Introduction
Abusive use of anabolic-androgenic steroids (AAS) is based on supraphysiological doses
that are 10 to 100 times greater than therapeutic doses,[1 ]
[2 ] and which have been associated with a wide spectrum of adverse physical and psychic
effects. Little is known about the effect of AAS in the human brain, with reports
of changes in behavior, such as aggressiveness, anxiety and depression, among others.[3 ]
[4 ]
[5 ]
Some studies have shown that AAS may have deleterious effect on the central nervous
system (CNS).[6 ] The few in vitro data obtained suggest that the influence of androgenic drugs on
neurodegeneration depends critically on the treatment regimen, concentration and cell
phenotype. A single application of testosterone in supraphysiological concentration
promoted cell death by apoptosis in nerve cells by means of altering their Ca2+ signaling.[7 ]
Recent animal models have demonstrated that chronic use and abuse of stanozolol have
reduced levels of brain-derived neurotrophic factor (BDNF) and dopamine in the hippocampus
and prefrontal cortex of rodents, in addition to the reduction in glucocorticoid receptor
expression in the hippocampus and in plasma, and an increase in basal morning plasma
cortisol levels. These metabolic changes have been related to mood disorders, such
as depression.[8 ]
[9 ]
As the monoaminergic system regulates aggression, sexual behavior, fear and anxiety,
one can suggest a possible correlation between changes in the monoamines generated
by AAS and behavioral alterations and mood disorders.[9 ]
[10 ]
From the above mentioned, it is evident that there are considerable negative neurological
effects due to abusive and/or chronic use of AAS that can lead to serious complications
and damages, both physical and behavioral. Regarding the lack of knowledge of the
neural mechanisms involved in the wide range of deleterious effects that may involve
the CNS, it is necessary to carry out new research aiming to elucidate the underlying
brain mechanisms associated to them.
Material and Methods
Ninety-two Swiss mice from the Central Biotery of the Universidade Federal de Alfenas
(UNIFAL-MG) were used, with 46 being male and 46 female, ∼ 90 days old (young adults),
weighting between 40 and 50 g. All animals were fed with commercial ration and water
ad libitum (at will) and kept in a 12-hour light-dark cycle. The animals were divided as follows:
CT group - Animals receiving testosterone cypionate (SigmaPharm Laboratories, Philadelphia,
Pensilvania, USA) at a dose of 0.8 mg/kg/day.
ST group - Animals receiving stanozolol (Winstrol Depot [Winthrop Chemical Company,
New York, NY, USA]) at a dose of 1.8 mg/kg/day.
Control group - Animals receiving sterile saline solution at 0.9% at a dose of 0.05
ml/day.
The treatment consisted of intraperitoneal (IP) injection of the drugs to be tested
for 33 days, with applications performed twice a week. On intercalated days, they
were submitted to swimming for an initial period of 5 minutes, gradually increasing
up to 15 minutes. The swimming was performed in a container measuring 43 × 34 × 26
cm, containing in its interior water in the temperature of 24 to 26°C to the edge.
Swimming training was performed in an attempt to generate the same stress conditions
as bodybuilding. From the 33rd day of treatment, the elevated plus maze (EPM) test was performed in all groups to
study the effects of these substances on the anxiety level of the animals.[11 ]
[12 ]
After the days of behavioral testing, the animals were euthanized by inhalation of
isoflurane for later craniotomy. The brains were removed entirely, washed in saline
solution and fixed in 4% paraformaldehyde in phosphate buffer for 24 hours. After
this period, the material underwent the conventional histological processes: alcohol
dehydration, xylol diaphanization and paraffin inclusion.
From each brain so obtained, serial and homotypic samples were taken in frontal sections[13 ]
[14 ] with a thickness of 7 μm in a Yidi YD-315 microtome. To evaluate the areas selected
for the study, the material was stained with Cresyl violet.[14 ]
[15 ] The determination of the studied areas matches the representation of plate 43 of
Franklin and Paxinos' mouse brain stereotactic atlas.[15 ]
[16 ] For the quantitative analysis of neuron body cells, the simple random sample counting
method was used.[17 ]
[18 ]
[19 ] Each division of the amygdala (basolateral amygdala [BLA] or ventral posterior nucleus
[VPN]) had two areas quantified per hemisphere, totaling four areas per section. To
avoid counting the same cell more than once, 3 semi-serial sections per animal were
analyzed, totaling 12 histological fields per nucleus studied for each animal. As
2 divisions (BLA) and (VPN) were quantified, a total of 24 areas per animal were obtained.
Thus, for the sample used, a total of 960 areas was quantified at a 400X magnification.
All analyzes were performed by means of the software Axiovision 4 Module Interactive
Measurement (Carl Zeiss Microscopy LLC, Thornwood NY, USA), an analyzing image system,
coupled to the AxioScope A1 microscope (Carl Zeiss Microscopy LLC).
The study represents a completely randomized design (CRD); therefore, the statistical
analysis was performed through a one-way analysis of variance (ANOVA) followed by
Tukey averages comparison test. Values of p < 0.05 were considered as indicative of significance.
Ethics Statement
This work is in accordance with the ethical principles of experiment animal use and
has been approved by the Commission for Ethics in Animal Experimentation of UNIFAL-MG,
under the registration number 505/2013.
Results
The effects of chronic administration of AAS related to anxious behavior (determined
by the EPM test) in male animals are shown in [Fig. 1 ]. A lower number of entries (< 45.25%) and a shorter staying time spent in the open
arms (< 41.9%) of the EPM were observed in the stanozolol (Winstrol Depot) group when
compared with the control group ([Table 1 ]). These data indicate a greater anxiogenic response to stanozolol (Winstrol Depot)
in the male animals group in relation to the control.
Fig. 1 Graphs illustrating analyzed elevated plus maze test parameters (male animals). Effect
of testosterone cypionate (Deposteron), stanozolol (Winstrol Depot) or saline solution
administration in relation to the number of entries in the closed arm (A), staying
time in the closed arm (B), number of entries in the open arm (C) and staying time
in the open arm (D). Means and respective standard deviations (*= p < 0.01) are depicted in relation to the control group.
Table 1
Number of entries and staying time in the arms of the elevated plus maze (EPM)test
Number of entries
(open arms)
Number of entries
(closed arms)
% staying time
(open arms)
% staying time
(closed arms)
Male
Control
8.11a
6.88a
11.34a
70.28a
Deposteron
5.57a
9.28a
8.2a
63.81a
Winstrol
4.44b (< 45.25%)
8.00a
6.58b (< 41.9%)
68.12a
Female
Control
6.2a
7.1a
8.04a
65.46a
Deposteron
6.00a
7.5a
3.85b (< 52.1%)
75.64b (> 15%)
Winstrol
5.7a
8.1a
6.66b (< 17.1%)
70.63a
Different letters in superscript refer to statistical difference using Tukey test
at the significance level of 5%.
The effects of chronic administration of AAS related to anxious behavior (EPM) in
female animals are shown in [Fig. 2 ]. A longer staying time spent in the closed arms of the EPM was observed for the
testosterone cypionate (Deposteron) group (> 15%) in relation to the control one,
as well as a shorter staying time spent in the open arms of the EPM for the testosterone
cypionate (Deposteron) group (< 17.1%) when compared with the control and the stanozolol
(Winstrol Depot) groups (< 52.1%). These data indicate a higher anxiogenic response
to testosterone cypionate (Deposteron) in the female animals group when compared with
the control group ([Table 1 ]).
Fig. 2 Graphs illustrating analyzed elevated plus maze test parameters (female animals).
Effect of testosterone cypionate (Deposteron), stanozolol (Winstrol Depot) or saline
solution administration in relation to the number of entries in the closed arm (A),
staying time in the closed arm (B), number of entries in the open arm (C) and staying
time in the open arm (D). Means and respective standard deviations (*= p < 0.01) are depicted in relation to the control group (• = p < 0.05 is in relation to stanozolol group).
In relation to neuronal density in the basolateral amygdala (ABL), a decrease in the
number of neuron body cells in the testosterone cypionate (Deposteron) (17.55%) male
animals group was observed when compared with the control group, as shown in [Fig. 3A ] and [Table 2 ]. However, in female animals, significant differences were found in both testosterone
cypionate (Deposteron) (19.16%) and stanozolol (Winstrol Depot) (18.36%) groups when
compared with the control group, as shown in [Fig. 3B ] and [Table 2 ].
Fig. 3 Neuron body cells counting in the basolateral amygdala (BLA) of male and female animals.
(A) shows comparative graphs of neuronal density in BLA of male mice in each analyzed
experimental group (*= p < 0.001) for testosterone cypionate in relation to the control group. (B) shows comparative
graphs of neuronal density in BLA of female mice in each analyzed experimental group
(*= p < 0.001) for testosterone cypionate and stanozolol in relation to the control group.
Table 2
Mean of basolateral amygdala and percentage of reduction neuron cells quantification
Groups
Mean
BLA neuron cell
Percentage of reduction
Male
Control
30.07a
−
Deposteron
30.79a
−
Winstrol
24.79b
17.55%
Female
Control
31.36a
−
Deposteron
25.35b
19.16%
Winstrol
25.60b
18.36%
Abbreviation: BLA, basolateral amygdala.
BLA neuron cells quantification exhibited as mean value.
Different letters in superscript refer to statistical difference using Tukey test
at the significance level of 5%.
The decrease in neuronal density in the ventral posterior nucleus (VPN) of the medial
amygdala was observed in both testosterone cypionate (Deposteron) and stanozolol (Winstrol
Depot) groups when compared with the control group, for male (Deposteron-13.55% and
Winstrol Depot-17.68%) and female animals (Deposteron 13.53% and Winstrol Depot 14.32%),
as shown in [Fig. 4 ] and [Table 3 ].
Fig. 4 Neuron body cells counting in the ventral posterior nucleus (VPN) of male and female
animals. (A) shows comparative graphs of neuronal density in VPN of male mice in each
analyzed experimental group (*= p < 0.001) for testosterone cypionate and stanozolol in relation to the control group.
(B) shows comparative graphs of neuronal density in VPN of female mice in each analyzed
experimental group (*= p < 0.001) for testosterone cypionate and stanozolol in relation to the control group.
Table 3
Mean of ventral posterior nucleus and percentage of reduction neuron cells quantification
Groups
Mean
BLA neuron cell
Percentage of reduction
Male
Control
45.00a
−
Deposteron
38.90b
13.55%
Winstrol
37.04b
17.68%
Female
Control
48.03a
−
Deposteron
41.53b
13.53%
Winstrol
41.15b
14.32%
Abbreviation: BLA, basolateral amygdala.
Ventral posterior nucleus neuron cells quantification exhibited as mean value.
Different letters in superscript refer to statistical difference using Tukey test
at the significance level of 5%.
Discussion
From the general analysis of the results in the EPM test, we can suggest that both
testosterone cypionate (Deposteron) and stanozolol (Winstrol Depot) are associated
with the generation of anxiogenic responses. The stanozolol (Winstrol Depot) male
animals group, as well as the testosterone cypionate (Deposteron) female animals group
presented responses indicative of greater anxiety. In the first group, the animals
presented a lower number of entries and a shorter staying time spent in the open arms
of the EPM (more aversive area). In the second group, the animals had a longer staying
time spent in the closed arms of the EPM (less aversive area) and a shorter staying
time spent in the open arms of the EPM (more aversive area). No significant results
were found for the testosterone cypionate (Deposteron) male animals group and stanozolol
(Winstrol Depot) female animals group.
The generation of an anxiogenic response is in agreement with the results obtained
in a study in which adult male C57Bl/6J mice receiving a daily subcutaneous injection
of 15 mg/kg of nandrolone decanoate presented an anxiogenic behavior in the EPM test
after 19 days of treatment.[20 ] Anxiogenic response in the EPM test was also observed in a study in which male Wistar
rats received nandrolone decanoate (5 mg/kg) twice a week for 6 weeks and were submitted
to the EPM test at the end of this period.[21 ]
In another study, albino male mice receiving daily subcutaneous injections of nandrolone
decanoate in the supraphysiological concentration of 15 mg/kg did not present changes
in anxiety levels when submitted to the EPM test on the 14th day of treatment[6] . Some authors have reported that the treatment of Long-Evans rats with subcutaneous
testosterone propionate through silicone implants for prolonged administration of
supraphysiological doses (6 days) promoted an increase in the open arms exploration
of the EPM, in relation to the control, indicating reduction on the level of anxiety.
However, in the same study, treatment for 14 days did not promote anxiogenic effects.
According to these authors, this variation of responses is due to the development
of tolerance to AAS, a very common phenomenon in chronic users of psychoactive drugs.[22 ]
It has been demonstrated that serotonin activates inhibitory GABAergic interneurons
in the amygdala, exerting a generalized inhibitory tonus on neuronal excitability,
which suggests that serotonin functions as a brake mechanism that limits neuronal
excitability.[23 ] There is a possibility that an inhibitory effect of the AAS on the serotonergic
system may contribute to a greater excitability of the amygdala, which, in turn, could
be responsible for the behavioral responses observed in the AAS prolonged use.[20 ]
Since a high dose (1 mg/kg) of AAS binds to glucocorticoid receptors,[21 ]
[24 ] the effects of these drugs could be related to their actions on these receptors.
Glucocorticoids may act in the modulation of anxiety by their actions in the CNS,
especially in the hippocampus.[25 ]
Some studies have attempted to correlate behavioral changes to the AAS effects on
central serotonergic and GABAergic neurotransmission, among others, in several areas
of the brain.[26 ]
In view of fact that there are synergistic interactions between glutamate and gonadal
steroids coordinating several hypothalamic and limbic functions, the amygdaloid structures
of treated groups, even with a lower number of neurons in relation to control group,
may, through high local concentrations of AAS, stimulate glutamatergic transmission,
increasing the excitatory signals in these neurons and keeping the aggressiveness
responses statistically equal to those of control group. At the same time, it is also
necessary to take into account the capacity of AAS to affect the gabaergic and serotoninergic
transmissions, and may also result in a greater excitation of these neurons. In view
of this situation, it is necessary to carry out new studies seeking to highlight how
the AAS have led to these changes in behavioral responses.
In one study, it was observed that the AAS chronic use in mice has been shown to induce
dose-, sex- and age-dependent changes in the GABA-receptor subunit gene expression
in anterior brain areas.[27 ] As observed in the present study, when evaluating neuronal density, it can be noted
that both stanozolol and testosterone cypionate may lead to a decrease in the number
of neuron body cells in mice. A statistically significant decrease in this number
was observed in BLA of the stanozolol male animals group, testosterone cypionate and
stanozolol female animals groups when compared with their respective control ones.
We also observed the same result in the VPN of all experimental groups. These results
are in agreement with previously published outcomes.[28 ]
[29 ]
In general, it seems that AAS physiological doses have the ability to play a neuroprotective
role; however, when AAS is administered in high concentrations, they appear to exert
a deleterious effect on neuron body cells, and most studies show an excitotoxic effect
when using concentrations. The same was observed by some other authors, who found
that pre-treatment with high doses of testosterone increased N-methyl-D-aspartate
(NMDA) toxicity. Low testosterone concentrations, on other hand, exerted neuroprotective
role, but became neurotoxic in presence of aromatase inhibitors.[7 ]
[30 ]
Another study has shown that hippocampal neurogenesis in adult male rats occurs via
dihydrotestosterone (DHT) mediation,[31 ] while other authors have demonstrated that in mouse hippocampal cell culture, high
concentrations of testosterone increased the toxicity induced by millimolar concentrations
of glutamate, whereas dehydroepiandrosterone (DHEA), a metabolite precursor of testosterone,
exerted a neuroprotective role.[32 ]
[33 ]
In vivo studies, androgenic neurosteroids, such as DHEA, testosterone and DHT, play
a protective role in the hippocampus against damage induced by excitotoxins, kainic
acid and domoic acid.[34 ]
[35 ] Neuroprotection was totally dependent on the availability of the enzyme aromatase,
which converts testosterone to stradiol.[36 ]
Surprisingly, little is known about how androgens affect excitotoxic neuronal death,
despite the large numbers of data obtained with female steroids. Excitotoxicity refers
to a particular mechanism of neuronal death triggered by excessive stimulation of
glutamatergic receptors.[37 ]
[38 ]
N-methyl-D-aspartate receptors are located on the postsynaptic membrane of excitatory
synapses and exhibit greater permeability to Ca2+ than α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid
(KA) receptors, a feature that gives them a more active role in neurotoxic mechanisms.
When the postsynaptic membrane is in resting potential, the NMDA channels are blocked
by a magnesium (Mg2+ ) ion that prevents the influx of Ca2+ to the postsynaptic receptors. However, in postsynaptic depolarization (which may
be caused by the activation of AMPA receptors, among others), Mg2+ ions are expelled from the NMDA channels, which thus allow the influx of Ca2+ in favor of its concentration gradient.[39 ] In the cytosol, Ca2+ is an important second messenger and influences a large number of cellular functions,
exerting a regulatory role in cellular proliferation and survival processes, as well
as in cell death due to necrosis or apoptosis.[40 ] The Ca2+ influx regulates membrane excitability and the intensity of synaptic transmission
through the activation of intracellular signaling cascades dependent on this ion.
Excessive concentrations of L-Glu in the synaptic cleft result in hyperstimulation
of its receptors and an excessive Ca2+ entrance at the postsynaptic receptors, which, together with the release of Ca2+ from intracellular reserves, raise the Ca2+ concentration above the triggering threshold of regulatory mechanisms by activating
the intracellular mechanisms of excitotoxicity that culminate in neuronal death.[41 ]
Other published data have shown that neuron body cells regulated by glutamate in the
hypothalamus are direct targets of gonadal steroids, both androgens and estrogens.
Thus, they can easily influence excitatory neurotransmission in these areas in a sexually
dimorphic manner.[42 ] The present work may explain the fact that testosterone cypionate led to a reduction
in the number of neuron body cells in the BLA of female subjects but not in that of
males.
Conclusion
The results obtained in the quantitative analyzes show that testosterone cypionate
and stanozolol in supraphysiological doses are able to cause a significant reduction
in the number of neuron body cells in the BLA and in the VPN of the medial amygdala
of mice, and these results, added to the behavioral outcomes, may stimulate directly
related anxiety-related emotional responses.
