Deleterious effects of prepubertal corticosterone treatment on rat prostate 1

PURPOSE: To investigate the structural and functional changes induced by corticosterone (CORT) in the ventral prostrate (VP) of rats in order to study chronic stress effects in the prepubertal phase. METHODS: Wistar rats received daily saline or CORT injections during the pubertal period from the 5th to 25th day of postnatal life. The animals were distributed into four groups: 1 Control (n=5); 2 Control 99mTc-P (n=5); 3 Treated with CORT (n=14); 4 Treated with CORT and 99mTc-P (n=10). All rats were sacrificed at two months of age. Technical tissue uptakes of 99mTc-P were used to evaluate the functional and stereological methods for morphological analysis. RESULTS: Acini distribution in the group treated with CORT differed significantly (p<0.0001) from the control. The control group’s epithelial average height (10.01±0.24 microns) was statistically significant (p<0.0001) from rats treated with CORT (19.27±0.73microns). The collagen distribution was lower in the treated group (2.79%) when compared to control (3.97%). The radioactivity percentage in the groups marked with 99mTc-P (%Ati/g) did not demonstrate a statistically significant difference (p=0.285897). CONCLUSION: Chronic administration of corticosterone in prepubertal rats causes changes in their acinar structure and their ventral prostate stroma, indicating possible deleterious effects of this hormone.


Introduction
Glucocorticoids (GC), produced by the adrenal cortex regulate the metabolism of carbohydrates and hemodynamic functions, and play an important role in physiological regulation and adaptation to stressful situations.Most of the effects are mediated by the glucocorticoid receptors and are essential to life 1 .
Although high levels of glucocorticoids are essential in an acute reaction to stress, chronically increased levels may have unwanted effects 2,3 .
In prenatal and pubertal stages, GCs help sex hormones in the formation and maturation of the genitals.However, at increased levels, GCs tend to inhibit hormone secretion 4 .Clinical and experimental studies have shown adverse effects of excess GC on testicular testosterone production 5 .
The prostate is the only accessory sex gland found in all mammalian species but may have appreciable morphological differences, even in closely related species within the same family.
It is transversed by the urethra and the ejaculatory ducts, has a conical shape with the apex down, and surrounded by a capsule rich in blood vessels.In rats the prostate is not a compact structure, lobes are divided into functional parts (anterior lobe or coagulating gland, dorsal and the ventral lobe) on the basis of differing responsiveness of hormones of the glandular tissue located ventral to the urethra as compared to those situated dorsally 6,7 .Both human and rodent prostates are composed of glandular acini dispersed in a fibromuscular stroma with excretory ducts lined by epithelial cells, glandular myoepithelial cells, interstitial and subepithelial bullae 8 .
There is a possibility that Technetium-99m pertechnetate (Tc-99m) might be captured by the genital organs, concentrating in the fallopian tubes, kidneys, ovaries, testes, penis and possibly the prostate 9,10 .But there is no data in the literature comparing the uptake capacity for these organs.This paper evaluates the structural and functional changes induced by corticosterone (CORT) in the ventral prostate (VP) of Wistar rats submitted to chronic stress effects in the prepubertal stage, using stereological methods and tissue uptake of 99mTc-P.

Animal model and experimental design
The experimental protocol was approved by the ethical committee of UERJ (CEA/250/2008), and follows the recommendations of the conventional guide to animal testing (Guide for the care and use of laboratory animals, 1996).

Virgin female Wistar rats (Rattus norvegicus albinus
Wistar) mated for 24 hours.Copulation was confirmed the following morning by the presence of a vaginal plug.All animals were maintained in individual cages under controlled temperature conditions, humidity (ca.70%) and light-dark cycle (12 hours each), with free access to feed (Nuvital ® , Nuvilab Ltda., Curitiba-PR, Brazil) and water ad libitum.
After pregnancy and during lactation, the animals were kept separated by brood in plastic cages covered with sawdust.
Up to weaning, mothers remained in cages with their pups.Five days after birth, the pups were divided (by marks on the tail) into two groups.One group (n= 10) received daily injections of saline during the pubertal period, from the 5th to the 25th day of postnatal life, and the other (n= 24) in the same period, received daily intraperitoneal injection of CORT at a 2 mg/100g body weight dose as suggested by the adapted protocol 5 .On the 25th postnatal day, injections of CORT and saline were suspended.The evolution of animal body weight was monitored daily until sacrifice.
On the 60th day prior to sacrifice, five rats in the control group and 10 in the group treated with CORT were separated and each rat received 0. All rats were sacrificed after two months (60 days) by the forced inhalation of carbon dioxide (CO 2 ) and a fraction of blood was collected from each by cardiac puncture.

Stereological analysis
Using a stereoscopic microscope (DF Vasconcelos ® SA, Sao Paulo, Brazil), we immediately extracted the prostates from all the rats in groups 1 and 3.These were weighed and fixed (1.27mol/l formaldehyde in 0.1M phosphate buffer, pH 7.2).The material was dissected following laboratory histological routine.
The following were analyzed: number of acini, area of the acini, and height of the epithelium of the prostate acini.In all analyses, were used section thicknesses of 5mm, stained with hematoxylin and eosin (HE).For the morphometric analysis of collagen density, 5mm thick sections, stained with Sirius Red Picro without bias were also used.
The images were captured using Image pro-Express ® (1993-2004 Media Cybernetics, Inc., version 5.0) and processed using Adobe Photoshop 6.0 ® (Technique Computed Histofotometria) 11 .The Image J ® program (Image Processing and Analysis in Java, version 1:37, National Institutes of Health, USA) was used for analysis of the captured images.
The sections were examined using an optical microscope (BX51 -Olympus, Japan) and images were captured using a camera (DP70 -Olympus, Japan) connected to monitor (LG Flatron, Brazil).As for the area, the acini were classified according to to size using this criteria: small (S), the area between 100 and 10.000 µm 2 ; Medium (M), the area between 10.000 and 100.000 µm 2 ; and large (L) µm 2 area above 100.000.

Study of uptake of Technetium (Tc-99m pertechnetate)
The animals in groups 2 and 4 were sacrificed 10 minutes after an injection of Na99mTcO4 (7.

Results
An analysis of animal body mass data from the 5th to the 60th day found no significant difference (p= 0.3226) in body weight gain in rats treated with CORT (Groups 3 and 4) and control (groups 1 and 2).In the period of treatment (from the 5th to the 25th day) weight gain differences were not significant (p= 0.8493).There was no significant prostate weight difference (p= 0.5648) between the average weights of the rats treated (1.08 ± 0.20 g) and untreated (1.03 ± 0, 09 g) with CORT (Table 1).Values represent the mean ± standard deviation (SD).In all analyzes, the difference was considered statistically significant when p<0.05.

Weight (g)
The acini distribution (%) by size (small, medium and large) in the group treated with CORT differed significantly (p<0.0001)compared to the control.In the control group, the growth percentages of the small acini (18.24%) and large (31.76%) were higher than in CORT (0.57% small acinar and 18.00% of large acini).Medium acini were higher in the treated rats (81.43%) than in the control (50.00%) (Table 2, Figures 1 A and B).
The collagen distribution was lower in the treated group (2.79%) when compared to control (3.97%) but was not considered significantly different (p= 0.1746) (Table 2).Size using this criteria: small (S); Medium (M); large (L).Data are expressed as mean ± SD and percentages (%).For percentage values Chi-square was applied.Student t test was performed for analysis of acinar epithelial height and area.Wilcoxon signed rank test was used to determine distribution of collagen.For the analysis of Tissue Uptake of 99mTc-P, ANOVA and multiple comparisons of Tukey-Kramer were used.In all analyses, the difference was considered statistically significant when p<0.05, a≠ control.The radioactivity percentage in the groups marked with 99mTc-P (% Ati/g) was 0.938917 ± 0.392 149% Ati/g for Group 2 and 0.805935 ± 0.455481% Ati/g for Group 4, showing no statistically significant difference (p= 0.285897).

Discussion
GCs are widely used in prostate cancer treatment owing to their palliative effects.Reported effects are linked to decreased prostate specific antigen levels and amelioration of symptoms 12 .
However, there is little information in the literature about normal CG effects on the prostate.
The present study found an approximately 10% body weight decrease in the group treated with CORT (Table 1), compared to the control, from the 5th to the 60th postnatal day, but this difference was not considered significant.
Acute treatment of rats with dexamethasone (Decadron, intraperitoneally 1mg/kg body weight for five days) was able to reduce animal body weight significantly 13 .However, dexamethasone is a synthetic corticosteroid with a glucocorticoid effect much higher (>25 fold) than that observed with cortisol and CORT 14,15 .
In contrast to these results, in a previous study, the mean body weight of rats increased during daily treatment with cortisol (1 mg/kg/day) administered from the 8th to the 36th day of postuterine life 16 .The authors attributed the increase of the cortisol to mineralocorticoid effect, which causes renal retention of Na + and K + loss when chronically administered in high doses, resulting in water retention.The corticosterone in rats regulates energy metabolism and stress responses, similar to cortisol effects on the human body.Cortisol is about 95% of the GC content secreted by human adrenal glands, while CORT is almost 100% of the content in rats 17,18 .
The main factor responsible for the body weight decrease which accompanied the dexamethasone treatment would be the serum insulin increase 13 .Previously, some authors 19,20 have suggested that insulin resistance could be the explanation for body weight reduction in rats treated with CORT.
Recently this effect has been attributed to lower feed intake, motivating a decrease of approximately 30% in body weight compared to control, accompanying the chronic high dose treatment with CORT.This hypothesis has been already raised in previous studies 21 .
Suggestions that GC chronic treatment is behind weight loss corroborate each other, since insulin acts in the hypothalamus to regulate the activity of some neuropeptides involved in food intake control, maybe being an indirect mechanism of CORT action on body weight 13 .
One hypothesis concerning the lack of significant data relative to the control, given the body weight of the animals in our research, contrasting with other works, is that the dose of CORT administered was not high enough to cause notable metabolic changes, besides the fact that CORT is much less powerful than dexamethasone.
Although no significant differences were found in the weight of rat prostates, those treated compared to control, the prostate structure analysis showed a significant difference (p<0.0001) in acini distribution of treated group compared to control.The acini classified as large (Table 2) in the treated group showed a reduction size of about 37% (152.500µm 2 ) compared to the control (241.300µm 2 ).Furthermore, a significant reduction in acinar epithelium height (Table 2) was observed in the group receiving CG, confirming other literature results.Dexamethasone treatment was observed to lead to atrophy and decreased proliferative activity of epithelial cells of the prostate 13 .
For collagen distribution (Table 2), the group treated with CORT had a reduction, which can be associated with the action of GC on ECM (Extracellular Matrix), especially on fibroblasts.
Morphological alterations were observed in stromal cells caused by GC activity, suggesting the activation of fibroblasts and atrophy of smooth muscle cells 13 .Consistent with that hypothesis, another study shows a decrease in the order of 43% and 80% in the formation of collagen gel in human fibroblast cultures that had received dexamethasone and cortisol, respectively 22 .
The collagen is known to be one of the most active components in the modulation of biological activities occurring in the ECM.Therefore, an appropriate balance between synthesis and degradation is required for normal tissue functioning.Improper balance, both quantitative and qualitative, can produce a decrease in compliance of the injured tissue, causing functional changes and therefore clinical problems.
The normal development of the VP rat goes through three distinct phases: (1) the initial growth within the first three weeks, (2) at rest and (3) the pubertal growth, the latter being coincident with the rise in testosterone plasma.Thus, the postnatal VP growth results from a combination of epithelial proliferation and / or differentiation and the synthesis and / or accumulation of secretory products into the lumen 23 .
The prostate is sensitive to androgen action, which plays a key role in the metabolism and functioning of this gland.
Testosterone influences glandular epithelium which converts to dihydrotestosterone (DHT) in the secretory cells of the glandular epithelium.DHT is about 30 times more potent than testosterone in this activity.Because of the functional testosterone dependence on it, deprivation of this hormone results in marked decline in the gland, with loss of epithelial cells by apoptosis and a prominent reorganization of the ECM [24][25] .
As already mentioned one of the effects of an excess of GC is testosterone secretion reduction.Independent of any direct GC effect on the prostatic epithelium, it has an indirect effect on the testosterone.
Alterations observed in the epithelium and the remaining collagen synthesis on the 20th day after the end of treatment with CORT may indicate danger to the regenerative capacity, and contractile prostate tissue function which would have been established during the pubertal phase.
The significant increase in medium acini with CORT treatment appears to have occurred mainly at expense of the number of small acini, although the order of the charge by size of the lobes has not changed: number of medium acini>large>small.
This significant reduction of small and large acini may also indicate less ability to rebuild tissue and functional changes of the acinar epithelium.
Prostate epithelial cells produce enzymes, including prostate specific antigen (PSA), prostatic acid phosphatase (regulates cell growth and metabolism of the prosthetic glandular epithelium) and fibrolisina (increases the fluidity of the semen) 8 .
The prostatic secretion makes up 70% of semen volume and an important function of this discharge is due to its high bicarbonate content, which confers an alkali content, capable of neutralizing the acidic vaginal environment, making it hospitable to spermatozoa 26 .
The analysis of prostate function was performed by tissue activity of 99mTc-P.There are no previous reports using this radioisotope for this assessment after GC administration.
The tissue radioactivity measurements showed that CORT decreased the tumor uptake of 99m Tc-P.This result contributes to the hypothesis that the prostatic secretory function decreases with chronic GC administration, although it was not possible to measure the significance of this change, owing to inaccuracy of the valuation at 99mTc-P radioisotope.

Conclusion
The chronic administration of corticosterone in prepubertal Wistar rats results in changes in their acinar structure and stroma of the ventral prostate, indicating a possible functional impairment of this gland in adulthood.

TABLE 1 -
Body weight gain among the groups.