Renin angiotensin system and gender differences in dopaminergic degeneration.

Abstract

There are sex differences in dopaminergic degeneration. Men are approximately two times as likely as premenopausal women of the same age to develop Parkinson's disease (PD). It has been shown that the local renin angiotensin system (RAS) plays a prominent role in sex differences in the development of chronic renal and cardiovascular diseases, and there is a local RAS in the substantia nigra and dopaminergic cell loss is enhanced by angiotensin via type 1 (AT1) receptors.
In the present study, we observed that intrastriatal injection of 6-hydroxydopamine induced a marked loss of dopaminergic neurons in the substantia nigra of male rats, which was significantly higher than the loss induced in ovariectomized female rats given estrogen implants (i.e. rats with estrogen). However, the loss of dopaminergic neurons was significantly lower in male rats treated with the AT1 antagonist candesartan, and similar to that observed in female rats with estrogen. The involvement of the RAS in gender differences in dopaminergic degeneration was confirmed with AT1a-null mice lesioned with the dopaminergic neurotoxin MPTP. Significantly higher expression of AT1 receptors, angiotensin converting enzyme activity, and NADPH-oxidase complex activity, and much lower levels of AT2 receptors were observed in male rats than in female rats with estrogen.
The results suggest that brain RAS plays a major role in the increased risk of developing PD in men, and that manipulation of brain RAS may be an efficient approach for neuroprotective treatment of PD in men, without the feminizing effects of estrogen.

Full text

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RESEARCH ARTICLE Open Access
Renin angiotensin system and gender differences
in dopaminergic degeneration
Ana I Rodriguez-Perez, Rita Valenzuela, Belen Joglar, Pablo Garrido-Gil, Maria J Guerra and
Jose L Labandeira-Garcia*
Abstract
Background: There are sex differences in dopaminergic degeneration. Men are approximately two times as likely
as premenopausal women of the same age to develop Parkinson’s disease (PD). It has been shown that the local
renin angiotensin system (RAS) plays a prominent role in sex differences in the development of chronic renal and
cardiovascular diseases, and there is a local RAS in the substantia nigra and dopaminergic cell loss is enhanced by
angiotensin via type 1 (AT1) receptors.
Results: In the present study, we observed that intrastriatal injection of 6-hydroxydopamine induced a marked loss
of dopaminergic neurons in the substantia nigra of male rats, which was significantly higher than the loss induced
in ovariectomized female rats given estrogen implants (i.e. rats with estrogen). However, the loss of dopaminergic
neurons was significantly lower in male rats treated with the AT1 antagonist candesartan, and similar to that
observed in female rats with estrogen. The involvement of the RAS in gender differences in dopaminergic
degeneration was confirmed with AT1a-null mice lesioned with the dopaminergic neurotoxin MPTP. Significantly
higher expression of AT1 receptors, angiotensin converting enzyme activity, and NADPH-oxidase complex activity,
and much lower levels of AT2 receptors were observed in male rats than in female rats with estrogen.
Conclusions: The results suggest that brain RAS plays a major role in the increased risk of developing PD in men,
and that manipulation of brain RAS may be an efficient approach for neuroprotective treatment of PD in men,
without the feminizing effects of estrogen.
Keywords: angiotensin, estrogen, menopause, NADPH-oxidase complex, neurodegeneration, oxidative stress, Par-
kinson, sex differences
Background
There are sex differences in dopaminergic (DA) degen-
eration, as observed in animal models as well as clinical
and epidemiological reports on Parkinson’s disease (PD).
The higher risk of developing PD in men than in pre-
menopausal women of the same age is well-established;
men are approximately two times as likely as women to
develop the disease [1-3]. However, the mechanisms
responsible for this difference have not been clarified
[4]. It has been shown that the renin angiotensin system
(RAS) plays a prominent role in sex differences in the
development of chronic renal and cardiovascular
diseases. The peptide angiotensin II (AII), via type 1
(AT1) receptors is one of the most important known
inflammation and oxidative stress inducers, and pro-
duces reactive oxygen species (ROS) by activation of the
NADPH-oxidase complex [5-7], which is the most
important intracellular source of ROS apart from mito-
chondria [8,9]. Interestingly, RAS activity is higher in
kidneys and cardiovascular tissues from males than in
the same tissues from females [10-13], and males and
females respond differently to stimulation and inhibition
of RAS [14,15]. Furthermore, it has been shown that
expression of vascular and renal AT1 receptors, as well
as the balance between AT1 and AT2 receptors may be
modulated by sex hormones, and a major role for RAS
in the gender differences in the development of chronic
renal and cardiovascular diseases has been proposed
* Correspondence: joseluis.labandeira@usc.es
Department of Morphological Sciences, Networking Research Center on
Neurodegenerative Diseases (CIBERNED), University of Santiago de
Compostela, Santiago de Compostela, E-15782 Spain
Rodriguez-Perez et al. Molecular Neurodegeneration 2011, 6:58
http://www.molecularneurodegeneration.com/content/6/1/58
© 2011 Rodriguez-Perez et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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[10-12]. Several studies have revealed that estrogen-
mediated down-regulation of the renin-angiotensin sys-
tem (RAS) mediates beneficial effects of estrogen (E2) in
several tissues [16-18]. Furthermore, there is substantial
evidence that androgens may upregulate RAS activity
and therefore amplify gender-related differences
[10,19,20].
The brain possesses a local RAS [21,22]. We have pre-
viously shown that there is a local RAS in the substantia
nigra and that DA cell loss is enhanced by AII via AT1
receptors and activation of the microglial NADPH-oxi-
dase complex in several animal models of PD [23-25].
However, it is not known if there are differences
between males and females in RAS activity in the sub-
stantia nigra, which may also be involved in the higher
risk of developing PD in men than in premenopausal
women. In the present study, we compared the effects
of the DA neurotoxin 6-hydroxydopamine (6-OHDA)
on DA neuron degeneration in male rats and rats with
high stable levels of E2 (i.e. similar to proestrus), and
investigated the nigral RAS in both groups of animals.
Several studies have shown that the risk of developing
several E2-related diseases varies with the menstrual
cycle in women [26,27]. However, normal rat females
have a 4-day estrous cycle, with a very short proestrus
period (i.e. only 12 hours with high levels of E2). It is
therefore expected that most of these rats will have low
levels of E2 when killed, and during most of the 6-
OHDA lesion period (two weeks), and are thus not sui-
table for comparison with male rats as regards under-
standing gender differences in
neurotoxins in humans. Finally, the involvement of RAS
in the observed gender differences in DA neuron sus-
ceptibility to the neurotoxin was confirmed by inhibition
of AT1 receptors with the AT1 receptor antagonist can-
desartan in 6-OHDA treated male rats, and by a second
experimental approach using AT1 deficient mice
lesioned with the DA neurotoxin MPTP (1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine).
vulnerability to
Results
Intrastriatal injection of 6-OHDA induced a similar and
marked loss of DA neurons in the substantia nigra of
male rats and female rats without estrogen (i.e. ovx
rats), which was significantly higher than that induced
by 6-OHDA in female rats with estrogen (ovx + E2).
Interestingly, however, the loss of DA neurons was sig-
nificantly lower in male rats treated with the AT1
antagonist candesartan, and similar to that observed in
female rats with estrogen. The present results therefore
show that candesartan induced neuroprotection in male
rats against 6-OHDA similar to that induced by estro-
gen in female rats. There was no significant difference
in the number of TH-ir neurons between control males
injected with vehicle and male rats treated with cande-
sartan alone (Figure 1). In order to confirm that 6-
OHDA induced cell death and not only phenotypic
down-regulation of tyrosine hydroxylase (TH) activity,
series of sections through the entire substantia nigra of
control rats and rats treated with 6-OHDA were coun-
terstained with cresyl violet, and the total number of
neurons in the SNc was estimated. The number of neu-
rons in unlesioned females (ovx+E2; 12688 ± 472) and
males (13067 ± 540) was much higher than in 6-
OHDA-lesioned ovx females (4318 ± 441) or males
(4856 ± 317), which was significantly lower than in
females with estrogen (ovx+E2+6-OHDA; 7732 ± 346)
or males treated with candesartan (males+6-OHDA
+cande; 8421 ± 417). As expected, the number of Nissl-
stained neurons counted in Cresyl-violet stained sections
was slightly higher than that of TH-immunoreactive
(TH-ir) neurons since some non-dopaminergic neurons
located in the area of the SNc were also included.
The involvement of the RAS with regard to gender
differences in DA degeneration was confirmed by using
a second experimental approach in which AT1a-null
mice were lesioned with the DA neurotoxin MPTP.
Administration of MPTP induced a similar and marked
loss of DA neurons in the substantia nigra of wild type
(WT) male mice and WT female mice without estrogen
(i.e. ovx WT mice), and was significantly higher than
that induced by MPTP in female WT mice with estro-
gen (ovx WT+E2). However, the loss of DA neurons
was significantly lower in male AT1a-null mice, and
similar to that observed in female WT mice with estro-
gen (Figure 2). In order to confirm that MPTP induces
cell death and not only phenotypic down-regulation of
TH activity, series of sections through the entire sub-
stantia nigra of different groups of mice were counter-
stained with cresyl violet, and the total number of
neurons in the SNc was estimated. The number of neu-
rons in unlesioned females (ovx+E2; 14760 ± 876), WT
males and AT1a-null mice (14209 ± 947 and 13976 ±
756, respectively) was much higher than in MPTP-
lesioned ovx females (5815 ± 698) or WT males (6070 ±
598), which was significantly lower than in females with
estrogen (ovx+E2+MPTP; 8911 ± 908) or AT1a-null
males (AT1-/-+ MPTP; 10553 ± 681), confirming that
MPTP induced cell death and not TH-downregulation
in the present experimental conditions.
Real time RT-PCR analysis revealed significantly
higher expression of AT1 receptor mRNA (around
160%) and much lower levels of AT2 mRNA (about
70% reduction) in male rats than in female rats with
estrogen (Figure 3A). Similarly, WB studies revealed a
significantly higher expression of AT1 receptors in male
rats than in female rats with estrogen, and the expres-
sion of AT2 receptors was significantly lower (about
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Figure 1 Dopaminergic neurons in the substantia nigra compacta (SNc) of rats. (A): Dopaminergic (TH-ir) neurons were counted in the SNc
two weeks after intrastriatal injection of vehicle (i.e. controls; males and ovx+E2 females) or 6-OHDA in the different experimental groups. The
dopaminergic neurons were quantified as the total number of TH-ir neurons in the SNc. Data are means ± SEM. *p < 0.05 relative to the
corresponding saline-treated group (males or ovx+E2 females),#p < 0.05 relative to the group treated with 6-OHDA alone (i.e. males+6OHDA
and ovx+6-OHDA). One-way ANOVA and Bonferroni post-hoc test. TH-ir, tyrosine hydroxylase immunoreactive. Representative photomicrographs
of TH-ir neurons in non lesioned (i.e. control) males (B), females with estrogen (ovx+E2; C), 6-OHDA lesioned males (D) and 6-OHDA lesioned
females without (E) and with estrogen (G), as well as male rats lesioned with 6-OHDA and treated with the AT1 antagonist candesartan (cand; F).
Scale bar: 500 μm.
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Figure 2 Dopaminergic neurons in the substantia nigra compacta (SNc) of mice. (A): Dopaminergic (TH-ir) neurons were counted in the
SNc one week after the last intraperitoneal injection of saline (i.e. controls; WT and AT1a-null males and WT ovx+E2 females) or MPTP in the
different experimental groups. The dopaminergic neurons were quantified as the total number of TH-ir neurons in the SNc. Data are means ±
SEM. *p < 0.05 relative to the corresponding saline-treated group (males or ovx+E2 females),#p < 0.05 relative to the group treated with MPTP
alone (i.e. WT males+MPTP and WT ovx+MPTP). One-way ANOVA and Bonferroni post-hoc test. (B-G): representative photomicrographs of TH-ir
neurons in non lesioned WT females with estrogen (ovx+E2; B), MPTP lesioned WT females with (C) and without (D) estrogen, as well as non-
lesioned AT1a-null male mice (AT1a-/-; E), AT1a-null male mice lesioned with MPTP (F), and MPTP lesioned WT males (G). AT1-/-, AT1a-null mice;
TH-ir, tyrosine hydroxylase immunoreactive; WT, wild type mice. Scale bar: 100 μm.
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65% reduction) in males (Figure 3B). ACE activity was
significantly lower in female rats with estrogen than in
males, which indicates increased AII production in
males (Figure 4). In accordance with this, male rats
showed significantly higher NADPH complex activity
than female rats (Figure 5A). In previous studies, we
observed the presence of several NADPH complex subu-
nits, including p47phox, in DA neurons and glial cells
(microglia and astrocytes; see references 24 and 25 for
details). The increased NADPH complex activation was
confirmed by the increase in the expression of the
NADPH-oxidase subunit p47phoxin males (Figure 5B).
Expression of the NADPH-oxidase cytosolic subunit
p47phoxis an indicator of the level of activation of the
NADPH-oxidase complex. The NADPH-oxidase com-
plex is composed of membrane-bound subunits and
cytosolic subunits such as p47phox, which is considered a
key subunit for NADPH-oxidase activation [28]. Trans-
location of cytosolic subunits to the membrane, which
leads to generation of ROS is a necessary step for
NADPH-oxidase activation. The level of the NADPH-
oxidase subunit p47phoxexpression is correlated with
NADPH-oxidase activity and NADPH-derived superox-
ide formation [7,29]. Finally, we confirmed that inhibi-
tion of AT1 receptors with candesartan induced a
decrease in RAS activity in male rats. Candesartan
induced a significant increase in the expression of AT2
receptors, as well as a decrease in the expression of the
NADPH-oxidase subunit p47phox. Changes in ACE activ-
ity were statistically not significant (Figure 6).
In previous studies, we observed AT1 and AT2 recep-
tors in DA neurons and glial cells (astrocytes and micro-
glia), and that AII induces microglial activation and DA
cell death, via AT1 receptors and activation of the
NADPH complex (see references 24 and 25 for details),
which may explain the greater effect of DA neurotoxins
observed in males than in female rats with estrogen. In
order to confirm that the different response to 6-OHDA
was associated with inhibition of the 6-OHDA-induced
microglial response, we analyzed the expression of OX6
in the substantia nigra, as a marker for activated micro-
glia. Control rats (i.e. ovx+E2 females and males injected
Figure 3 Angiotensin receptor expression in male and female
rats. Real-time quantitative RT-PCR (A) and Western blot (WB; B)
analysis of AT1, AT2 receptor expression in male rats as compared
with female rats with estrogen (ovx+E2). Protein expression was
obtained relative to the GAPDH band value and the expression of
each gene was obtained relative to the housekeeping transcripts (b-
Actin). The results were then normalized to ovx+E2 values (100%).
Data are mean values ± SEM. *p < 0.05 (Student’s t test).
Figure 4 Activity of the angiotensin converting enzyme (ACE)
in male and female rats. ACE activity in the ventral
mesencephalon of males and female rats with estrogen (ovx +E2).
ACE activity was significantly higher in male rats. Data were
obtained as nmoles of his-leu produced per milligram of protein
per minute and the results were then normalized to the values for
ovx+E2 females (100%). Data are means ± SEM. *p < 0.05 (Student’s
t test).
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with vehicle) showed minimal microglial activation. In
male rats and ovx female rats injected with 6-OHDA,
microglial activation was much higher than in controls.
However, 6-OHDA-induced microglial activation was
significantly lower in male rats treated with candesartan
(males+cand+6-OHDA) and in female rats with estrogen
(ovx+E2+6-OHDA; Figure 7).
Discussion
PD is usually considered a multifactorial process in
which low and apparently non-toxic doses of several
pathogenic factors can act synergistically to cross the
threshold of the DA cell degeneration [30] and oxidative
stress and inflammation play major roles in the synergis-
tic process [31-33]. Factors that increase the oxidative
and inflammatory state of DA neurons may therefore
Figure 5 Activity of the NADPH oxidase complex in male and
female rats. Lucigenin-enhanced chemiluminescence (A) and
western blot (B) analysis of the NADPH complex activation in the
ventral mesencephalon of males and female rats with estrogen (ovx
+E2). Chemiluminescence analysis revealed significantly higher
NADPH activity in males (A). Western blot analysis revealed that
males had significantly higher expression of NADPH oxidase subunit
p47phox(B). Data, expressed as relative light units (RLU/min/mg
protein; A) and p47phoxprotein expression (B) was obtained relative
to the GAPDH band value and then normalized to ovx+E2 values
(100%). Data are means ± SEM. *p < 0.05 (Student’s t test).
Figure 6 Analysis of changes induced by treatment
candesartan in male rats. Real-time quantitative RT-PCR (A) and
Western blot (WB; B) analysis, and activity of the angiotensin
converting enzyme (ACE; B) in male rats treated with candesartan.
Blockage of AT1 receptors induced a significant increase in the
expression of AT2 receptor and significant decrease in the
expression of NADPH oxidase subunit p47phoxin comparison with
the corresponding untreated male rats; the activity of the
angiotensin converting enzyme (ACE) was not significantly lower
than in untreated rats. Protein expression was obtained relative to
the GAPDH band value and the expression of each gene was
obtained relative to the housekeeping transcripts (b-Actin). The
results were then normalized to the values for control male rats
(100%). Data are means ± SEM; *p < 0.05 (Student’s t test).
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increase the risk of developing PD. In the present study
we observed higher RAS activity in male rats and mice
than in females with stable high levels of E2 (i.e. similar
to proestrus); the observed upregulation of RAS activity
may contribute to increased DA cell vulnerability in
males. Male rats showed increased ACE activity,
increased AT1 expression and decreased AT2 expres-
sion, as well as increased NADPH activity and p47phox
Figure 7 Activated microglial cells in the substantia nigra compacta (SNc). (A): density of OX6-positive cells in the SNc of female and male
rats of different experimental groups treated with vehicle or 6-OHDA. The microglial cells were quantified as the number cells per mm3, and the
data are means ± SEM. *p < 0.05 compared with rats treated with vehicle,#p < 0.05 compared with rats treated with 6-OHDA alone (One-way
ANOVA and Bonferroni post-hoc test). (B-G): photomicrographs showing activated microglial cells at central levels of the substantia nigra in
different experimental groups. Microglial activation was significantly higher in male rats and ovx female rats treated with 6-OHDA alone (D, G).
Scale bar: 100 μm.
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expression. It is known that AII acts via AT1 receptors
to induce inflammatory responses and to release high
levels of ROS mainly by activation of the NADPH com-
plex in vascular degenerative disease and other diseases
mediated by oxidative stress and chronic inflammation
[7,34]. In the nigrostriatal system of animal models of
PD (i.e. rats lesioned with 6-OHDA and mice lesioned
with MPTP), we have previously shown that brain AII
induces activation of the NADPH complex via AT1
receptors, leading to increased neuroinflammation, oxi-
dative stress and DA cell death [23-25].
The increased ACE activity in male rats leads to
increased AII production. The observed upregulation of
AT1 receptors in male rats may also contribute to
NADPH activation and increased DA cell vulnerability.
This is supported by the present experiments, in which
we have observed that the enhanced susceptibility of
DA neurons was significantly decreased by AT1 receptor
inhibition with candesartan or deletion of AT1a recep-
tors. Furthermore, the decrease in DA neuron suscept-
ibility to MPTP observed in AT1a-null mice shows that
the neuroprotective effect is related to the blockage of
AT1 receptors and not to any other possible pharmaco-
logical effect of candesartan. It is particularly interesting
that male rats also showed significantly fewer AT2
receptors than female rats, which may further enhance
DA cell loss. AT1 and AT2 receptors have opposing
effects and AT2 receptors counterbalance the deleter-
ious effect of AT1 receptor stimulation, so that func-
tional interactions between the two receptor subtypes
and their specific distribution determines the AII-
induced effects [35], which in the case of male rats
resulted in a pro-oxidative state as suggested by
increased NADPH activity and p47phoxexpression.
Interestingly, treatment with the AT1 antagonist cande-
sartan induced a significant increase in AT2 receptor
expression in males, which may also contribute to the
decrease in NADPH activity and the neuroprotective
effects of candesartan. An increase in AT2 expression
after treatment with AT1 antagonists has also been
observed in previous studies [36,37]. Furthermore, AT1
receptor blockage may also lead to preferential activa-
tion of the unopposed and upregulated AT2 receptors
by similar levels of AII, as no significant change in ACE
activity was observed.
We have previously shown NADPH expression in
dopaminergic neurons and microglial cells. However, it
appears that the AII-induced increase in microglial
NADPH-oxidase activity plays a major role. It is known
that in non-inflammatory cells, such as neurons, the
NADPH complex produces only low rates of ROS for
signaling function. In inflammatory cells such as micro-
glia, NADPH activation produces high concentrations of
ROS that are released extracellularly to kill invading
microorganisms or cells [8,9]. In accordance with this,
we observed that AII was not able to increase DA neu-
ron death in the absence of microglial cells [24,25]. The
present data showing differences in 6-OHDA-induced
microglial activation in male and female rats confirmed
the involvement of the microglial response in the sexual
dimorphism of 6-OHDA neurotoxicity, and that inhibi-
tion of RAS activity with candesartan inhibits the
enhanced microglial response in male rats.
The results of the present study (i.e. upregulation of
RAS activity in male rats) suggest that RAS plays a
major role in the higher risk in men than in premeno-
pausal women of developing PD. It is known that neu-
roinflammation and microglial activation play a major
role in the progression of PD [32,33,38]. AII via AT1
receptors is one of the most important inflammation
and oxidative stress inducers, and a number of recent
studies suggest that anti-inflammatory actions are at the
core of estrogen-induced protective actions on different
tissues [16,39,40]. Similarly, several studies have also
shown that modulation of the glial neuroinflammatory
response by estrogen is involved in the neuroprotective
effects exerted by this hormone [41,42], and the present
results suggest that the brain RAS is also involved. The
exact mechanism of interaction between E2 and RAS
has not been clarified. It has also been suggested that E2
may inhibit the effects of AII by inhibiting NADPH-
derived ROS production [43], and that E2 may modulate
Rho kinase signaling or other downstream pathways
involved in RAS signaling [44].
In addition to the lack of estrogen, additional factors
may increase RAS activity and the risk of developing PD
in men. Several studies have shown that testosterone
may upregulate RAS activity and therefore amplify gen-
der-related differences [10,19,20]. It has been suggested
that testosterone may modulate downstream pathways
involved in RAS signaling such as Rho kinase [45]. Sex
chromosomal complement may also influence AT2
receptor expression since the AT2 gene is located on
the X chromosome [46]. Thus, it has been reported that
AT2 receptors are absent from the kidneys of adult
male rats [47], or are detected at lower levels than in
females [48]. However, some studies have observed that
sex differences in some RAS components in the kidney
are E2-dependent and sex chromosome-independent
[13]. As commented above, AT2 receptor stimulation
acts in opposition to and in equilibrium with AT1
receptors, and exerts anti-inflammatory and antiproli-
ferative effects in several tissues [35,49]. Furthermore,
additional factors can further increase RAS activity and
DA vulnerability in males. Firstly, aging is a particularly
important factor, since advancing age itself is one of the
most significant risk factors for the development of neu-
rodegenerative diseases such as PD, and we have shown
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that RAS activity and oxidative stress and inflammation
markers are significantly higher in aged male rats than
in young male rats [50]. Secondly, we recently observed
that an extensive DA denervation (i.e. 6-OHDA lesion)
or functional DA depletion induced an increase in RAS
activity and NADPH activity in the substantia nigra
[51]. Similarly, several recent studies have revealed a
counterregulatory interaction between the dopaminergic
and RAS systems in several peripheral tissues, which
plays a major role in degenerative changes in renal and
cardiovascular systems [52-54]. This suggests that in
males, an initial higher susceptibility to DA cell death
and greater loss of DA terminals and DA depletion may
lead to further increase in RAS activity and contribute
to increased progression of PD in males.
Conclusion
The results suggest that brain RAS plays a major role in
the increased risk of developing PD in men, and that
manipulation of the brain RAS may be an efficient
approach for neuroprotective or coadjutant treatment of
PD in men since estrogen-like effects can be obtained
without the feminizing effects of estrogen.
Methods
Experimental design
Young adult female and male Sprague-Dawley rats (ten
weeks old at the beginning of the experiments; n = 79)
and male C57BL-6 mice weighing 20-25 g (i.e. 7 weeks
old; n = 43) were used. Mice were wild type (Charles
River, France) or homozygous mice deficient for AT1a
(the major mouse AT1 isoform and the closest murine
homolog to the single human AT1; [55]; Jackson
Laboratory, Bar Harbor, ME, USA). All experiments
were carried out in accordance with the “Principles of
laboratory animal care” (NIH publication No. 86-23,
revised 1985) and approved by the corresponding com-
mittee at the University of Santiago de Compostela. The
animals were anesthetized with ketamine/xylazine
anesthesia prior to surgery, and were fed with 2014S
Teklad Rodent Maintenance Diet (Harlan Laboratories)
to minimize the occurrence of natural phytoestrogens.
The animals were divided into 3 groups. Rats or mice in
group A were females, which were ovariectomized (ovx)
and given empty implants (see below; n = 4 rats and 6
mice). Rats or mice in group B were females, which
were ovariectomized and given implants containing 17b-
estradiol (ovx + E2; see below; n = 27 rats and 12 mice).
Group C were male rats or mice (wild type or AT1a-
null mice) given empty implants (n = 48 rats and 25
mice).
In the first series of experiments female or male rats
(n = 35; 15 females and 20 males) and female or male
mice (wild type and AT1a-null mice; n = 43; 15 AT1a-
null males, 10 WT males, and 18 WT females) were
used to determine the effect of the presence of E2 or
the AT1 receptor antagonist candesartan or AT1 dele-
tion (i.e. inhibition of RAS activity by AT1 blockage) on
the DA degeneration induced by the neurotoxin 6-
OHDA in rats and MPTP in mice in comparison with
the corresponding controls injected with vehicle. A sec-
ond series of experiments was carried out to investigate
the levels of RAS components and markers of NADPH-
oxidase activity in male rats and female rats with estro-
gen (ovx+E2; n = 44; 28 males and 16 females; 2 nigral
areas per rat). Rats and mice in the first series of experi-
ments were injected intrastriatally with 6-OHDA (rats)
or intraperitoneally with MPTP (mice) or vehicle (con-
trols), then killed for immunohistochemical studies (i.e.,
quantification of dopaminergic cell death and activated
microglia), as described below. Rats in the second series
of experiments were killed by decapitation three weeks
after ovariectomy and/or treatment with implants. The
brains were rapidly removed and coronal slices of the
mesencephalon were cut with a tissue chopper set to 1
mm. To obtain substantia nigra compacta (SNc), the
individual 1 mm tissue sections were dissected on a pre-
cooled glass plate under a stereoscopic microscope
(Leica M220). Both SNc was dissected according to Pax-
inos and Watson [56], frozen on dry ice, and stored
separately (two SNc per rat) at -80°C until processed.
75% of the nigras were used for expression of AT1 and
AT2 receptors and expression of the NADPH-oxidase
cytosolic subunit p47phoxby Western Blot (WB) and
RT-PCR studies, or Angiotensin converting enzyme
(ACE) activity; 25% of the nigras were processed for
NADPH oxidase activity by lucigenin-enhanced chemi-
luminescence (see below).
Estrogen and Candesartan administration
Female rats or mice (groups A and B) were bilaterally
ovariectomized through a dorsal incision and received
Silastic implants placed subcutaneously in the midscapu-
lar region [57,58]. Rats received a single silastic implant
prepared with Silastic®tubing (1.47 mm ID × 1.95 mm
OD, Dow Corning 508-006; VWR Scientific, Bridgeport,
NJ), as described by Febo et al. [58]. Briefly, 5-mm-long
sections of tubing were sealed at one end with Silastic
silicone sealant (Dow Corning 732; VWR) and left to
dry for 30 min. The implants were then either filled
with crystalline 17-b- estradiol (17-b- estradiol benzoate;
Sigma-Aldrich; group B) or were left empty (groups A
and C); the open end was then sealed in the same way
as the other end. Implants were air-dried and incubated
in sterile saline for at least 12-16 h to allow the initial
surge of high estradiol levels to be released before use.
It has been observed that such implants achieve stable
levels of plasma estradiol over 30 d, with a release rate
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of 75-100 pg/ml per 24 h [58], as confirmed in our pre-
vious studies [59]. However, stable levels of E2 have also
been found to persist for only 7-24 days [60]. Therefore,
rats were killed 3 weeks after implantation (i.e., 2 weeks
after 6-OHDA injection, see below). Mice received
implants comprising a single 5-mm-long silastic tube
prepared as described above and filled with 17- b- estra-
diol:cholesterol (1:1) or empty silastic implants (con-
trols). This treatment provides plasma levels of 17- b-
estradiol of 87 ± 9 pg/ml. (i.e., similar to proestrus in
normal mice) [61].
In addition, some male rats (n = 16) received cande-
sartan in their drinking water (Astra-Zeneca; 3 mg/kg/
day) from 7 days before the empty implants were fitted
until they were killed for immunohistochemistry or
determination of levels of different RAS components. It
has been reported that candesartan is the most effective
AT1 antagonist in crossing the blood-brain barrier, and
that low doses have little effect on blood pressure and
block brain AII effects [62].
Intrastriatal injection of 6-OHDA and intraperitoneal
injection of MPTP
One week after receiving empty or E2 implants, some
rats in the different groups (n = 35) were injected
intrastriatally with 6-OHDA or vehicle. Thirty minutes
prior to intrastriatal injection with 6-OHDA or vehicle,
rats were treated with the selective inhibitor for the nor-
epinephrine transporter desipramine (Sigma, 25 mg/kg i.
p.) to prevent uptake of 6-OHDA by noradrenergic
terminals. The rats were injected in the right striatum
with 7 μg of 6-OHDA (in 3 μl of saline containing 0.2%
ascorbic acid; Sigma, USA). Stereotaxic coordinates were
1 mm anterior to bregma, 3.0 mm right of midline, and
5.5 mm ventral to the dura; tooth bar at -3.3. Control
animals were injected with 3 μl of sterile saline alone.
Rats were killed by chloral hydrate overdose 2 weeks
post-lesion (i.e. 3 weeks post-implant). Previous studies
on the time course of 6-OHDA lesions have shown that
the loss of TH-immunoreactive (TH-ir) neurons is com-
plete [63] or practically complete [64] 2 weeks after
administration of intrastriatal injections. Although a few
DA neurons may degenerate after the two-week period,
we considered it more important to kill the rats before
any possible loss of E2 levels (i.e. 3 weeks after implan-
tation and 2 weeks after 6-OHDA injection).
One week after receiving empty or E2 implants, some
mice in the different groups (n = 43) were injected with
MPTP (Free base, Sigma; 30 mg/kg/day in saline, intra-
peritoneally; for 5 days; n = 24) or intraperitoneal vehi-
cle (n = 19). The mice were killed by chloral hydrate
overdose one week after treatment with MPTP or vehi-
cle (i.e. when the DA lesion is complete or practically
complete) and then processed for histology.
RNA extraction and real-time quantitative RT-PCR
Total RNA from the nigral region was extracted with
Trizol (Invitrogen), according to the manufacturer’s
instructions. Total RNA (2.5 μg) was reverse-transcribed
to cDNA with dNTPs, random primers, and Moloney
murine leukemia virus reverse transcriptase (M-MLV;
200U; Invitrogen). Real-time PCR was used to examine
relative levels of angiotensin receptors type 1 (AT1a)
and type 2 (AT2) mRNA. Experiments were performed
with a real-time iCyclerTM PCR platform (BioRad). b-
Actin was used as housekeeping gene and was amplified
in parallel with the genes of interest. The comparative
Ct method was used to examine the relative mRNA
expression. The expression of each gene was obtained as
relative to the housekeeping transcripts. The data were
then normalized to the values of the female group (ovx
+E2) of the same batch (100%) to counteract any possi-
ble variability among batches. Finally, the results were
expressed as mean ± SEM. Primers sequences were as
follows: for AT1a, forward 5’-TTCAACCTCTACGC-
CAGTGTG-3’, reverse 5’-GCCAAGCCAGCCATCAGC-
3’; for AT2, forward 5’-AACATCTGCTGAAGACCAA-
TAG-3’, reverse 5’-AGAAGGTCAGAACATGGAAGG-
3; for p47phox, forward 5’-CCACACCTCTTGAA
CTTCTTC-3’, reverse 5’- CTCGTAGTCAGCGATGGC
-3’; for b-actin, forward 5’-TCGTGCGTGACATTAAA-
GAG-3’, reverse 5’-TGCCACAGGATTCCATACC-3’.
Western blot analysis (WB) and ACE activity
For WB, tissue was homogenized in RIPA buffer con-
taining protease inhibitor cocktail (P8340, Sigma) and
PMSF (P7626, Sigma). The homogenates were centri-
fuged and protein concentrations were determined with
the Bradford protein assay. Equal amounts of protein
were separated by 5-10% Bis-Tris polyacrylamide gel,
and transferred to nitrocellulose membrane. The mem-
branes were incubated overnight with primary antibo-
dies (1:200) against AT1 receptor (sc-31181), AT2
receptor (sc-9040), and p47phox(sc-7660 all from Santa
Cruz Biotechnology. The HRP conjugated secondary
antibodies used were Protein A (NA9120V, GE Health-
care) and Protein G (18-161, Upstate-Millipore). Immu-
noreactivity was detected with an Immun-Star HRP
Chemiluminescent Kit (170-5044, BioRad) and imaged
with a chemiluminescence detection system (Molecular
Imager ChemiDoc XRS System, BioRad). Blots were
stripped and reprobed for anti-GAPDH (G9545, Sigma;
1:25000) as loading control. In each animal, protein
expression was measured by densitometry of the corre-
sponding band and expressed as relative to the GAPDH
band value. The data were then normalized to the values
of the female-group of the same batch (100%) to coun-
teract any possible variability among batches. Finally,
the results were expressed as means ± SEM.
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ACE and NADPH oxidase activity
ACE activity in ventral mesencephalic tissue was assayed
with hippuryl-L-histidyl-L-leucine (Hip-His-Leu; Sigma)
as substrate, as described by Hemming et al. [65]. Fluor-
escence was assayed in 96-well plates in an Infinite
M200 multiwell plate reader (TECAN; excitation, 355;
emission, 535) and determined as nmoles of his-leu pro-
duced per milligram of protein per minute. The data
were then normalized to the values of the female-group
of the same batch (i.e., expressed as a percentage of the
female values; 100%). NADPH oxidase activity in ventral
mesencephalic tissue was measured by lucigenin-
enhanced chemiluminescence with an Infinite M200
multiwell plate reader (TECAN), as described by
Griendling et al. [66] and Hong et al. [67], respectively.
Chemiluminescence was expressed as relative light units
(RLU/min/mg protein).
Immunohistochemistry. Dopaminergic neuron and
microglia quantification
The animals used for immunohistochemistry (i.e. those
injected 6-OHDA or MPTP or vehicle) were first per-
fused with 0.9% saline and then with cold 4% parafor-
maldehyde in 0.1 M phosphate buffer, pH 7.4. The
brains were removed and subsequently washed and
cryoprotected in the same buffer containing 20%
sucrose, and finally cut into 40 μm sections on a freez-
ing microtome. The sections were incubated for 1 h in
10% normal swine serum with 0.25% Triton X-100 in 20
mM potassium phosphate-buffered saline containing 1%
bovine serum albumin (KPBS-BSA) and then incubated
overnight at 4°C with antibodies anti-tyrosine hydroxy-
lase (TH) as DA marker (mouse monoclonal anti-TH
for rat sections, Sigma, 1:10 000; rabbit polyclonal anti-
bodies to TH for mouse sections, Peel-Freez, 1:500), or
anti-OX6 (a mouse monoclonal antibody directed
against a monomorphic determinant of the rat major
histocompatibility complex class II antigens, expressed
by activated microglia but not by resting cells; 1:200;
Serotec) as a marker of reactive microglia/macrophages.
The sections were subsequently incubated, first for 60
min with the corresponding biotinylated secondary anti-
body, and then for 90 min with avidin-biotin-peroxidase
complex (ABC, 1:100, Vector). Finally the labeling was
revealed by treatment with 0.04% hydrogen peroxide
and 0.05% 3-3’diaminobenzidine (DAB, Sigma). In all
experiments the control sections, in which the primary
antibody was omitted, were immunonegative for these
markers.
The total number of TH-immunoreactive (TH-ir)
neurons in the substantia nigra compacta was estimated
by an unbiased stereological method (the optical fractio-
nator). The stereological analysis was carried out with
the Olympus CAST-Grid system (Computer Assisted
Stereological Toolbox; Olympus, Denmark). Uniform
randomly chosen sections through the substantia nigra
(every fourth section) were analyzed for the total num-
ber of TH-ir cells by means of a stereological grid (frac-
tionator), and the nigral volume was estimated
according to Cavalieri’s method [68]. To confirm that
6-OHDA induces cell death, series of sections through
the entire substantia nigra of control rats and rats trea-
ted with 6-OHDA were counterstained with Cresyl vio-
let, and the total number of neurons in the substantia
nigra was estimated by the unbiased stereology method
described above for TH-ir cells. Neurons were distin-
guished from glial cells on a morphological basis, and
neurons with visible nuclei were counted as above for
TH-ir neurons. The number of OX6-ir cells (i.e. reactive
microglia) was estimated using the Olympus CAST-Grid
system and the unbiased stereological method described
above for counting TH-ir neurons. At least four sec-
tions through the central SNc of each animal were mea-
sured. The density of OX6-ir cells (cells/mm3) was
determined by dividing the number of labeled cells by
the volume that they occupied (see references 23 and
24 for details).
Statistical analysis
All data were obtained from at least three independent
experiments and were expressed as means ± SEM. Two-
group comparisons were analyzed by a Student’s t test
and multiple comparisons were analyzed by one-way
ANOVA followed by a post-hoc Bonferroni test. The
normality of populations and homogeneity of variances
were tested before each ANOVA. Differences were con-
sidered significant at p < 0.05. Statistical analyses were
carried out with SigmaStat 3.0 from Jandel Scientific
(San Rafael, CA, USA).
List of abbreviations
(6-OHDA): 6-hydroxydopamine; (ACE): Angiotensin converting enzyme; (AII):
Angiotensin II; (AT1): Angiotensin type 1 receptors; (AT1-/-): AT1a-null mice;
(AT2) Angiotensin receptors type 2; (DA): Dopaminergic; (E2): Estrogen;
(MPTP): 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; (ovx): Ovariectomized;
(PD): Parkinson’s disease; (RAS): Renin angiotensin system; (ROS): Reactive
oxygen species; (TH-ir): Tyrosine hydroxylase immunoreactive; (WB): Western
Blot; (WT): wild type mice.
Acknowledgements
The authors thank Pilar Aldrey, Iria Novoa and Jose A. Trillo for their
excellent technical assistance. The authors are thankful to Astra Zeneka for
providing candesartan for the experiments. Funding: Spanish Ministry of
Science and Innovation, Spanish Ministry of Health (RD06/0010/0013 and
CIBERNED), Galician Government (XUGA) and FEDER.
Authors’ contributions
AIR-P conducted the experiments with rats and performed quantitative PCR
and statistical analysis; RV performed Western Blot and enzyme activity
analysis; BJ performed the experiments with mice; PG-R performed the
histological analysis, MJG and JL-G conceived the study and its design,
supervised the project and edited the manuscript preparation. All authors
have read and approved the manuscript.
Rodriguez-Perez et al. Molecular Neurodegeneration 2011, 6:58
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Authors’ information
Laboratory of Neuroanatomy and Experimental Neurology, Dept. of
Morphological Sciences, Faculty of Medicine, University of Santiago de
Compostela. Networking Research Center on Neurodegenerative Diseases
(CIBERNED, Instituto de Salud Carlos III), IDIS (Instituto de Investigaciones
sanitarias de Santiago). Santiago de Compostela, Spain.
Competing interests
The authors declare that they have no competing interests.
Received: 7 April 2011 Accepted: 16 August 2011
Published: 16 August 2011
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doi:10.1186/1750-1326-6-58
Cite this article as: Rodriguez-Perez et al.: Renin angiotensin system and
gender differences in dopaminergic degeneration. Molecular
Neurodegeneration 2011 6:58.
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