nilton Alves
Faculdade de Odontologia
Universidade Estadual Paulista Júlio de Mesquita Filho
R. Humaitá, 1680
Caixa Postal ; C.P. 331
Araraquara - São Paulo
Recibido : 10-12-2001
Aceptado : 06-03-2002
2MINI-MOP kontron Bildanalyse coupled to a binocular microscope equipped with a clear camera, both of CARL ZEISS. The parameter studied was the area, and the data obtaines was sent from a MINI-MOP for a PC-AT/386 through a serial interface RS232 for storage and subsequent analysis.