The effect of finasteride on the male rat brain

doi:10.21203/rs.3.rs-3554857/v1

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The effect of finasteride on the male rat brain

https://doi.org/10.21203/rs.3.rs-3554857/v1

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Introduction

5AR (5-alpha reductase) inhibitors may have negative effects on psychosexual activity, but there is still debate.

Objectives

We aimed to observe the effect of 5-alpha reductase type 2 inhibition on the brain by using finasteride.

Methods

Eight fourteen-week-old male rats were assigned to one of three groups (Group 1 as the control group, Group 2 as the finasteride group, and Group 3 as the finasteride withdrawal group). Rats in Groups 2 and 3 were administered finasteride for 4 weeks, and then rats in Group 2 were sacrificed, whereas rats in Group 3 were sacrificed 4 weeks thereafter. RT‒PCR and western blot for brain hemisphere, and immunohistochemistry for hippocampus and VMPOA were performed to evaluate the effect of finasteride on 5-alpha reductase type 2 and c-Fos.

Results

Dihydrotestosterone (ng/dl) and the dihydrotestosterone to testosterone ratio (%) decreased after 1 month of finasteride administration (Group 2), but the changes were abrogated after discontinuation of the drug (Group 3). Generally, these results were generally in accordance with those from the western blot and immunohistochemistry experiments; however, western blot analysis of 5-alpha reductase type 2 remained downregulated in Group 3. ThemRNAexpression of 5AR and c-Fos did not significantly change but tended to increase during finasteride administration.

Conclusions

Administration of finasteride exerted an effect on the brain, including the hippocampus and VMPOA, via 5AR-2 inhibition, resulting in a decrease inc-Fos protein activation, which may disappear after long-term cessation of the drug.

Health sciences/Medical research/Experimental models of disease

Health sciences/Diseases/Urogenital diseases/Sexual dysfunction

5-alpha reductase

Finasteride

Erectile dysfunction

Persistent finasteride syndrome

Finasteride, a 5-alpha reductase (5AR) inhibitor, is widely used for the treatment of benign prostatic hyperplasia and male pattern baldness known as androgenetic alopecia. However, side effects caused by finasteride are creating serious societal issues [1]. Now that many studies have shown the possibility of serious adverse effects, such as decreased libido, erectile dysfunction, sexual anhedonia, decreased sperm count, diabetes, cognitive impairment, fatigue, anxiety, depression, and suicidal ideation even after discontinuing finasteride treatment, it is worth noticing whether the effects are persistent or not. Melcangi et al. [2] implied that the levels of dihydrotestosterone in the cerebrospinal fluid of postfinasteride syndrome patients are decreased. Irwig et al. [3] reported that in male pattern baldness patients who were treated with finasteride, 94% developed low libido and 92% developed erectile dysfunction. Additionally, the mean duration of persistent sexual side effects was 40 months after finasteride cessation.

The chemosensory inputs from the olfactory system are known to be the most important stimuli for sexual behaviours in male rodents. These stimuli are eventually transmitted to the hippocampus and ventromedial paraoptic area (VMPOA), regulating male sexual behaviour [4, 5]. Furthermore, the c-Fos protein is involved in various biological processes, such as learning, memory, stress response, and sexual behaviour, in rats. The expression of c-Fos in the MPOA and hippocampus of rats has been used as a marker for assessing neural activity.

Therefore, the aim of this study was to investigate the biochemical and changes occurring in the brain after finasteride treatment, watching whether it would be persistent even after the discontinuation of treatment, in a rat model. Understanding the effect of finasteride on rat brain tissue could provide valuable insight into the underlying mechanisms of the persistent side effects of the drug.

Ethical approval

All experimental protocols were approved by the Institutional Animal Care and Use Committee of St. Vincent’s Hospital, The Catholic University of Korea (approval no. IACUC-20-08, date: Feb 20, 2020). All experiments were performed in a laboratory at St. Vincent’s Hospital under standard conditions. The experiments adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [6].

Experimental setting

Twenty-four male Wistar rats (14~15 weeks old, 300~350 g, KOATECK, Pyeongtaek, Korea) were used. The animals were housed in standard polypropylene cages in a temperature-controlled room (25 °C ± 1 °C) with a 12:12-h light/dark cycle (lights on at 7 AM) and were allowed free access to food and water for 1 week of acclimation. During this period, the mean water intake was 30 ml per day. Eight rats were assigned to each group (Group 1 as the control group, Group 2 as the finasteride group, and Group 3 as the finasteride withdrawal group). Previous research used finasteride 3 mg/kg/day to evaluate the effect of finasteride on brain tissue [7]. To prevent water depletion, 10 mg of finasteride was dissolved in 300 ml water using 0.6 ml DMSO (dimethyl sulfoxide), and the solution was freely fed for 5 days (rats in Groups 2 and 3 were supposed to have 5 mg of finasteride with 150 ml water for 5 days, it was equal to finasteride 3 mg/kg/day considering that the weight of each rat was 300~350 g). Then, we checked the residual solution volume while changing the solution every 5 days. The same amount of DMSO without finasteride was applied to the water for Group 1. For 4 weeks, solutions were given to all groups, and Group 3 was observed for an additional four weeks after the discontinuation of treatment.

Mating and Attracting

Previous sexual experience enhances the neuroendocrinal response to sexual stimuli, for which reason, after a certain period of time after mating, it is recommended to place bedding stained with female secretions into the breeding cage immediately before sacrifice [8]. Twenty-four female Wistar rats (14~15 weeks old, 250~300 g) were used for 1:1 mating during the adjustment period before finasteride administration. The relevant female bedding was offered to matched male rats just before sacrifice.

Sacrifice, blood sampling and tissue retrieval

Rats were anaesthetized with isoﬂurane in N2O-O2 (70%:30%) in a small plastic chamber. The gas ﬂow rate was 500 mL/min, and the isoﬂurane ﬂow rate was set to 4% during induction. Once the rats lost consciousness, the gas ﬂow was switched from the chamber to an anaesthetic nose cone, and the cone was placed over the rats’ noses on the experimental table where the isoﬂurane ﬂow rate used for maintenance was set to 1.5% - 2%, with a gas ﬂow rate of 200 mL/min. Blood samples were obtained by a direct puncture of the right atrium after the chest wall was opened immediately before decapitation and the retrieval of brain tissues. The brain was retrieved using a protocol reported by Jaszczyk et al. [9]. Once the brain was retrieved, one hemisphere was immediately fixed with 10% formalin to evaluate histology, while the other hemisphere was used to evaluate RNA and protein expression.

Hormone assay

Plasma was obtained by centrifuging blood samples for 10 minutes at 8,000 rpm, and samples were stored at -20 °C using a standard method. Enzyme-linked immunosorbent assays (ELISAs) were performed to determine the serum levels of testosterone and dihydrotestosterone (E05100r, Cusabio Biotech) using commercial kits.

Immunohistochemistry

Fixed tissues were embedded in paraffin, cut into 4-μm sections, and placed onto glass slides. Since finasteride is a 5AR-2 (5-alpha reductase type 2) inhibitor [10], a primary antibody targeting 5AR-2 (Biorbyt, Cat#orb100170, Cambridge, UK) was used to evaluate the treatment effect. c-Fos is induced by a broad range of stimuli and is a reliable marker for neural activity [11], and c-Fos has been used to investigate the neural pathway of libido in rodents [12]. Therefore, we also used another primary antibody targeting c-Fos (Abcam, Cat# ab190289, Cambridge, UK). Heat-induced antigen retrieval was performed by heating tissues in a citrate buffer solution in a microwave for 2 min [13]. ImmPress kit (Vector Laboratories, Burlingame, CA, USA) secondary antibodies were used, and Tris-buffered saline was used to wash the specimens. We focused on the VMPOA and the hippocampus according to previous studies [14, 15] (Supplementary Figure 2). In addition, it has been recognized that sexual behaviour in rodents is dependent on the vomeronasal pathway, and signal transmission from the pathway continues into the hippocampus [16]. A 3,3'-diaminobenzidine tetrahydrochloride (DAB) kit was used for visualization. Three sections from each rat were bilaterally analysed by a pathologist who was blinded to the groups. Semiquantitative evaluations were performed by measuring the staining intensity (0, 1+, 2+, 3+, or 4+) in a fixed field.

RNA extraction (real-time PCR)

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat# 74004, Valencia, CA, USA). The RNA was reverse transcribed using a cDNA synthesis kit (PrimeScript RT Reagent Kit, Takara Bio, Cat#RR037A, Kusatsu, Japan) according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was performed using LC SYBR Green (Roche Diagnostics, Cat# 03 003 230 001, Mannheim, Germany) on a LightCycler 2.0 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany). Reactions for each sample were run in triplicate, cycle thresholds were normalized to glyceraldehyde-3-phosphate dehydrogenase expression, and comparative quantitation was performed using LightCycler 167 software (version 4.1, Roche). RT‒PCR was performed using the conditions described in Supplementary Table 1. We used the GAPDH gene as a housekeeping gene, and the National Center for Biotechnology Information accession number was NG028301.

Western blotting

Brain tissue was frozen and homogenized in protein extraction buffer (Intron, Seongnam, Korea) containing protease inhibitors (Sigma, MO, USA). The homogenates were centrifuged at 12,000 rpm for 20 minutes. The total protein concentration in the supernatant was measured using a Bradford assay (Bio-Rad, CA, USA). Equal amounts of protein (10 μg) were electrophoretically separated on 10% SDS‒PAGE gels and transferred to a PVDF membrane (Bio-Rad, CA, USA). The membrane was blocked with 5% skim milk (Bio-Rad, CA, USA) containing 0.1% Tween 20 for 1 hour and incubated overnight with antibodies against 5AR-2 (LifeSpan BioSciences, Cat# LS-C780518, WA, USA), c-Fos (Abcam, Cat# ab190289, Cambridge, UK), phospho-c-Fos (Ser374) (Invitrogen, Cat# PA5-104728, MA, USA) and β-actin (Cell Signalling, Cat# 4967S, MA, USA). The membrane was incubated with a secondary antibody at room temperature for 1 hour. The expression of each protein was visualized using enhanced chemiluminescence (ECL) (Bio-Rad, CA, USA). Western blot images was captured using an LAS 4000 mini instrument (GE Healthcare, IL, USA), and the results were quantified using densitometry.

Statistical analysis

The Kruskal‒Wallis test was performed to compare the 3 groups. The Mann‒Whitney U test with Bonferroni correction as a post hoc test was also performed. The number of experimental animals needed (a total of twenty-four, with eight in each group) was determined based on the ‘resource equation’ approach because of the exploratory nature of the current study [16]. Data from statistical analyses are presented as the mean ± standard error. Values of p < 0.05 were considered statistically significant.

For 4 weeks, the estimated consumption of DMSO (ml) was 1.58 ± 0.17, 1.69 ± 0.11 and 1.63 ± 0.05 in Groups 1, 2 and 3, respectively (Kruskal‒Wallis test, p=0.111). During the experiment, there were no deaths in any of the groups. Testosterone levels (ng/dl) did not differ among groups (p=0.423, Kruskal‒Wallis test). Dihydrotestosterone levels (ng/dl) and the dihydrotestosterone to testosterone ratio (%) decreased after 1 month of finasteride administration (Group 2), and the decrease was abrogated after discontinuation of the drug (Group 3). The mean difference in the mean value between Group 1 and Group 3 did not reach statistical significance (p=0.195, Mann‒Whitney test with Bonferroni correction) (Figure 1).

Considering western blotting, 5AR-2 expression differed among the 3 groups. 5AR-2 levels sharply decreased in Group 2 (p<0.001) and showed a tendency to increase in Group 3, but the increase did not reach statistical significance (p=0.315, Mann‒Whitney test with Bonferroni correction). The mean value of 5AR-2 between Groups 1 and 3 was still different (p=0.015, Mann‒Whitney test with Bonferroni correction), which means that 1 month of drug discontinuation may not be sufficient to recover 5AR-2 function (Figure 2A). c-Fos protein expression also differed among the 3 groups (p<0.001). c-Fos protein was downregulated in Group 2 compared to Group 1 (p<0.001, Mann‒Whitney test with Bonferroni correction), but c-Fos was upregulated in Group 3 compared to Group 2 (p=0.030, Mann‒Whitney test with Bonferroni correction). The mean value of c-Fos protein expression in Group 3 did not differ from that in Group 1 (p=0.063, Mann‒Whitney test with Bonferroni correction) (Figure 2B). Regarding the RT‒PCR analysis, the mean value of each group did not significantly differ (p=0.089 for 5AR-2 mRNA and p=0.071 for c-Fos mRNA). However, both 5AR-2 mRNA and c-Fos mRNA levels showed a common tendency to increase, which was reflected in the protein expression levels (Figure 2C and 2D).

Figure 3 shows the immunohistochemical staining of 5AR, where the mean density was significantly lower in Group 2 than in Group 1 and Group 3, but the difference between Group 1 and Group 3 was negligible (p=0.150 in VMPOA, p=0.177 in Hippocampus, Mann‒Whitney test with Bonferroni correction).

In regard to c-Fos immunohistochemical staining, the results were similar to those of 5AR-2 immunohistochemical staining (Figure 4). The mean density was obviously lower in Group 2 than in Group 1 and Group 3. The density in Group 3 was similar to that in Group 1 (p=1.000 in VMPOA, p=0.275 in Hippocampus, Mann‒Whitney test with Bonferroni correction).

Finasteride has a relatively short half-life. The plasma half-life of finasteride is approximately 5-6 hours in men between 18-60 years of age and 8 hours in men more than 70 years of age, and tissue binding is 4-5 days [18]. However, there has been a concern that adverse events such as decreased libido may persist up to a couple of years after cessation of finasteride treatment [2]. Considering that almost 12 days in an adult rat is equivalent to 1 year in humans according to a model comparing the lifespan of rats with the normal human lifespan [19], we conducted the present experiment at least for 4 weeks after cessation of finasteride, as shown in other studies [20].

Administering finasteride at a dosage of 3 mg/kg/day to rats for 4 weeks induced a decrease in 5AR-2 protein expression levels, which subsequently caused a decrease in serum dihydrotestosterone, as previously confirmed in other studies [7, 20]. Giatti et al. [7] showed that exposure to finasteride for 20 days decreased dihydrotestosterone levels in plasma, and the decreased dihydrotestosterone levels tended to persist after withdrawal of the drug. However, based on the present study, the dihydrotestosterone level was restored after the discontinuation of finasteride and the expression of both 5AR protein and c-Fos protein in Group 3 were not inferior to those in Group 1 according to the immunohistochemistry experiment. As a result, it is hard to say that the effect of finasteride is persistent after its discontinuation. However, the western blot results showed that 5AR-2 expression in Group 3 remained downregulated compared to that in the control group despite the tendency of increase in 5AR-2 RNA levels. Given that immunohistochemistry was performed in localized areas (hippocampus and VMPOA) whereas western blot was performed using a hemisphere of the brain, and that the results were evaluated using a semiquantitative method for immunohistochemistry, the discrepancy between the western blot and immunohistochemistry results could occur. Although the western blot and RT-PCR for 5AR-2 did not statistically differ between Group 1 and Group 3, the trend of western blot was U-shape whereas that of RT-PCR was inverted U-shape (Figure 2), which infer the recovery process after discontinuation of the drug. Overall, it is reasonable to say that the recovery of whole brain tissue from long-term 5AR-2 inhibition by finasteride may require a relatively long time.

Although there are substantial data supporting postfinasteride syndrome, the underlying causes driving this phenomenon remain poorly understood. In male rats, volatile odours emitted by females play an important role in eliciting sexual behaviours via the hippocampus and VMPOA [4,5,15,16]. These rats detect such odours via the accessory olfactory system, leading to the activation of granule cells in the accessory olfactory bulb (AOB) in a distinct pattern [21, 22]. Yoshikage et al. [23] explored how and which hormones modulate the signals of these volatile odours within the AOB utilizing c-Fos immunohistochemistry. After intact male rats were exposed to volatile female odours, there was an increase in c-Fos-positive cells in the AOB. This elevation was noticeable 3 days postcastration but was not evident by the 7th day. Subsequently, castrated male rats that were administered dihydrotestosterone showed a higher number of c-Fos-positive cells than castrated male rats without any treatment. In line with this study, we can infer that in the male rat brain, the expression of c-Fos can be modulated by sex hormones. The administration of finasteride led to a reduction in dihydrotestosterone levels, correlating with a downregulation in c-Fos expression. Upon discontinuation of the drug, we observed a trend where both dihydrotestosterone and c-Fos expression levels began to recover.

The authors want to note the limitations of this study. Due to animal ethical issues, only a minimal number of animals (8 males for evaluation and 8 females for mating in each group) were sacrificed. Therefore, the standard errors were relatively large in several metrics. In addition, a limitation of this kind of animal study is that the genetic responses to a drug are not always similar between animals and humans. Finasteride inhibits the action of 5AR-2, preventing the conversion of testosterone to dihydrotestosterone. As a result, the serum level of dihydrotestosterone decreases. Some studies have shown that this decrease can lead to an increase in serum testosterone levels [24]. In this animal study, there was no difference in testosterone levels, and the exact reason for this is unclear. However, as previously mentioned, it is suspected that the absence of testosterone level changes might be due to the study being conducted with a limited number of animals. Finally, we did not evaluate the 5AR type 1 response in brain tissue. We initiated our research hypothesizing that a 5AR inhibitor hinders the expression neurotransmitters such as c-Fos. In the early stages of our work, we opted for dutasteride, knowing its inhibitory effects on both 5AR type 1 and type 2. However, a challenge arose when a higher concentration of DMSO was needed to dissolve the drug in water. Ultimately, we turned to finasteride to inhibit 5AR type 2 and found that it could suppress c-Fos, potentially indicative of psychosexual activity in response to appropriate stimuli.

Finasteride exerted an influence on brain tissue, including the hippocampus and VMPOA, via 5AR-2 inhibition, resulting in a decrease in c-Fos protein expression. However, the negative impact of finasteride on the rat brain may disappear after long-term cessation of the drug. Further studies using other biomarkers and different withdrawal periods are required to clarify the detailed role of 5AR in brain tissue.

Acknowledgements

The authors wish to acknowledge the financial support of St. Vincent’s Hospital, Research Institute of Medical Science (SVHR-2020-08). We are particularly grateful to Eun Yong Shin (The Institute of Medical Science, St. Vincent’s Hospital) for assisting with all experiments.

Conflict of Interest

Nothing to declare.

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There is NO conflict of interest to disclose.

SupplementaryFigure1.tif
Supplementary Figure 1. Study design 24 male rats were randomly assigned into three groups and then 1:1 mating was performed before finasteride administration.
SupplementaryFigure2.tif
Supplementary Figure 2. Sagittal section of rat brain Red rectangular area was evaluated in the present study. Ventromedial preoptic area is located between anterior commissure and optic chiasm.
Supplementarytable1.docx

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The effect of finasteride on the male rat brain

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