Testosterone supplementation's effects on age-related bladder remodeling – experimental study in rats

Abstract

Objective: This study was designed to evaluate the effect of testosterone replacement on the fibrotic process of the detrusor bladder muscle during the normal aging process.

Methods: 15 Wistar senile rats, aged between 18 and 20 months were divided into two groups: testosterone group – 11 animals submitted to the administration of testosterone undecanoate (50 mg/kg intramuscular), once per month; and, Control group – four animals underwent a sham procedure. At the end of eight weeks, animals from both groups were sacrificed; bladders were removed and subsequently stereologically evaluated to determine the volumetric density of collagen fibers. The success of testosterone administration was confirmed by the measurement of serum testosterone at the beginning and end of the experiment.

Results: In the replacement group, testosterone average was 3.2 ng/ml, whereas in the control group, the mean testosterone at the end of the experiment was 0.64 ng/ml (p < 0.05). Analysis of stereological collagenous fiber showed higher density in the control group compared to the testosterone group I (56% versus 37.02%, respectively). The difference of volume concentration of collagen between both groups was statistically significant (p < 0.000).

Conclusion: Bladder wall fibrosis was reduced in senile rats subjected to testosterone replacement.

• Aging
• bladder
• rats
• testosterone

Introduction

Lower urinary tract symptoms (LUTS) are directly related to age, and aging is considered the main well established risk factor for the appearance of urinary changes in men and women [1]. This clinical finding may be explained by altering the composition and distribution of bladder wall fibers during this process [2].

Disequilibrium in the pro- and anti-apoptosis mechanism is critical in the aging process [3]. Apoptosis plays a vital role in various mitotic tissues, as in the liver and leucocytes, as it prevents tumorigenesis and keeps track of immunocompetent cells. However, in post-mitotic cells – as those in the detrusor muscle – it has a negative effect since there is destruction of essential and sometimes irreplaceable cells [4].

The literature describes it as a possible pathophysiological mechanism of the onset of bladder disease, which occurs after chronic bladder outlet obstruction, leading to recurrent ischemia cycles followed by reperfusion in the detrusor generating free radicals [2]. An experimental model of chronic urethral obstruction showed partial correlation between the increase in involuntary detrusor contractions associated with increased oxidative stress in the bladder muscles of mice studied [5].

Dambros et al. [6] found that bladder strips subjected to repeated electrical stimulation evolve with reduction of contractile force and no apparent increase of oxidants, suggesting that hydrogen peroxide forms induced cell damage depending on the concentration of the oxidants generated. Frequently, antioxidant mechanisms are able to limit or prevent the adverse effects of hydrogen peroxide, but with age these mechanisms decrease, thereby making oxidative damage more prevalent with aging [7].

Studying bladders of young rats with orchiectomy and senescent rats, Lorenzetti [8] showed that fibrosis of the bladder wall was higher within the orchiectomy group, probably resulting from the sharp drop and not adaptive levels of testosterone, thus losing the ability to differentiate into smooth muscle stroma and anti-inflammatory power. However, apoptosis in the detrusor muscle was stronger in senescent rats, perhaps by greater involvement of free radicals when androgen decline is slow.

The genitourinary tract is especially sensitive to changes in serum testosterone levels. The versatility of testosterone and its interference in various chains of action in the bladder was first observed by Holgmäng et al. [9]. This researcher and his collaborators confirmed an increase in the peak urinary flow in men with testosterone administration compared to those with placebo administration. This initial finding prompted studies in the basic science that demonstrated the close relationship between DHT (dihydrotestosterone) receptor in the bladder muscle and suppression of detrusor activity [10].

The importance of testosterone on bladder physiology and its decrease with aging have encouraged research on the impact of these phenomena on the detrusor muscle apoptosis. Nakazawa et al. [11], demonstrated, for the first time, increased expression of angiotensin-converting enzyme and angiotensin receptor type II (AII) in the bladder of rats undergoing bilateral orchiectomy. Since angiotensin type II acts through the rho/rho kinase via, this means that there are increasing pro-apoptotic factors associated with declining levels of testosterone. This phenomenon has unquestionable relevance, because it showed that environmental androgenic changes have directly affected the bladder.

Today it is known that the production of Nitric Oxide (NO) in the urinary tract depends on the regulation of testosterone, and its potential relaxing effect of the detrusor muscle and the bladder neck can be impaired in hypogonadic patients [12]. Moreover, a clinical trial suggested that there is improvement in the IPSS (International Prostate Symptoms Score) and better control of the metabolic syndrome after testosterone replacement [13]. Regarding the above discussed, this paper aims to study the relationship between late onset hypogonadism and structural remodeling of the senescent bladder, and whether this process can be influenced by exogenous testosterone replacement, by quantifying fibrosis of the bladder wall of senile rats with and without testosterone supplementation.

Materials and methods

Fifteen male senescent Wistar senile rats (18–20 months old), weighing 380–530 g were kept in a controlled environment (25 °C ± 2 °C) with exposure to light for 12 hours a day, water available ad libitum and Labina® animal chow (Purina®, São Paulo, Brazil). The study was carried out according to the guidelines of the Brazilian College for Animal Experimentation (COBEA) under approval of the Institutional Committee for Ethics in Animal Research.

Groups were formed as follows: Testosterone Group – 11 senile animals subjected to testosterone supplementation. Control Group – four senile animals subjected to intramuscular injection of 0.9% saline solution.

The animals were anesthetized with a solution composed of xylazine (0.87 mg/kg) and ketamine (43.3 mg/kg) injected intraperitoneally, and after anesthesia was reached, they were weighed and later the venous blood was held through puncture of the retro-orbital venous plexus with a Pasteur pipette, transferring the blood sample directly to an Eppendorf tube. Subsequently, the sample was centrifuged and only the serum was frozen at −20 °C.

Testosterone undecanoate (TU) was administered intramuscularly (50 mg/kg) into the animal’s dorsum using a fine insulin type needle, on the first and 28th day of the experiment. In the control group, 0.9% saline was administered at the same volume of drug solution in the testosterone group, on the same days.

On the last day of the experiment (56th day), the animals were anesthetized and then intracardiac punctures were performed with a 23 G needle, for blood collection, keeping the needle in the puncture site for further sacrifice with potassium chloride.

The bladder was approached by median abdominal incision of 1.5 cm and sectioned at the level of the bladder neck. After removal, the bladder was stored in formalin for 48 h and then in 70% alcoholic solution for further paraffinization and study of stereological collagen fibers.

Stereology was the method chosen in order to evaluate morphometrically the muscle and collagen fibers of the urethral sphincter. The fiber analysis was performed using preparations from seven-micrometer thin sections. The modified picrosirius red staining technique was used. The slides were analyzed via optical microscopy with polarized light at a 40× magnification. Ten fields per slide and 10 slides per animal were evaluated. The volumetric density of the collagen fibers and muscle fibers was analyzed by overlaying the M-42 grid system on the computed morphological image of the slides. The volumetric density was the relative density taken up by fibers in the tissue under examination. The stereological method determined quantitatively the parameters of the anatomical structural base on the two-dimensional thin sections, in three dimensions [14].

The equation was used to calculate the volume density of the collagen fibers, where: Vv = volumetric density, Ps = the number of structure points studied (collagen) and Pp = the number of possible test points (42 in this case).

The evaluation of testosterone levels was performed using the radioimmunoassay kit DSL-4100 Testosterone ®, manufactured by Diagnostics Systems Laboratories, Inc. (Webster, TX, USA) and imported by Genesis Diagnostics Products Ltd (São Paulo, Brazil). All samples were analyzed in duplicate with theoretical sensitivity or 0.05 ng/ml minimum detection limit.

The data obtained were analyzed using the BioEstat 5.0 (Belém, Brazil) and were expressed as means ± standard deviation. The results were validated by analysis of variance (multivariate analysis) and the non-parametric Kruskal–Wallis test was used to assess the differences between the independent samples. The significance criterion used was two-sided p < 0.05.

Results

The mean body weights of rats between the two groups throughout the duration of the experiment were compared using the Kruskal–Wallis test at 95% of probability, showing that there was no change in body weight between the groups (Table 1).

Table 1. Average body weights in grams of the mice over time and groups involved in the experiment.

	Average weights (g)
Groups	D0	D28	D56
Testosterone	438*	427.9*	418.9*
Control	449.5*	420*	405*

*(p > 0.05), (Kruskal–Wallis test).

The mean serum testosterone of the testosterone group on the first day (D0) of the experiment was 1.227 ng/ml. After hormone replacement therapy, this index reached a value of 3.242 ng/ml, measured on the 56th day (D56). On the other hand, the control group had an average of 1.133 ng/ml on D0, which decreased to 0.64 ng/ml of serum testosterone at the end of the study (D56). According to the analysis of variance (ANOVA), by Tukey’s test, the testosterone group's replacement therapy was effective and satisfactory, while the androgenic decline continued by the time of experiment in the control group. This demonstrated that there was a significant hormone deficit at the time of sacrificing these control animals (p < 0.05) (Figure 1).

Figure 1. Box-plot of the values of serum testosterone in the testosterone and control groups on D0 (beginning of the experiment) and D56 (animal sacrifice).

Table 2 presents the descriptive analysis of the volumetric density of the collagen fibers in the bladder wall in both groups of the study. It was observed that the testosterone group had a volumetric density of collagen (37.02%), which was lower than that of the control group (56%) showing statistical significance (Figure 2) (t test, p < 0.000).

Figure 2. Volumetric density of the collagen fibers of the bladder wall in different groups. p < 0.000; t test.

Table 2. Descriptive analysis on collagen fibers of bladder wall in both groups.

	Volumetric density (Vv) -- %
	Testosterone	Control
Mean	37.02	55.95
SD	8.3	9.45
Minimum	19.05	35.71
25th Percentile	33.33	47.62
Median	35.71	55.95
Maximum	54.76	71.43
75th Percentile	42.26	62.49
Standard Error	1.86	2.11
95% CI	33.33–40.48	51.80–60.09

Figures 3 and 4 show picrosirius stained bladder wall highlighting collagen fibers distributed among muscle fibers, ranging mainly from an intense yellow to orange. In the testosterone group, the presence of less collagen fibers between the detrusor is observed when compared to the control group; however, it was noted a gap between the muscle fibers, as expected for an aging bladder wall [15].

Figure 3. Picrosirius stained bladder wall of the control group analyzed under polarized light microscopy (400×).

Figure 4. Picrosirius stained bladder wall of the testosterone group analyzed under polarized light microscopy (400×).

Discussion

The presence of androgen receptor in rat urinary bladder (urothelium and detrusor) [16] and the modulation of autonomic pelvic plexus by testosterone [17] reinforce indirectly the influence of androgenic hormone in the lower urinary tract, even if this influence is not the protagonist of the normal micturition mechanism. Furthermore, the polymorphic nature of these receptors may justify the heterogeneity of the results in studies attempting to correlate LUTS and plasma testosterone levels [18].

Takyu [19] observed that castration decreased the function of alpha1-adrenergic and muscarinic receptors, restoring their functions after testosterone replacement. Moreover, the existence of receptors of angiotensin II (AII) in the bladder, especially type 2, are related to inflammatory and apoptotic stimuli. Nakazawa et al. [11] using castrated rats, observed increased expression of caspase-3 in the bladder mediated by type 2 AII receptor, when compared to control and testosterone replacement groups.

Filippi et al. [20] found that the expression of phosphodiesterase-5 (PDE 5) in the bladder – an important enzyme that inhibits the NO cycle – is dependent on the levels of circulating androgens. Furthermore, the RhoA/Rho-kinase system has been investigated for years as a cause of urinary tract disorders, including participation in the overactive bladder, due to its activation of actin-myosin complex that causes muscle contractions, regardless of the levels of cytosolic free calcium. This RhoA/Rho-kinase route is also modulated by sex hormones, however when the ratio estrogen/testosterone gets imbalanced, as occurs in aging and obesity, it seems to be the most important pathway's trigger [21].

From the histological point of view, normal aging bladder has some important structural features: the absence of vacuoles on the surface of muscle cells, which in normal cells are related to the process of cell differentiation and activation; increase in the interstitial space, populated mainly by collagen; cell dedifferentiation, represented by the inactivation of mature muscle cells; and limited areas of cellular degeneration [15]. This process can be explained by an animal model study conducted by Traish et al. [22], which showed that testosterone has a regulatory effect on stromal cell growth and differentiation, with decreased smooth muscle in androgen deprivation, replaced by connective tissue collagen.

Moreover, Bhasin et al. [23] and Singh et al. [24] believe that androgens stimulate concomitantly the pluripotent cells to differentiate into muscle lineages including smooth muscle, inhibiting strains for the differentiation of adipocytes. To support this finding, Lorenzetti [8], in assessing the influence of testosterone on the bladder wall fibrosis in rats, observed that the sudden drop of testosterone by castration of young rats caused more muscle replacement by collagen fibers when compared to senile rats, whose process of hormonal decline is slow and gradual. Although the orchiectomy model is usually used to assess the influence of testosterone on the bladder, it does not seem appropriate to mimic the process of late onset hypogonadis. Therefore, we decided to use only senile animals (18–28 months) in this work, which represents more risks and higher costs, but portrays adequately the physiological aging process, avoiding the sample selection bias.

The average baseline levels of testosterone in both groups (Group testosterone group – 1.22 ng/dl; control group – 1.13 ng/dl) were below the levels described by Kinoshita et al. [25] in seven-month-old rats (2.2 ± 0.3 ng/dl average testosterone), but very similar to the 21-month-old rats (1.1 ± 0.2 ng/dl average testosterone). This fact demonstrates the physiologic decrease of testosterone in rats in this study.

Early studies using the replacement model with 50 mg/kg of testosterone undecanoate (TU) per month have been conducted for about 12 years, showing that this choice is simple, reproducible and effective for hormone replacement in rats [26]. Another variation of the experimental model of hormone supplementation, described more recently, uses 100 mg/kg of TU, in a single dose, and its effects can be evaluated after two months of administration [27]. A monthly dosage was chosen in this study because it is consolidated in the literature. This method of supplementation caused a mean serum testosterone of about 3.2 ng/dl in the group who underwent hormone replacement therapy, which was not too superior as the values of young rats (2.2 ± 0.3 ng/dl average testosterone) [25].

For quantitative analysis of fibrotic reaction in the bladder wall, we performed the stereological study of collagen fibers in both groups [14]. The volumetric density (Vv%), magnitude used in this study is a stereological parameter that produces reliable results with minimal variation. At the same time, it is not dependent on histologic complex blade or experience of the researcher, being employed in the quantification of fibrous component of the extracellular matrix, particularly in collagen and elastic fibers of various tissues [28].

Despite the number of individuals in each group was different, the number of fields assessed was high. Each animal had 10 histological fields counted, representing 420 possible points, being the means in each group identified, which were assessed by Kruskal–Wallis. In this way, it was possible to increase the replication with improved statistical power for the result of the Vv%. Also, the magnitude of the effect (d) calculated for this work was 2.1, representing that in future studies we can expect that about 99% of the subjects in the experimental group will exceed the mean value of the control group. Stereology has advantages inherent to the method, as for example, turning the results into numerical values, with easy reproducibility and comparison between groups. And most importantly, stereology presents a well-defined theoretic basis, which makes it a method widely accepted by the scientific community [14].

Under polarized light, type I collagen fibers appear as being thick, strongly birefringent, with colors ranging from yellow to red. The use of polarized light for the analysis of Picrosirius Red stained samples is a special procedure for histological analysis of type-specific collagen, and it is not necessary to evaluate fibrotic processes, especially due to the prevalence of type I collagen in these processes. However, Picrosirius Red polarization facilitates the identification and counting of collagen mass in relation to the detrusor muscle, improving the accuracy of the method [29].

The time between the intervention and evaluation of the final result in this issue was two months and this interval has been the most used to assess changes in rat muscles, showing that it is a sufficient time for the analysis of preventive actions or for stimulating the bladder fibrosis and remodeling [27,30].

The process of muscle fibers replacement and bladder remodeling associated with changes in sex hormones has been largely studied. Fraga et al. [31] observed that there was an increase in volumetric density of type IV collagen in bladder basal membrane of ovariectomized rats. Ludwig et al. [30] found oxidative stress and apoptosis increased in the bladder of rats subjected to castration; however, it could be reduced by supplementation with alpha-tocopherol. Still, it was not clear whether the increase of the apoptotic process increases the risk of bladder dysfunction. In fact, oxidative stress is one of the most important factors involved in the pathogenesis of age-related detrusor dyskinesia [32] and may be aggravated by diabetes mellitus [33] and by the hypogonadism [30], as described above. On the other hand, vitamin E produces protective effect against free radicals, especially when used at an early stage of the process of tissue injury [33].

Çayan et al. [34] showed that rats with bilateral orchiectomy, testosterone replacement associated with estradiol had better results than androgen replacement alone regarding the preservation of the muscle/collagen ratio, demonstrating the stroma modulating role of testosterone, regardless of gender. Tek et al. [27] used the model of castrated ten-month-old rats to evaluate the effect of testosterone replacement (100 mg/kg testosterone undecanoate dose) on bladder function and histology. They concluded that hormone replacement in these animals produced an improvement in the smooth muscle/collagen ratio, including the development of bladder capacity. Our study was a pioneer in demonstrating that testosterone, even lower than the doses used in the above-mentioned work, favored the gradual development of smooth muscle cells in bladder of rats with senile hormonal decline and testosterone supplementation in aged rats protected them against the process of remodeling/fibrosis of the bladder.

Based on the discussion above, it can be stated that: the decline in testosterone levels occurred with age is associated with the process of fibrosis in the bladder wall of rats in this study. The senile rats that are undergoing hormone replacement with testosterone have less fibrosis of the bladder wall compared with senescent rats not subjected to this replacement.

Declaration of interest

The authors report no declarations of interest.