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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/S0717-95022003000300006 

Int. J. Morphol., 21(3):221-226, 2003.

MORPHOMETRICAL AND STEREOLOGICAL ANALYSIS OF THE
SUPERIOR CERVICAL GANGLION OF Rattus norvegicus SUBMITTED
TO CHRONIC TREATMENT WITH CORTISOL

ANÁLISIS MORFOMÉTRICO Y ESTEREOLÓGICO DEL GANGLIO CERVICAL SUPERIOR
DE Rattus norvegicus SOMETIDO A TRATAMIENTO CRÓNICO CON CORTISOL
** Cortez, C. M.; *Costa, W. S.; *,***Babinski, M. A. & *,***Chagas, M. A.


CORTEZ, C. M.; COSTA, W. S.; BABINSKI, M. A. & CHAGAS, M. A. Morphometrical and stereological analysis of the superior cervical ganglion of Rattus norvegicus submitted to chronic treatment with cortisol. Int. J. Morphol., 21(3):221-226, 2003.

SUMMARY: The present work investigates the corticoid action on the growth of the superior cervical ganglion of the rat and describes the cortisol effect during early stages of development. The study was based on morphometric and stereological analysis of the perikarya. Eight rats were treated intraperitoneally with cortisol (1mg/Kg/day) during 36 days. Treatment was initiated in the 8th day after birth and was withdrawn one day before the sacrifice. There was a significant difference (£P0,05) for the neural mean diameter between the control group (16.78 ± 1.11mm) and treated animals (15.84 ± 0.99mm). The decrease of perikarya neuronal diameter was also demonstrated by stereological methods. Morphometrical findings may suggest alterations in superior cervical ganglion neuronal activity in rats treated for long term with cortisol.

KEY WORDS: 1. Morphometry; 2. Stereology; 3. Glucocorticoid; 4. Sympathetic ganglion; 5. Rat. 


INTRODUCTION

The sympathoadrenal cell lineage originated from the neural crest comprises the neurons of sympathetic ganglia and adrenal cells. In the superior cervical ganglion (SGC) of the rat this cell lineage includes the principal sympathetic neurons and the small intensely fluorescent (SIF) cells (Meyer, 1985; Ross et al., 1995). The SIF cells are easily distinguished from the other components by their high nucleocytoplasmatic ratio and their small size (Eränkö & Eränkö, 1971; Borghini et al.,1991 ; Heppner et al.,1996).

Cortisol is a natural glucocorticoid hormone. It represents the greatest part of adrenal secretion (Cortez-Maghelly, 1991; Axelrod & Reisine, 1984; Ramey & Goldstein, 1957). Lymphocytolytic and antinflamatory functions, inhibition on rate of growth or involution of many of the somatic tissues are well known effects of glucocorticoids (Pratt & Aronow, 1966). The most important action of glucocorticoids is exemplified by its effects that give rise to catecholaminergic characteristics of cells derived from the neural crest (Wurtman & Axerold, 1965).

On the nervous system, glucocorticoids have a crucial action on the regulation of several basic growth processes and cellular differentiation (Bohn, 1983; Doupe and Patterson, 1982). There is substantial evidence indicating that, among other substances present in the cellular microenvironment, corticoids are critical determinants of cellular morphological and biochemical differentiation (Benda et al., 1968). This event is frequently studied by means of neurotransmitter expression (Smith and Fauquet, 1984; Elfvin et al., 1975).

It was observed that cellular differentiation was maintained during continuous cortisol administration, and disappeared after treatment withdrawal. It has been suggested that these effects, which are time and dose dependent, could be due to death of this cell population, diminished expression of catecholaminergic characteristics, or loss of glucocorticoid responsively (Wurtman & Axerold, 1966; Benda et al.).

Despite the existence of different types of neurons, small and large, in the SCG of the rat, the present work aimed to study cortisol action on cellular growth considering these different cell types as only one cell population and to describe the cortisol effect in the SCG, by means of morphometric and stereological analysis of the neuronal soma.

MATERIAL AND METHOD

This study was performed on 8 male Wistar rats treated with intraperitoneal injections of cortisol (1mg/Kg/day). Treatment was initiated on the 8th day after birth and withdrawn one day before rats were sacrificed (36 days of treatment). The injections of cortisol were performed in the morning, always after 9:00 am and before 11:00 am. The animals have been kept with their mothers until they were sacrificed. To serve as controls, two other animals were kept under the same conditions during the same period, and were injected intraperitoneally with NaCl solution (0,9%). Animals were sacrificed by exposure to ether. The ganglia were dissected under binocular microscope and every ganglion was weighted. The ganglia were then immersed in Bouin fixative by a period not superior than 12 h. The ganglia were embedded in paraplast, 5mm sections were obtained and stained by the Gomory trichromic.

It was analyzed 500 neural soma in 10 randomize sections obtained from 6 ganglia. The morphometric data of every soma were measured semi automatically by the software system Image Pro (Media Cybernetics). The image was seized by a light microscopy Olympus BH and a color camera CCD on the screen of a computer Pentium and processed automatically. The selection and analysis of the object were done by the intervention of the researcher. The measure of mean soma diameter was obtained by circumscribing exclusively the neurons with visible nucleus and nucleolus. The data was transferred to statistic software, Graph Pad Instat and then processed.

We determined by stereological methods the relation surface/volume (Sv) and the volumetric density (Vv= total surface area / surface of perikarya circumscribed in every field) (Weibel and Gomez, 1962; Weibel, 1979; Mandarim-de-Lacerda, 1998,1999). ANOVA test was used to verify statistical differences between treated and control group.

RESULTS

The SCG of the rats presented two kinds of cell population: small and large neurons (Fig. 1). Animal body weight at the end of treatment, and ganglion weight was shown in Table I. There was a significant difference (P0,05) on neuronal mean diameter between the control and treated group (Table II, Figs. 1, 2). Fig. 3 shows the neuronal diameter of SCG on control and treated animals. The diameter values of SCG for control group presented the highest frequency between 17 and 19µm; however for cortisol treated group the highest frequency for this parameter was between 15 and 17µm (Fig. 4). The decrease in perikarya neuronal diameter was also demonstrated by stereological methods; mean Sv was 0,21µm-1 in control group and 0,32µm-1 in cortisol treated group. Stereological data are expressed in the Table III.


Fig. 1. Photomicrography showing the two kinds of cell population (control group): small and large neurons, Haematoxylin-Eosin, X400.


Fig. 2. Photomicrography showing neurons of the treated group. Haematoxylin-Eosin, X400.

DISCUSSION

The administration of glucocorticoids to immature animals results in a striking suppression of somatic growth (Henderson et al., 1971). In the present work it was found an increase in animal body weight for cortisol treated group. This increase in body weight is normally due to the mineralocorticoid effect of the glucocorticoid that, when administrated in superior doses to the physiologic quantities, causes renal retention of Na+ and loss of K+ (Katzung, 1992). It was also observed that in treated animals the ganglion volume is greater than that of control group. Since Vv value is inversely proportional to the ganglion volume (Weibel), we may say that our stereological results corroborate this statement. For control group, that shows a smaller ganglion volume, we found 23% of the total volume occupied by the perikarion whereas for treated animals, that shows a greater volume, we found 17%.

Table I. Animal weight at the end of treatment and ganglion weight for control and treated group.


  Animal weight (g) Ganglion weight (mg)
Data Control Cortisol Control Cortisol
Mean 115.44 119.13 1.08 1.28
Max 156.28 146.30 1.20 1.55
Min 067.90 109.83 1.15 1.15
SME 025.73 013.81 0.07 0.15

Table II. Morphological results of soma mean diameter for control and cortisol treated group.


  Diameter (µm)
  Control Cortisol
Mean 16.78 15.84
Max 28.84 27.30
Min 08.25 08.28
SEM 01.11 00.99

Several authors have discribed the presence of two types of neuron in the ganglia of autonomic nervous system: small and large, respectively cells with diameter smaller than 13µm, SIF cells, and those with diameter between 13 and 50 µm, ortosympathetic neurons (Eränkö & Eränkö; Gabella & Trigg, 1984; Borghini et al.; Flett & Bell, 1991 and Costa et al., 1992 ). Studies concerning the effects of GC in the sympathetic ganglia are mainly related to SIF cells and point out the effect of this hormone on the number of these cells during early stages of development. According to some experimental studies (Eränkö et al., 1971; Ciaranello et al., 1973; Eränkö et al., 1982; Paivarinta & Eränkö, 1982; Paivarinta et al., 1984 ) chronic treatment started at birth, led to the appearance of numerous small cells in SCG. On the other hand, this effect has not been observed neither for adult rats nor for animals of more than 1 week of age (Koslow et al., 1975; Bohn et al., 1982; Bohn and Friederich, 1982 and Paivarinta & Eränkö). The mechanism by which additional cells are formed, in the case of neonatal rats treated of corticoid, has alredy been discussed. Immunocytochemical techniques suggested that the new small cells are formed from the small and previously nonfluorescent cells present in the ganglion by large increases in catecholamine synthesis (Eränkö et al., 1982).


In the present study although the beginning of cortisol treatement was performed on the 8th day posnatal, morphometrical and stereological evaluations, show modifications in neuronal cells diameter induced by long term cortisol action. Data concerning the perycarion of all ganglionic neurons (SIF cells and ortosympathetic neurons) showed that three is a significant difference (P<0.05) for the mean diameter between control and cortisol treated animals (Table II). Also stereological data concerning the relation between surface and volume showed that Sv values clearly indicate a decreasing in mean neuronal volume (Table III). However it is possible that the lower mean values of morphometric and stereological parameters found for cortisol treated animals are due to the increase in number of the small cells above mentioned.

Table III. Stereological data for control and cortisol treated groups.


  Vv (%) Sv (µm-1)
Data Control Cortisol Control Cortisol
Mean 23.33 17.86 0.22 0.32
Max 28.57 28.57 0.64 0.35
Min 11.70 07.14 0.08 0.35
SME 01.78 01.95 0.05 0.03

There is a well-established relation between nerve cell diameter, Nissl substance and cellular activity (Cormack,1987). Studies in vitro about glucorticoid action showed an inhibitory effect on the growth of mouse fibroblast with reduced rate of thymidine and uridine incorporation into macromolecular material (Pratt & Aronow). Despite morphological and stereological results do not permit an evaluation of the functional activity of the orthosympathic neurons or SIF cells, these morphological findings may suggest a decreasing in the sympathetic neuronal activity of the SCG. This can arise even when the treatment began in rats of more than one week of age treated for long term with cortisol.

ACKNOWLEDGMENTS

The present study was supported by Grants from the National Council of Scientific and Technological Development (CNPq-Brazil), and from the Foundation for Research Support of Rio de Janeiro (FAPERJ). The authors wish to thank Mss Ana Cristina Moreira da Costa for her technical support. 


CORTEZ, C. M.; COSTA, W. S., BABINSKI, M. A. & CHAGAS, M. A. Análisis morfométrico y estereológico del ganglio cervical superior de Rattus norvegicus ssometidos a tratamiento crónico con cortisol. Int. J. Morphol., 21(3):221-226, 2003.

RESUMEN: Se investigó la acción de corticoides en el crecimiento del ganglio cervical superior de rata y se describieron los efectos del cortisol durante las fases tempranas de desarrollo. El estudio se basó en los análisis morfométrico y estereológico del pericario. Fueron inyectadas intraperitonealmente 8 ratas con cortisol (1mg/Kg/día) durante 36 días. El tratamiento se comenzó en el día 8 después del nacimiento, finalizándose un día antes del sacrificio. Hubo una diferencia significativa (£P0,05) en el promedio del diámetro neuronal entre el grupo control (16.78 ± 1.11 µm) y los animales tratados (15.84 ± 0.99 µm). La disminución del diámetro neuronal del pericario también fue demostrada a través de la estereología. Los hallazgos morfométricos pueden hacer pensar en alteraciones de la actividad neuronal del ganglio cervical superior, en ratas tratadas por un largo periodo con cortisol.

PALABRAS CLAVE: 1. Morfometría; 2. Estereología; 3. Glicocorticoide; 4. Ganglio simpático; 5. Rata. 


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Correspondence to:
Prof. Dr. Waldemar Silva Costa
Departamento de Anatomia (IB)
Universidade Estadual do Rio de Janeiro
Av. 28 de Setembro 87 (Fds)
CEP 20 551-030
Rio de Janeiro
BRASIL

E-mail: waldemar@uerj.br

Received : 07-06-2003
Accepted : 18-07-2003 


* Department of Anatomy and
**Physiology, Institute of Biology, State of Rio de Janeiro University (UERJ) - Rio de Janeiro, Brazil.
*** Department of Morphology, Federal Fluminense University (UFF) -Rio de Janeiro, Brazil.

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