Dopaminergic neuroprotection of hormonal replacement - Brain

BRAINA JOURNAL OF NEUROLOGY

Dopaminergic neuroprotection of hormonalreplacement therapy in young and agedmenopausal rats: role of the brainangiotensin systemAna I. Rodriguez-Perez,1,2 Rita Valenzuela,1,2 Begona Villar-Cheda,1,2 Maria J. Guerra1,2 andJose L. Labandeira-Garcia1,2

1 Laboratory of Neuroanatomy and Experimental Neurology, Department of Morphological Sciences, Faculty of Medicine, University of Santiago de

Compostela, 15782 Santiago de Compostela, Spain

2 Networking Research Centre on Neurodegenerative Diseases (CIBERNED), 28031, Spain

Correspondence to: Jose L. Labandeira-Garcia MD, PhD,

Department of Morphological Sciences,

Faculty of Medicine,

University of Santiago de Compostela,

15782 Santiago de Compostela, Spain

E-mail: [email protected]

There is a lack of consensus about the effects of the type of menopause (surgical or natural) and of oestrogen replacement

therapy on Parkinson’s disease. The effects of the timing of replacement therapy and the female’s age may explain the observed

differences in such effects. However, the mechanisms involved are poorly understood. The renin-angiotensin system mediates

the beneficial effects of oestrogen in several tissues, and we have previously shown that dopaminergic cell loss is enhanced by

angiotensin via type 1 receptors, which is activated by ageing. In rats, we compared the effects of oestrogen replacement

therapy on 6-hydroxydopamine-induced dopaminergic degeneration, nigral renin-angiotensin system activity, activation of the

nicotinamide adenine dinucleotide phosphate oxidase complex and levels of the proinflammatory cytokine interleukin-1b in

young (surgical) menopausal rats and aged menopausal rats. In young surgically menopausal rats, the renin-angiotensin system

activity was higher (i.e. higher angiotensin converting enzyme activity, higher angiotensin type-1 receptor expression and lower

angiotensin type-2 receptor expression) than in surgically menopausal rats treated with oestrogen; the nicotinamide adenine

dinucleotide phosphate oxidase activity and interleukin-1b expression were also higher in the first group than in the second

group. In aged menopausal rats, the levels of nigral renin-angiotensin and nicotinamide adenine dinucleotide phosphate oxidase

activity were similar to those observed in surgically menopausal rats. However, oestrogen replacement therapy significantly

reduced 6-hydroxydopamine-induced dopaminergic cell loss in young menopausal rats but not in aged rats. Treatment with

oestrogen also led to a more marked reduction in nigral renin-angiotensin and nicotinamide adenine dinucleotide phosphate

oxidase activity in young surgically menopausal rats (treated either immediately or after a period of hypo-oestrogenicity) than in

aged menopausal rats. Interestingly, treatment with the angiotensin type-1 receptor antagonist candesartan led to remarkable

reduction in renin-angiotensin system activity and dopaminergic neuron loss in both groups of menopausal rats. This suggests

that manipulation of the brain renin-angiotensin system may be an efficient approach for the prevention or treatment of

Parkinson’s disease in oestrogen-deficient females, together with or instead of oestrogen replacement therapy.

Keywords: ageing; angiotensin; oestrogen; menopause; Parkinson’s disease

doi:10.1093/brain/awr320 Brain 2012: 135; 124–138 | 124

Received March 25, 2011. Revised September 7, 2011. Accepted October 3, 2011. Advance Access publication December 20, 2011

� The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

For Permissions, please email: [email protected]

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IntroductionSex steroids have been shown to have a neuroprotective role in

several models of neurological diseases, although the effects of the

loss of ovarian function and hormonal replacement therapy in

humans are controversial. Numerous observational studies have

supported the concept that oestrogen therapy in postmenopausal

females protects against ageing-related diseases, including cardio-

vascular diseases, stroke and neurodegeneration. This was not

confirmed in several randomized controlled trials that reported

no or even detrimental effects (Rossouw et al., 2002;

Chlebowski et al., 2010). Several possible explanations have

been proposed for the discrepant results (Clarkson and

Mehaffey, 2009). However, the age of the females receiving the

treatment appears to be a major factor. The vast majority of fe-

males participating in these trials were on average 565 years, and

had started oestrogen therapy 12 years after undergoing meno-

pause (Turgeon et al., 2004, 2006); on the contrary, in observa-

tional studies that reported beneficial effects, most females had

initiated oestrogen therapy in their perimenopausal period

(Harman et al., 2005; Miller et al., 2005, 2009). The mechanisms

underlying the differences in the effects of oestrogen therapy be-

tween young perimenopausal females (including early surgical

menopause) and senescent females are still poorly understood

(Sohrabji and Bake, 2006; Suzuki et al., 2007).

Experiments with animal models (Dluzen, 1997; Murray et al.,

2003), epidemiological and clinical evidence (Shulman, 2002;

Ragonese et al., 2006a, b; Liu and Dluzen, 2007) suggest that

sex hormones have a beneficial influence on the risk, onset and

severity of Parkinson’s disease, and that oestrogen modulates

dopaminergic function. The anti-inflammatory effects of oestrogen

appear to play a major role in the neuroprotective effects on

Parkinson’s disease (Suzuki et al., 2007; Vegeto et al., 2008).

However, conflicting findings have been reported from some clin-

ical trials and there is still a lack of consensus about the effects of

the type of menopause (i.e. surgical versus natural) and of the

oestrogen therapy on Parkinson’s disease (Ragonese et al.,

2006a, b; Miller et al., 2009). As already mentioned for other

diseases, the effects of the timing of postmenopausal oestrogen

therapy and the age of the females receiving the treatment may

be a major factor.

The renin-angiotensin system mediates the beneficial effects of

oestrogen in several tissues (Nickenig et al., 1998; Dean et al.,

2004; Chen et al., 2008), and interactions between oestrogen and

angiotensin receptors have also been observed (Liu et al., 2002;

Tasuda et al., 2005; Xue et al., 2007; Hoshi-Fukushima et al.,

2008). In addition, the local renin-angiotensin system has been

reported to be involved in age-related degenerative changes in

several tissues (Heymes et al., 1998; Basso et al., 2005; Benigni

et al., 2009). The peptide angiotensin, via type 1 (AT1) receptors,

is one of the most important known inducers of inflammation and

oxidative stress, and produces reactive oxygen species by activa-

tion of the NADPH oxidase complex (Seshiah et al., 2002; Touyz

et al., 2002; Cai et al., 2003), which is the most important intra-

cellular source of reactive oxygen species apart from mitochondria

(Babior, 1999, 2004). The brain possesses a local renin-angiotensin

system (Mckinley et al., 2003; Savedra et al., 2005), and we have

previously shown that the dopaminergic cell loss is enhanced by

angiotensin via AT1 receptors (Rey et al., 2007; Rodriguez-Pallares

et al., 2008; Joglar et al., 2009). In addition, we have observed

that oestrogen decreases and ageing increases the effect of the

nigral renin-angiotensin system on enhancing dopaminergic

neuron degeneration in young female rats and aged male rats,

respectively (Rodriguez-Perez et al., 2010; Villar-Cheda et al.,

2010b). It is therefore possible that ageing modifies the beneficial

effects of oestrogen therapy in postmenopausal females, and that

the nigral renin-angiotensin system plays a major role. In the pre-

sent study, we compared the effects of oestrogen therapy on

dopaminergic degeneration, nigral renin-angiotensin system activ-

ity, activation of the NADPH oxidase complex and levels of the

pro-inflammatory cytokine interleukin-1b (IL-1b) in rat models of

untreated and treated (either immediate or delayed oestrogen

therapy) surgical (young ovariectomized rats) and natural (aged

menopausal rats) menopause, and investigated if renin-angiotensin

system manipulation may improve or replace the effects of oes-

trogen therapy.

Materials and methods

Experimental designYoung adult and aged acyclic female Sprague-Dawley rats

(10 weeks and 24 months old at the beginning of the experiments,

respectively) were used. All experiments were carried out in ac-

cordance with the ‘Principles of laboratory animal care’ (NIH

publication No. 86-23, revised 1985) and approved by the corres-

ponding committee at the University of Santiago de Compostela.

All surgery was performed under ketamine/xylazine anaesthesia,

and rats were fed with 2019S Teklad Rodent Maintenance Diet

(Harlan Laboratories) to minimize the occurrence of natural phyto-

estrogens. Five rat models corresponding to five major possibilities

in clinical menopause were studied: untreated surgical menopause

(young ovariectomized rats), surgical menopause immediately

treated with oestrogen therapy, surgical menopause treated with

oestrogen therapy after a period of hypo-oestrogenicity, untreated

natural menopause (untreated aged rats), and natural menopause

treated with oestrogen therapy. In addition, a group of aged rats

were ovariectomized and treated with oestrogen to study the po-

tential effects of the presence of ovaries on renin-angiotensin

system activity in aged rats.

The rats were divided into six groups. Rats in Group A were

young rats (10-weeks-old at the beginning of the experiments),

which were ovariectomized (i.e. menopause was induced surgi-

cally) and given empty implants (n = 38). Rats in Group B were

young rats that were ovariectomized and simultaneously received

implants containing 17b-oestradiol (n = 31). Rats in Group C were

treated as Group B rats but the oestrogen implants were given

3 weeks after ovariectomy. Rats in Group D were aged rats

(24 months old; aged; n = 31) given empty implants (i.e. natural

senescent menopause). Rats in Group E were aged rats given im-

plants containing 17b-oestradiol (n = 25). Rats in Group F were

aged rats that were ovariectomized and given implants containing

Menopause, angiotensin and dopaminergic degeneration Brain 2012: 135; 124–138 | 125

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17b-oestradiol (n = 5). Two series of experiments were carried out

with the different groups of rats (Groups A–F). In the first series of

experiments, young ovariectomized or aged menopausal rats

(n = 55) were used to determine the effect of oestrogen and can-

desartan (i.e. inhibition of renin-angiotensin system activity by

this AT1 receptor antagonist) or treatment with oestro-

gen + candesartan on the dopaminergic degeneration induced by

the neurotoxin 6-hydroxydopamine, relative to rats in the different

groups treated with 6-hydroxydopamine alone and compared with

the corresponding controls injected with vehicle. A second series

of experiments was carried out to investigate the effect of treat-

ment with oestrogen or candesartan on markers of NADPH oxi-

dase activity/oxidative stress, inflammation and renin-angiotensin

system activity in young ovariectomized and aged menopausal rats

(n = 87). Rats in the first series of experiments were injected

intrastriatally with 6-hydroxydopamine or vehicle (controls) and

treated with oestrogen and/or candesartan or vehicle (controls),

then killed for immunohistochemical studies (i.e. quantification of

dopaminergic cell death), as described below. Rats in the second

series of experiments were killed by decapitation 3 weeks after

ovariectomy and/or treatment with implants. The brains were rap-

idly removed and coronal slices of the mesencephalon were cut

with a tissue chopper set to 1 mm. To obtain substantia nigra

compacta, the individual 1-mm tissue slides were dissected on a

pre-cooled glass plate under a stereoscopic microscope (Leica

M220). The substantia nigra compacta was dissected according

to Paxinos and Watson (1985), frozen on dry ice, and stored at

�80�C until processed for investigation of expression of AT1 and

AT2 receptors, expression of the NADPH oxidase cytosolic subunit

p47phox and expression pro-inflammatory cytokine IL-1b by west-

ern blot and reverse transcriptase–polymerase chain reaction

studies.

Expression of the NADPH oxidase cytosolic subunit p47phox is

an indicator of the level of activation of the NADPH oxidase com-

plex. The NADPH oxidase complex is composed of membrane-

bound and cytosolic subunits such as p47phox, which is considered

key for NADPH oxidase activation (Li and Shah, 2003).

Translocation of cytosolic subunits to the membrane, which

leads to reactive oxygen species generation, is a necessary step

for NADPH oxidase activation. The level of the NADPH oxidase

subunit p47phox is correlated with NADPH oxidase activity and

NADPH-derived superoxide formation (Rueckschloss et al., 2002;

Touyz et al., 2002). Finally, angiotensin converting enzyme activ-

ity was determined by lucigenin-enhanced chemiluminescence (see

below).

Oestrogen and candesartanadministrationRats were bilaterally ovariectomized through a dorsal incision and

simultaneously (Groups A, B and F) or 3 weeks later (Group C)

received Silastic implants, placed subcutaneously in the midscapu-

lar region (Dziuk and Cook, 1966; Febo et al., 2005). Aged acyclic

rats (Groups D and E) received similar implants without prior ovari-

ectomy. Silastic implants were prepared with Silastic� tubing

(1.47 mm inner diameter � 1.95 mm outer diameter, Dow

Corning 508-006; VWR Scientific), as described by Febo et al.

(2005). 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 crys-

talline 17-b-oestradiol (17-b-oestradiol benzoate; Sigma-Aldrich;

Groups B, C, E and F) or were left empty (Groups A and D);

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 oestradiol levels to

be released before use. It has been observed that such implants

achieve stable levels of plasma oestradiol over 30 days, with a

release rate of 75–100 pg/ml per 24 h (Febo et al., 2005), as con-

firmed in our previous studies (Rodriguez-Perez et al., 2010).

However, stable levels of oestrogen have also been found to per-

sist for only 7–24 days (Mannino et al., 2005). Therefore, rats

were killed 3 weeks after oestrogen implantation (i.e. 2 weeks

after 6-hydroxydopamine injection).

Some rats in the different groups received candesartan in

their drinking water (candesartan cilexetil, AstraZeneca; 3 mg/kg/

day) from 7 days before the empty or oestrogen implants

were fitted until they were killed. The oral bioavailability of

candesartan cilexetil is 5–10% oral (Kondo et al., 1996; Zorad

et al., 2006); the drug was diluted (0.025 mg/ml) following

the protocol suggested by AstraZeneca and administered to the

rats in their drinking water (25–30 ml/day/rat). It has been re-

ported 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 angiotensin effects

(Unger, 2003).

Intrastriatal injection of6-hydroxydopamineOne week after receiving empty or oestrogen implants, some rats

in the different groups were injected intrastriatally with

6-hydroxydopamine or vehicle. Thirty minutes prior to intrastriatal

injection with 6-hydroxydopamine or vehicle, rats were treated

with the selective inhibitor for the norepinephrine transporter

desipramine (Sigma, 25 mg/kg i.p.) to prevent uptake of

6-hydroxydopamine by noradrenergic terminals. The rats were in-

jected in the right striatum with 7 mg of 6-hydroxydopamine (in

3 ml of saline containing 0.2% ascorbic acid; Sigma). Stereotaxic

coordinates were 0.8 mm anterior to bregma, 3.0 mm right of

midline and 5.0 mm ventral to the dura; tooth bar at �3.3.

Control animals were injected with 3ml of vehicle alone. Rats

were killed 2-week post-lesion (i.e. 3 weeks post-implant).

Previous studies on the time course of 6-hydroxydopamine lesions

have shown that the loss of tyrosine hydroxylase immunoreactive

neurons is complete (Przedborski et al., 1995) or practically com-

plete (Sauer and Oertel, 1994) 2 weeks after administration of

intrastriatal injections. Although a few dopaminergic neurons

may degenerate after the 2-week period, we considered it more

important to kill the rats before any possible loss of oestrogen

levels (i.e. 3 weeks after implantation and 2 weeks after

6-hydroxydopamine injection).

126 | Brain 2012: 135; 124–138 A. I. Rodriguez-Perez et al.

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RadioimmunoassayIn order to confirm the efficiency of ovariectomies and implants,

blood samples were taken from the rats by cardiac puncture just

before the animals were killed. Blood was collected on ice and

serum samples immediately frozen at �70� until analysis for

17b-oestradiol by radioimmunoassay, with the Diagnostic

Products Corporation Coat-a-Count kit and protocol. The samples

were counted for 1 min in a gamma counter, and assayed in

triplicate.

RNA extraction and real-timequantitative reversetranscriptase–polymerase chain reactionTotal RNA from the nigral region was extracted with TRIzol�

(Invitrogen), according to the manufacturer’s instructions. Total

RNA (2.5 mg) was reverse-transcribed to complementary DNA

with deoxynucleotide triphosphates, random primers and

Moloney murine leukaemia virus reverse transcriptase (M-MLV;

200 U; Invitrogen). Real-time polymerase chain reaction was

used to examine relative levels of angiotensin receptors type 1

(AT1a) and type 2 (AT2) messenger RNA, p47 and IL-1b.

Experiments were performed with a real-time iCyclerTM polymer-

ase chain reaction platform (Bio-Rad). b-Actin was used as house-

keeping gene and was amplified in parallel with the genes of

interest. The comparative Ct method was used to examine the

relative messenger RNA expression. The expression of each gene

was obtained as relative to the housekeeping transcripts. The data

were then normalized to the values of the control group of the

same batch (i.e. expressed as a percentage of the control; 100%)

to counteract any possible variability among batches. Two house-

keeping genes [b-actin and GAPDH (glyceraldehyde 3-phosphate

dehydrogenase)] were simultaneously included in some experi-

ments to exclude any potential effect of oestrogen on the house-

keeping gene: no significant differences were detected. Finally, the

results were expressed as mean � SEM. Primers sequences were as

follows: for AT1a, forward 50-TTCAACCTCTACGCCAGTGTG-30,

reverse 50-GCCAAGCCAGCCATCAGC-30; for AT2, forward

50-AACATCTGCTGAAGACCAATAG-30, reverse 50-AGAAGGTC

AGAACATGGAAGG-30; for p47phox, forward 50-CCACAC

CTCTTGAACTTCTTC-30, reverse 50-CTCGTAGTCAGCGATGGC-30;

for b-actin, forward 50-TCGTGCGTGACATTAAAGAG-30, reverse

50-TGCCACAGGATTCCATACC-30; for and IL-1b forward 50-

ATCTCACAGCAGCATCTC, reverse 50-TAGCAGGTCGTCATCATC.

Western blot analysis and angiotensinconverting enzyme activityFor western blot analysis, tissue was homogenized in radioimmu-

noprecipitation buffer containing protease inhibitor cocktail

(P8340, Sigma) and phenylmethylsulphonyl fluoride (P7626,

Sigma). The homogenates were centrifuged and protein concen-

trations were determined with the Bradford protein assay. Equal

amounts of protein were separated by 5–10% bis–Tris polyacryl-

amide gel, and transferred to nitrocellulose membrane. The

membranes were incubated overnight with primary antibodies

(1:200) against AT1 receptor (sc-31181), AT2 receptor

(sc-9040), p47-phox (sc-7660) and IL-1b (sc-1252), all from

Santa Cruz Biotechnology. The horseradish peroxidase conjugated

secondary antibodies used were Protein A (NA9120V, GE

Healthcare) and Protein G (18-161, Upstate-Millipore).

Immunoreactivity was detected with an Immun-StarTM HRP

Chemiluminescent Kit (170-5044, Bio-Rad) and imaged with a

chemiluminescence detection system (Molecular Imager

ChemiDoc XRS System, Bio-Rad). Blots were stripped and

re-probed for anti-GAPDH (G9545, Sigma; 1:25 000) as loading

control. In each animal, protein expression was measured by

densitometry of the corresponding band and expressed as relative

to the GAPDH band value. The data were then normalized to the

values of the control group of the same batch (i.e. expressed as a

percentage of the control values; 100%) to counteract any pos-

sible variability among batches. GAPDH and b-actin were simul-

taneously included as loading control in some experiments to

exclude any potential effects of oestrogen on the internal control:

no significant differences were detected. Finally, the results were

expressed as means � SEM.

Angiotensin converting enzyme 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. (2007).

Fluorescence was assayed in 96-well plates in an Infinite M200

multi-well plate reader (TECAN; excitation, 355; emission, 535)

and determined as nmol his-leu/mg protein/min. The data

were then normalized to the values of the control group of

the same batch (i.e. expressed as a percentage of the control

values; 100%)

Immunohistochemistry anddopaminergic neuron quantificationThe animals used for immunohistochemistry (i.e. those injected

intrastriatally with 6-hydroxydopamine or vehicle) were first per-

fused with 0.9% saline and then with cold 4% paraformaldehyde

in 0.1 M phosphate buffer, pH 7.4. The brains were removed and

subsequently washed and cryoprotected in the same buffer con-

taining 20% sucrose, and finally cut into 40-mm sections on a

freezing microtome. The sections were incubated for 1 h in 10%

normal swine serum with 0.25% Triton X-100 in 20 mM potas-

sium phosphate-buffered saline containing 1% bovine serum al-

bumin and then incubated overnight at 4�C with a mouse

monoclonal anti-tyrosine hydroxylase (Sigma; 1:10 000), as dopa-

minergic marker. 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 labelling was revealed by treat-

ment with 0.04% hydrogen peroxide and 0.05% 3-

30diaminobenzidine (DAB, Sigma). In all experiments the control

sections, in which the primary antibody was omitted, were immu-

nonegative for these markers.

The total number of tyrosine hydroxylase immunoreactive

neurons in the substantia nigra compacta was estimated by

an unbiased stereological method (the optical fractionator).


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The stereological analysis was carried out with the Olympus

CAST-Grid system (Computer Assisted Stereological Toolbox;

Olympus). Uniform randomly chosen sections through the sub-

stantia nigra (every fourth section) were analysed for the total

number of tyrosine hydroxylase immunoreactive cells by means

of a stereological grid (fractionator), and the nigral volume was

estimated according to Cavalieri’s method (Gundersen et al.,

1988). To confirm that 6-hydroxydopamine induces cell death,

series of sections through the entire substantia nigra of control

rats and rats treated with 6-hydroxydopamine were counter-

stained with Cresyl violet, and the total number of neurons in

the substantia nigra was estimated by the unbiased stereology

method described above for tyrosine hydroxylase immunoreactive

cells. Neurons were distinguished from glial cells on a morpho-

logical basis, and neurons with visible nuclei were counted as

above for tyrosine hydroxylase immunoreactive neurons (see Rey

et al., 2007 for details).

Statistical analysisAll data were obtained in triplicate (i.e. from each rat at least three

samples were independently processed and the mean value for

each rat was used for statistical analysis). Data were expressed

as means � SEM. Two group comparisons were analysed by a

Student’s t-test and multiple comparisons were analysed 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 considered signifi-

cant at P50.05. Statistical analyses were carried out with

SigmaStat 3.0 from Jandel Scientific.

Results

Effect of the dopaminergic neurotoxin6-hydroxydopamine on different groupsof menopausal ratsIntrastriatal injection of 6-hydroxydopamine induced a marked loss

of dopaminergic neurons in the substantia nigra of aged rats and

young ovariectomized rats, which was significantly higher than

that induced by 6-hydroxydopamine in young ovariectomized

rats immediately treated with oestrogen, or young ovariectomized

rats treated with oestrogen 3 weeks after ovariectomy. The loss of

dopaminergic neurons induced by the neurotoxin in aged rats

treated with oestrogen tended to be lower than in untreated

aged rats, but the difference was not significant. Interestingly,

however, the loss of dopaminergic neurons was significantly

lower in both groups of menopausal rats treated with the

AT1 antagonist candesartan, and treatment with oestro-

gen + candesartan did not increase the neuroprotective effect

induced by treatment with candesartan alone (Fig. 1A–E).

Therefore, the present results indicate that either oestrogen or

candesartan induced significant neuroprotection against

6-hydroxydopamine in young menopausal rats (surgically induced

menopause). However, only candesartan induced significant neu-

roprotection in aged menopausal rats (natural menopause).

Renin-angiotensin system activity inyoung surgically menopausal and agedmenopausal ratsBoth groups of menopausal rats showed very low blood levels of

oestrogen. Blood levels of oestrogen in young ovariectomized rats

were 18.6 � 2.4 pg/ml, and similar to those observed in aged

menopausal rats (26.2 � 4.5 pg/ml). Comparison between both

groups of menopausal rats did not reveal any significant differ-

ences in the expression of AT2 and the NADPH subunit p47phox

and IL-b (Fig. 2A and B). However, differences were observed

between both groups of menopausal rats in AT1 receptor expres-

sion; AT1 messenger RNA expression was higher in aged rats.

Since the present and previous studies have shown that oestrogen

affects AT1 expression at post-transcriptional levels (see below),

the increased messenger RNA expression in aged rats must be

related to ageing-related factors other than low levels of oestro-

gen. Interestingly, AT1 protein expression was higher in young

ovariectomized rats, which suggests that the lack of oestrogen

affects the expression of AT1 receptors to a greater extent in

young ovariectomized rats than in aged rats. Similar levels of

AT2 messenger RNA and protein expression in young ovariecto-

mized and aged rats also suggest that ageing-related factors other

than low levels of oestrogen reduce the expression of AT2 recep-

tors in aged rats, because the lack of oestrogen reduced AT2 re-

ceptor levels in young rats but did not significantly change AT2

receptor levels in aged rats (see below). Angiotensin converting

enzyme activity was 0.0798 � 0.00723 nmol his-leu/mg protein/

min in ovariectomized rats and 0.0890 � 0.0117 in aged rats.

Effect of oestrogen therapy in youngsurgically menopausal and agedmenopausal rats compared with thecorresponding untreated menopausalratsIn another set of experiments, we tested the effects of oestrogen

therapy with respect to the corresponding untreated menopausal

rats. Blood levels of oestrogen in young ovariectomized rats that

received implants with oestrogen were 144.8 � 16.7 pg/ml, which

were similar to values observed in normal rats in proestrous (refer

to ‘Discussion’ section). Plasma oestrogen concentrations in aged

menopausal rats that received implants with oestrogen were not

significantly different from those of young ovariectomized rats

treated with oestrogen (151.1 � 22.4 pg/ml immediately before

sacrifice). However, the effects of oestrogen treatment on

renin-angiotensin system activity were different. In young surgi-

cally ovariectomized rats, treatment with oestrogen at the time of

ovariectomy induced a marked decrease in renin-angiotensin

system activity as well as in the expression of the NADPH oxidase

subunit and the pro-inflammatory cytokine IL-1b. The AT1 recep-

tor protein decreased by �80%. However, there were no


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Figure 1 Dopaminergic (tyrosine hydroxylase immunoreactive, TH-ir) neurons (A–E) in the substantia nigra compacta (SNc) 2 weeks after

intrastriatal injection of vehicle or 6-hydroxydopamine (6-OHDA) in young ovariectomized (ovx) rats or aged acyclic rats not treated or

treated with 17-b-oestradiol, either immediately (E2) or 3 weeks later ( + E2), and/or the AT1 antagonist candesartan (CAND).

Representative photomicrographs of the substantia nigra compacta of different groups of aged rats are shown in B–E. The estimated total

number of dopaminergic (tyrosine hydroxylase immunoreactive) neurons in the substantia nigra compacta of the different experimental

groups is shown in A. Data are means � SEM; n = 5. *P5 0.05 relative to the corresponding vehicle-treated group (ovariectomized or

aged rats), #P50.05 relative to the group treated with ovariectomized + 6-hydroxydopamine alone, &P50.05 relative to the group

aged + 6-hydroxydopamine alone, + P50.05 relative to the group ovariectomized + 6-hydroxydopamine + oestrogen and

ovariectomized + t + 6-hydroxydopamine + E2 (one-way ANOVA and Bonferroni post hoc test). Scale bar = 500 mm.


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significant changes in AT1 messenger RNA expression, which

suggests that oestrogen regulates AT1 expression at a post-

transcriptional level, possibly via cytosolic proteins that bind to

cis elements in the 50 leader sequence of the AT1 messenger

RNA, interfere with ribosomal scanning and inhibit translational

efficiency as observed in other cell types (Krishnamurthi et al.,

1999; Wu et al., 2003 b). In young menopausal rats, treatment

with oestrogen induced a marked increase in AT2 messenger

RNA and protein expression (�200%), and a significant decrease

in angiotensin converting enzyme activity. Angiotensin convert-

ing enzyme activity was 0.0798 � 0.00723 nmol his-leu/mg

protein/min in ovariectomized rats and 0.0562 � 0.00419 in rats

with surgical menopause immediately treated with oestrogen

therapy. In accordance, the results indicate that oestrogen induced

a significant decrease in NADPH-derived reactive oxygen species

and pro-inflammatory changes, as revealed by decreased p47phox

expression and IL-1b expression, respectively (Fig. 3A and B).

The effect of treatment with oestrogen in aged acyclic rats was,

however, different. Oestrogen induced a significant decrease in

AT1 protein expression although much less than in young ovar-

iectomized rats (i.e. 76.7% and 22.4% of the corresponding un-

treated menopausal rats, respectively), and no significant changes

were observed in AT2 and p47phox messenger RNA or protein

expression. Slight but significant decreases were observed in

angiotensin converting enzyme activity (0.0890 � 0.00117 and

0.0666 � 0.00182) and IL-1b expression (�70% of untreated

aged controls; Fig. 3C and D).

Effect of delayed oestrogen therapy inyoung surgically menopausal rats andin aged ovariectomized ratsAs previously observed in young rats immediately subjected to

oestrogen therapy, renin-angiotensin system activity and expres-

sion of the NADPH oxidase subunit and the pro-inflammatory

cytokine IL-1b were lower in young ovariectomized rats treated

with oestrogen 3 weeks after ovariectomy than in untreated ovar-

iectomized rats. The AT1 receptor protein was �80% lower,

although there were no significant changes in AT1 messenger

RNA expression (see above). In addition, AT2 messenger RNA

and protein expression were significantly higher (�140%), and

angiotensin converting enzyme activity (0.0798 � 0.00723 and

0.0564 � 0.00374, respectively), p47phox expression and IL-1b ex-

pression were significantly lower than in untreated ovariectomized

rats (Fig. 4A and B). Interestingly, however, comparison between

young ovariectomized rats subjected to either immediate or

delayed oestrogen therapy revealed significant differences in the

expression of AT2 receptors, which was significantly lower in the

rats subjected to a 3-week period of hypo-oestrogenicity (Fig. 4C

and D).

As it has been reported that significant levels of androgens are

produced in the ovaries of aged rats, and androgens may affect

renin-angiotensin system activity (see ‘Discussion’ section), a

group of aged rats were ovariectomized before oestrogen therapy

and compared with intact aged rats treated with oestrogen ther-

apy. However, no significant differences were observed in the

expression of AT1, AT2, p47, IL-1b and angiotensin converting

enzyme (0.0794 � 0.00667 and 0.0666 � 0.00182, respectively)

between the two groups of aged menopausal rats treated with

oestrogen therapy (Fig. 5A and B).

Effect of treatment with candesartanin young menopausal and agedmenopausal ratsContrary to what was observed after oestrogen therapy, treatment

with the AT1 antagonist candesartan induced similar effects in

Figure 2 Real-time quantitative reverse transcriptase–poly-

merase chain reaction (A) and western blot (B) analysis of AT1,

AT2, p47phox and IL-1b expression, and activity of the angio-

tensin converting enzyme (ACE; B) in young ovariectomized

(ovx) rats as compared with aged menopausal rats. Protein ex-

pression 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 nor-

malized to ovariectomized values (100%). Data are

means � SEM; n = 5. *P5 0.05 (Student’s t-test).


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young ovariectomized and aged menopausal rats. In young

menopausal rats, candesartan induced a significant increase in

the messenger RNA and protein expression of AT2 receptors,

as well as a decrease in the expression of the NADPH oxidase

subunit p47phox and the pro-inflammatory cytokine IL-1b.

Changes in angiotensin converting enzyme activity were statistic-

ally not significant (0.0798 � 0.00723 and 0.0650 � 0.0111;

Fig. 6A and B).

In aged menopausal rats, treatment with the AT1 blocker can-

desartan also induced a significant increase in the messenger RNA

and protein expression in AT2 receptors, as well as a marked de-

crease in the expression of the NADPH oxidase subunit p47phox

and the pro-inflammatory cytokine IL-1b. In aged rats, treatment

with candesartan, therefore, induced higher anti-inflammatory and

antioxidant effects than treatment with oestrogen. Changes in

angiotensin converting enzyme activity were statistically not sig-

nificant (0.0890 � 0.0117 and 0.0889 � 0.00805, respectively;

Fig. 6C and D).

DiscussionThe present results confirm that oestrogen therapy protects

against dopaminergic degeneration induced by neurotoxins in

young rats subjected to surgical menopause and immediate oes-

trogen therapy, and that this effect is lower in aged rats.

Interestingly, oestrogen administered to young rats after a

period of hypo-oestrogenicity was also neuroprotective, although

the rats showed some changes in renin-angiotensin system activity

that suggest that the effectiveness of oestrogen therapy may

Figure 3 Real-time quantitative reverse transcriptase–polymerase chain reaction (A and C) and western blot (B, and D) analysis of

changes induced by treatment with 17-b-oestradiol (E2) in the expression of AT1, AT2, p47phox and IL-1b, and the activity of the

angiotensin converting enzyme (ACE; B and D) in young ovariectomized rats (ovx + E2; A and B) and aged acyclic rats (aged + E2; C and

D) in comparison with the corresponding untreated rats (ovariectomized or aged rats). Protein expression was obtained as 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 untreated ovariectomized (A and B) or aged (C and D) rats (100%). Data are means � SEM; n = 5. *P50.05

(Student’s t-test).


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decrease after very long periods of hypo-oestrogenic state in

young animals or in older animals subjected to hypo-

oestrogenicity. The present results may explain discrepancies

among previous studies in which neither the age of the subjects

nor the time postmenopause prior to oestrogen therapy were

taken into account. The mechanism involved in the beneficial ef-

fects of oestrogen has not been totally clarified. However, a grow-

ing body of evidence from preclinical and clinical studies suggests

that anti-inflammatory actions are fundamental for the protective

actions of oestrogen (Turgeon et al., 2006; Suzuki et al., 2007;

Chen et al., 2008). Recent studies have also shown that modula-

tion of the glial neuroinflammatory response by oestrogen is

involved in the neuroprotective effects (Morale et al., 2006;

Tripanichkul et al., 2006); it is also known that neuroinflammation

and microglial activation play a major role in the progression of

Parkinson’s disease (Wu et al., 2002, 2003a; Rodriguez-Pallares

et al., 2007). Interestingly, however, recent studies have shown

that oestrogen attenuates inflammatory cytokine production fol-

lowing brain lesions in young adult female rats but not in acyclic

reproductive senescent females (Johnson et al., 2006), and that

oestrogen suppresses microglial activation and cytokine expression

in cultured microglia derived from neonatal or foetal sources, but

failed to do so in cultures of microglia from senescent females

(Sohrabji, 2005; Johnson et al., 2006). The present results support

this view, and suggest that the brain renin-angiotensin system

plays a major role in these effects.

Figure 4 Real-time quantitative reverse transcriptase–polymerase chain reaction (A and C) and western blot (B and D) analysis of changes

induced by treatment with 17-b-oestradiol (E2) in the expression of AT1, AT2, p47phox and IL-1b, and the activity of the angiotensin

converting enzyme (ACE; B and D) in young ovariectomized rats subjected to a 3-week period of hypo-oestrogenicity (ovx + t + E2) in

comparison with untreated young menopausal rats (ovx; A and B) and young menopausal rats immediately treated with oestrogen

(ovx + E2; C and D). Protein expression was obtained as 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 ovariectomized (A and B) or ovx + E2

(C and D) rats (100%). Data are means � SEM; n = 6. *P50.05 (Student’s t-test).


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In the present study, we observed increased renin-angiotensin

system activity in young rats subjected surgical menopause (ovar-

iectomized) relative to rats with stable high levels of oestrogen

(i.e. surgical menopause treated with oestrogen therapy).

Increased angiotensin converting enzyme activity, increased AT1

expression and decreased AT2 expression, as well as increased

NADPH activity and IL-1b expression were observed in untreated

surgical menopausal rats. Comparison between young surgical

menopausal rats and aged menopausal rats revealed that

renin-angiotensin system activity was also exacerbated in aged

menopausal rats. Both groups of menopausal rats showed similar

levels of NADPH activity and AT2 receptor expression. The AT1

messenger RNA expression was higher in aged rats than in young

ovariectomized rats. Since oestrogen affects AT1 expression at

post-transcriptional levels, this suggests that additional factors in-

crease renin-angiotensin system activity in aged rats (see below).

However, AT1 protein expression was higher in young ovariecto-

mized rats than in aged menopausal rats, suggesting that the

effect of the presence/absence of oestrogen is more important

in surgical menopause than in natural menopause, which was con-

firmed in menopausal rats treated with oestrogen. Similar levels of

AT2 messenger RNA and protein expression in young ovariecto-

mized and aged rats also suggest that ageing-related factors other

than low levels of oestrogen reduced the expression of AT2 re-

ceptors in aged rats, because the lack of oestrogen decreased AT2

receptor levels in young rats but did not significantly change AT2

receptor levels in aged rats.

The observed upregulation of renin-angiotensin system activity

may contribute to increased dopaminergic cell vulnerability. It is

well known that angiotensin acts via AT1 receptors inducing in-

flammatory responses and releasing high levels of reactive oxygen

species, mainly by activation of the NADPH complex in vascular

degenerative disease and other diseases mediated by oxidative

stress and chronic inflammation (Touyz, 2002; Qin et al., 2004).

In the nigrostriatal system in animal models of Parkinson’s disease,

we have previously shown that brain angiotensin induces activa-

tion of the NADPH complex via AT1 receptors, leading to

increased neuroinflammation, reactive oxygen species and dopa-

minergic cell death (Rey et al., 2007; Rodriguez-Pallares et al.,

2008; Joglar et al., 2009). We have shown NADPH expression

in dopaminergic neurons and microglial cells. However, it is

known that in non-inflammatory cells, such as neurons, the

NADPH complex produces only low rates of reactive oxygen spe-

cies for signalling function. In inflammatory cells such as microglia,

NADPH activation produces high concentrations of reactive

oxygen species that are released extracellularly to kill invading

microorganisms or cells (Babior, 1999, 2004). In accordance with

this, we observed that angiotensin was not able to increase dopa-

minergic neuron death in the absence of microglial cells

(Rodriguez-Pallares et al., 2008; Joglar et al., 2009). The increased

angiotensin converting enzyme activity in menopausal rats leads to

increased angiotensin production. Increased angiotensin convert-

ing enzyme activity has also been observed in other tissues in

postmenopausal females (Nogawa et al., 2001) and in experimen-

tal models of cardiovascular disease after ovariectomy (Wu et al.,

2003b; Dean et al., 2005). The observed upregulation of AT1

receptors may also contribute to NADPH activation and increased

dopaminergic cell vulnerability. This is supported by the present

experiments in which we have observed that the enhanced sus-

ceptibility of dopaminergic neurons was significantly decreased by

AT1 receptor inhibition with candesartan. Furthermore, it is par-

ticularly interesting that untreated surgically menopausal rats had

significantly fewer AT2 receptors than young rats treated with

Figure 5 Real-time quantitative reverse transcriptase–poly-

merase chain reaction (A) and western blot (B) analysis of

changes induced by treatment with 17-b-oestradiol (E2) in the

expression of AT1, AT2, p47phox and IL-1b, and the activity of

the angiotensin converting enzyme (ACE; B) in aged ovariecto-

mized rats (aged + ovx + E2) in comparison with intact aged

menopausal rats (aged + E2). No significant differences were

observed between both groups of aged menopausal rats.

Protein expression was obtained as 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 intact aged rats (100%). Data are

means � SEM; n = 5.


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oestrogen, which may also enhance dopaminergic cell loss. AT1

and AT2 receptors have opposing effects and AT2 receptors coun-

terbalance the deleterious effect of AT1 receptor stimulation, so

that functional interactions between the two receptor subtypes

and their specific distribution determine the angiotensin-induced

effects (Sohn et al., 2000), which in the case of untreated meno-

pausal rats resulted in a pro-oxidative and pro-inflammatory state,

as suggested by the increased NADPH activity and IL-1b expres-

sion in comparison with rats treated with oestrogen. The detailed

mechanisms involved in the above described regulation of

renin-angiotensin system components by oestrogen have not

been clarified for the brain or peripheral tissues. Oestrogen has

been suggested to inhibit the angiotensin-induced NADPH activa-

tion and production of reactive oxygen species (Xue et al., 2007,

2008), to inhibit AT1 receptor-mediated extracellular

signal-regulated kinase activation (Liu et al., 2002), and to inhibit

angiotensin-induced activation of mitogen-activated protein kin-

ases (Imanishi et al., 2005) in different tissues.

In the present study, it is particularly interesting that the re-

sponse to oestrogen therapy was different in young (i.e. surgically

induced menopause) and aged menopausal rats. The effects of

treatment with oestrogen on AT1, AT2 receptor expression and

NADPH activity were lower in aged rats than in young meno-

pausal rats. In accordance with this, oestrogen therapy signifi-

cantly reduced 6-hydroxydopamine-induced dopaminergic cell

loss in young rats but not in aged rats. Oestrogen regulation of

AT2 levels appears particularly important (Okumura et al., 2005;

Chakrabasty et al., 2008; Sakata et al., 2009). AT2 receptors

are particularly abundant in tissues during development and de-

crease after birth, although they are upregulated under patho-

logical conditions to counter-regulate pro-inflammatory effects

mediated by AT1 receptors. The present results suggest that

Figure 6 Real-time quantitative reverse transcriptase–polymerase chain reaction (A and C) and western blot (B and D) analysis of changes

induced by treatment with the AT1 antagonist candesartan in the expression of AT2, p47phox and IL-1b, and the activity of the angiotensin

converting enzyme (ACE; B and D) in young ovariectomized rats (ovx + candesartan; A and B) and aged acyclic rats (aged + candesartan;

C and D) in comparison with the corresponding untreated rats (ovx or aged 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 ovariectomized (A and B) or aged (C and D) rats (100%). Data are means � SEM; n = 6. *P50.05 (Student’s

t-test).


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ageing disrupts the ability of the cell to respond to oestrogen by

upregulation of AT2 receptors (Armando et al., 2002; Suarez

et al., 2004). Interestingly, the results observed in rats subjected

to delayed oestrogen therapy indicate that the treatment is able to

reverse changes induced by a period of hypo-oestrogenicity

in young animals (i.e. the increase in renin-angiotensin system

activity, and in markers of oxidative stress, neuroinflam-

mation and dopaminergic neuron vulnerability). However, the

oestrogen-induced upregulation of AT2 receptor expression was

significantly lower than in young ovariectomized rats treated im-

mediately with oestrogen therapy. It is therefore possible that very

long periods of hypo-oestrogenicity in young animals and/or

delayed oestrogen therapy in older rats decreases the neuropro-

tective effects of oestrogen therapy.

Different responses to oestrogen therapy in young menopausal

rats and aged rats have also been observed in other experimental

models. Thus, oestrogen therapy has been shown to protect

against other diseases such as atherosclerosis, although only

when the vessels are healthy and not when atherosclerosis is al-

ready established (Clarkson, 2007, 2009; Antonicelli et al., 2008).

However, the mechanism responsible for the different response to

oestrogen therapy in young and aged animals (i.e. surgical and

natural menopause) has not been clarified. In the present study,

young ovariectomized rats and aged rats treated with oestrogen

did not show significant differences in blood levels of oestrogen,

which were similar to those previously observed with similar im-

plants (Febo et al., 2002; Sandoval and Witt, 2011), and similar to

those observed in intact rats in proestrous (Nequin et al., 1979).

The above reported differences are therefore due to differences in

the response to oestrogen between young and aged rats. Several

recent studies have reported that significant levels of androgens

are produced in the ovaries of aged rats (Fogle et al., 2008; Korse

et al., 2009), but not in young ovariectomized rats (surgically

induced menopause), and it has been suggested that administra-

tion of androgens induce activation of renin-angiotensin system in

several tissues (Fisher et al., 2002; Henriques et al., 2008; Ojeda

et al., 2010). Androgens derived from aged ovaries may therefore

counteract the inhibitory effect of oestrogen therapy on the nigral

renin-angiotensin system. However, we have observed that

oestrogen therapy did not have significantly different effects in

aged ovariectomized rats and intact aged rats. Furthermore,

renin-angiotensin system activity has also been found to be

higher in aged males than in young males (Villar-Cheda et al.,

2012). Interestingly in the case of the dopaminergic system, it

has been shown that loss of oestrogen decreases striatal dopamin-

ergic levels and oestrogen administration increases dopaminergic

release (Becker, 1999; Ohtani et al., 2001; Shulman, 2002; Liu

and Dluzen, 2007), and that there is a counter-regulatory mech-

anism between the dopaminergic and renin-angiotensin system

(Gildea, 2009; Villar-Cheda et al., 2010), which may also explain

the increased renin-angiotensin system activity in menopausal rats

and the decrease in renin-angiotensin system activity by oestrogen

therapy. Decreased dopaminergic levels have also been observed

in aged male animals (i.e. independently of oestrogen depletion;

McCormack et al, 2004; Cruz-Muros et al., 2009), which may

also induce increased renin-angiotensin system activity in males.

However, different responses to oestrogen in young and aged

animals have also been observed in other tissues (independently

of dopaminergic levels; see above) and decreased levels of dopa-

mine with age is not the only factor involved.

It has been suggested that prolonged hypo-oestrogenicity dis-

rupts the ability of injured tissue to upregulate oestrogen receptors

that mediate the anti-inflammatory and neuroprotective actions of

oestrogen, thus eliminating the beneficial effects of oestrogen

treatment (Suzuki et al., 2007). However, it has also been sug-

gested that a prolonged period of oestrogen deprivation may

upregulate oestrogen receptors in senescent females, and exogen-

ous oestrogen therapy may be ineffective or neurotoxic when

acting on supraphysiological receptor substrates (Bake et al.,

2008; Selvamani and Sohrabji, 2010). The present results in

young ovariectomized rats subjected to delayed oestrogen therapy

reveal that a prolonged hypo-oestrogenic state may modify the

effects of oestrogen on renin-angiotensin system activity, particu-

larly the oestrogen-induced upregulation of AT2 receptors.

However, several studies in different tissues suggest that pro-

longed hypo-oestrogenicity is not the only factor responsible for

the different responses to oestrogen therapy in young and aged

rats. Normal ageing is associated with a proinflammatory,

pro-oxidant state that may favour an exaggerated response to

injury and degenerative diseases, in males as well as in females

(Csiszar et al., 2003; Ungvari et al., 2004; Choi et al., 2008);

particularly, we have observed increased renin-angiotensin

system activity in the substantia nigra of aged males in comparison

with young males (i.e. independently of oestrogen levels or

hypo-oestrogenic periods; Villar-Cheda et al., 2012). Factors

other than oestrogen are therefore involved in the upregulation

of renin-angiotensin system in aged rats, an effect that cannot be

counteracted by oestrogen therapy alone.

Parkinson’s disease 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 dopamin-

ergic cell degeneration (Gao et al., 2003), and oxidative stress and

inflammation play major roles in the synergistic process (Wu et al.,

2003a; Andersen, 2004). A pro-oxidative and proinflammatory

state in both groups of menopausal rats may therefore be related

to the increased risk of Parkinson’s disease reported in menopausal

females in several epidemiological studies (Currie et al., 2004;

Ragonese et al., 2006a, b). Furthermore, untreated surgical or

natural menopause may increase the progression of the dopamin-

ergic degeneration in patients with initial or moderate stages of

Parkinson’s disease.

In conclusion, the results suggest that oestrogen constitutes a

major factor for inhibition of renin-angiotensin system activity in

young females. The loss of oestrogen after ovariectomy (surgical

menopause) increases renin-angiotensin system activity and the

susceptibility to neuroinflammation oxidative damage and dopa-

minergic neuron neurodegeneration, and oestrogen therapy in-

duces pronounced renin-angiotensin system inhibition and

neuroprotection. In aged animals, other factors in addition to

the lack of oestrogen contribute to the increase in

renin-angiotensin system activity, and oestrogen therapy can

only partially counteract the renin-angiotensin system hyperactiv-

ity, leading to no significant neuroprotection in the present experi-

mental conditions. In addition, changes in oestrogen receptors or


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oestrogen-induced intracellular signalling with ageing may also

lead to partial or ineffective renin-angiotensin system inhibition

by oestrogen therapy in aged animals. In any case, it is

particularly interesting that direct manipulation of the

renin-angiotensin system with the AT1 antagonist candesartan

led to remarkable reduction in renin-angiotensin system activity

(increased either by the lack of oestrogen in young surgically

induced menopausal rats or by the lack of oestrogen and addition-

al factors in aged rats) and significantly protected against dopa-

minergic neuron loss in both types of menopausal rats. This

suggests that manipulation of the brain renin-angiotensin system

may be an efficient approach for the prevention or treatment of

Parkinson’s disease in oestrogen-deficient females together with or

instead of oestrogen replacement therapy.

AcknowledgementsThe authors thank Pilar Aldrey, Iria Novoa and Jose A. Trillo for

their excellent technical assistance. The authors are thankful to

AstraZeneca for providing candesartan for the experiments.

FundingSpanish Ministry of Science and Innovation; Spanish Ministry of

Health (RD06/0010/0013, TERCEL and CIBERNED); Galician

Government (XUGA) and European Regional Development Fund.

ReferencesAndersen JK. Oxidative stress in neurodegeneration: cause or conse-

quence. Nat Med 2004; 10: S18–25.

Antonicelli R, Olivieri F, Morichi V, Urbani E, Mais V. Prevention of

cardiovascular events in early menopause: a possible role for hormone

replacement therapy. Int J Cardiol 2008; 130: 140–6.Armando I, Jezova M, Juorio AV, Terron JA, Falcon-Neri A, et al.

Estrogen upregulates renal angiotensin II AT(2) receptors. Am J

Physiol Renal Physiol 2002; 283: F934–43.

Babior B. NADPH oxidase: an update. Blood 1999; 93: 1464–76.

Babior BM. NADPH oxidase. Curr Opin Immunol 2004; 16: 42–7.Bake S, Ma L, Sohrabji F. Estrogen receptor-alpha overexpression sup-

presses 17beta-estradiol-mediated vascular endothelial growth factor

expression and activation of survival kinases. Endocrinology 2008; 149:

3881–9.

Basso N, Paglia N, Stella I, de Cavanagh EM, Ferder L, del Rosario Lores

Arnaiz M, et al. Protective effect of the inhibition of the

renin-angiotensin system on aging. Regul Pept 2005; 128: 247–52.

Becker JB. Gender differences in dopaminergic function in striatum and

nucleus accumbens. Pharmacol Biochem Behav 1999; 64: 803–12.

Benigni A, Corna D, Zoja C, Sonzogni A, Latini R, Salio M, et al.

Disruption of the Ang II type 1 receptor promotes longevity in mice.

J Clin Invest 2009; 119: 524–30.

Cai H, Griendling KK, Harrison DG. The vascular NAD(P)H oxidases as

therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci

2003; 24: 471–8.Chakrabarty A, Blacklock A, Svojanovsky S, Smith PG. Estrogen elicits

dorsal root ganglion axon sprouting via a renin-angiotensin system.

Endocrinology 2008; 149: 3452–60.

Chen J, Yang S, Hu S, Choudhry MA, Bland KI, Chaudry IH. Estrogen

prevents intestinal inflammation after trauma-hemorrhage via

downregulation of angiotensin II and angiotensin II subtype I receptor.

Am J Physiol Gastrointest Liver Physiol 2008; 295: G1131–7.

Chlebowski RT, Anderson GL, Gass M, Lane DS, Aragaki AK, Kuller LH,

et al. Estrogen plus progestin and breast cancer incidence and mortal-

ity in postmenopausal women. JAMA 2010; 304: 1684–92.

Choi DY, Zhang J, Bing G. Aging enhances the neuroinflammatory re-

sponse and alpha-synuclein nitration in rats. Neurobiol Aging 2010;

31: 1649–53.

Clarkson TB. Estrogen effects on arteries vary with stage of reproductive

life and extent of subclinical atherosclerosis progression. Menopause

2007; 14: 373–84.Clarkson TB, Appt SE. Controversies about HRT–lessons from monkey

models. Maturitas 2005; 51: 64–74.Clarkson TB, Mehaffey MH. Coronary heart disease of females: les-

sons learned from nonhuman primates. Am J Primatol 2009; 71:

785–93.

Cruz-Muros I, Afonso-Oramas D, Abreu P, Perez-Delgado MM,

Rodrıguez M, Gonzalez-Hernandez T. Aging effects on the dopamine

transporter expression and compensatory mechanisms. Neurobiol

Aging 2009; 30: 973–86.

Csiszar A, Ungvari Z, Koller A, Edwards JG, Kaley G. Aging-induced

proinflammatory shift in cytokine expression profile in coronary

arteries. FASEB J 2003; 17: 1183–5.Currie LJ, Harrison MB, Trugman JM, Bennett JP, Wooten GF.

Postmenopausal estrogen use affects risk for Parkinson disease. Arch

Neurol 2004; 61: 886–8.

Dean SA, Tan J, O’Brien ER, Leenen FH. 17beta-estradiol downregulates

tissue angiotensin-converting enzyme and ANG II type 1 receptor in

female rats. Am J Physiol Regul Integr Comp Physiol 2004; 288:

R759–66.

Dluzen D. Estrogen decreases corpus striatal neurotoxicity in response to

6-hydroxydopamine. Brain Res 1997; 767: 340–4.

Dziuk PJ, Cook B. Passage of steroids through silicone rubber.

Endocrinology 1966; 78: 208–11.

Febo M, Ferris CF, Segarra AC. Estrogen influences cocaine-induced

blood oxygen level-dependent signal changes in female rats.

J Neurosci 2005; 25: 1132–6.Febo M, Jimenez-Rivera CA, Segarra AC. Estrogen and opioids interact

to modulate the locomotor response to cocaine in the female rat. Brain

Res 2002; 943: 151–61.

Fischer M, Baessler A, Schunkert H. Renin angiotensin system and

gender differences in the cardiovascular system. Cardiovasc Res

2002; 53: 672–7.

Fogle RH, Stanczyk FZ, Zhang X, Paulson RJ. Ovarian androgen produc-

tion in postmenopausal women. J Clin Endocrinol Metab 2007; 92:

3040–3.

Gao HM, Liu B, Zhang W, Hong JS. Synergistic dopaminergic neurotox-

icity of MPTP and inflammogen lipopolysaccharide: relevance to the

etiology of Parkinson’s disease. FASEB J 2003; 17: 1957–9.Gildea JJ. Dopamine and angiotensin as renal counterregulatory systems

controlling sodium balance. Curr Opin Nephrol Hypertens 2009; 18:

28–32.

Gundersen HJG. Bendsen, T.F., Korbo, L., Marcussen, N., Moller, A.,

Nielsen, K., Some new, simple and efficient stereological methods

and their use in pathological research and diagnosis. APIMS 1988;

96: 379–94.

Harman SM, Naftolin F, Brinton EA, Judelson DR. Is the estrogen con-

troversy over? Deconstructing the Women’s Health Initiative study: a

critical evaluation of the evidence. Ann NY Acad Sci 2005; 1052:

43–56.

Hemming ML, Selkoe DJ, Farris W. Effects of prolonged

angiotensin-converting enzyme inhibitor treatment on amyloid

b-protein metabolism in mouse models of Alzheimer disease.

Neurobiol Dis 2007; 26: 273–81.

Henriques T, Zhang X, Yiannikouris FB, Daugherty A, Cassis LA.

Androgen increases AT1a receptor expression in abdominal aortas to

promote angiotensin II-induced AAAs in apolipoprotein E-deficient

mice. Arterioscler Thromb Vasc Biol 2008; 28: 1251–6.


Dow



ovember 2021

Heymes C, Silvestre JS, Llorens-Cortes C, Chevalier B, Marotte F,

Levy BI, et al. Cardiac senescence is associated with enhanced expres-

sion of angiotensin II receptor subtypes. Endocrinology 1998; 139:

2579–87.

Hoshi-Fukushima R, Nakamoto H, Imai H, Kanno Y, Ishida Y,

Yamanouchi Y, et al. Estrogen and angiotensin II interactions deter-

mine cardio-renal damage in Dahl salt-sensitive rats with heart failure.

Am J Nephrol 2008; 28: 413–23.Imanishi T, Hano T, Nishio I. Estrogen reduces angiotensin II-induced

acceleration of senescence in endothelial progenitor cells. Hypertens

Res 2005; 28: 263–71.Joglar B, Rodriguez-Pallares J, Rodrıguez-Perez AI, Rey P, Guerra MJ,

Labandeira-Garcia JL. The inflammatory response in the MPTP

model of Parkinson’s disease is mediated by brain angiotensin: rele-

vance to progression of the disease. J Neurochem 2009; 109: 656–69.

Johnson AB, Bake S, Lewis DK, Sohrabji F. Temporal expression of

IL-1beta protein and mRNA in the brain after systemic LPS injection

is affected by age and estrogen. J Neuroimmunol 2006; 174: 82–91.

Kondo T, Yoshida K, Yoshimura Y, Motohashi M, Tanayama S.

Disposition of the new angiotensin II receptor antagonist candesartan

cilexetil in rats and dogs. Arzneimittelforschung 1996; 46: 594–600.

Korse CM, Bonfrer JM, van Beurden M, Verheijen RH, Rookus MA.

Estradiol and testosterone levels are lower after oophorectomy than

after natural menopause. Tumour Biol 2009; 30: 37–42.

Krishnamurthi K, Verbalis JG, Zheng W, Wu Z, Clerch LB, Sandberg K.

Estrogen regulates angiotensin AT1 receptor expression via cytosolic

proteins that bind to the 50 leader sequence of the receptor mRNA.

Endocrinology 1999; 140: 5435–8.Li JM, Shah AM. Mechanism of endothelial cell NADPH oxidase activa-

tion by angiotensin II. Role of the p47phox subunit. J Biol Chem 2003;

278: 12094–100.Liu B, Dluzen DE. Oestrogen and nigrostriatal dopaminergic neurodegen-

eration: animal models and clinical reports of Parkinson’s disease. Clin

Exp Pharmacol Physiol 2007; 34: 555–65.

Liu HW, Iwai M, Takeda-Matsubara Y, Wu L, Li JM, Okumura M, et al.

Effect of estrogen and AT1 receptor blocker on neointima formation.

Hypertension 2002; 40: 451–7.

Mannino CA, South SM, Inturrisi CE, Quinones-Jenab V.

Pharmacokinetics and effects of 17beta-estradiol and progesterone

implants in ovariectomized rats. J Pain 2005; 6: 809–16.

McCormack AL, Di Monte DA, Delfani K, Irwin I, DeLanney LE,

Langston WJ, et al. Aging of the nigrostriatal system in the squirrel

monkey. J Comp Neurol 2004; 471: 387–95.

McKinley MJ, Albiston AL, Allen AM, Mathai ML, May CN,

McAllen RM, et al. The brain reenin-angiotensin system: location

and physiological roles. Int J Biochem Cell Biol 2003; 35: 901–18.

Miller VM, Black DM, Brinton EA, Budoff MJ, Cedars MI, Hodis HN,

et al. Using basic science to design a clinical trial: baseline character-

istics of women enrolled in the Kronos Early Estrogen Prevention Study

(KEEPS). J Cardiovasc Transl Res 2009; 2: 228–39.Miller VM, Clarkson TB, Harman SM, Brinton EA, Cedars M, Lobo R,

et al. Women, hormones, and clinical trials: a beginning, not an end.

J Appl Physiol 2005; 99: 381–3.Morale MC, Serra PA, L’episcopo F, Tirolo C, Caniglia S, Testa N, et al.

Estrogen, neuroinflammation and neuroprotection in Parkinson’s dis-

ease: glia dictates resistance versus vulnerability to neurodegeneration.

Neuroscience 2006; 138: 869–78.

Murray HE, Pillai AV, McArthur SR, Razvi N, Datla KP, Dexter DT,

Gillies GE. Dose- and sex-dependent effects of the neurotoxin

6-hydroxydopamine on the nigrostriatal dopaminergic pathway of

adult rats: differential actions of estrogen in males and females.

Neuroscience 2003; 116: 213–22.Nequin LG, Alvarez J, Schwartz NB. Measurement of serum steroid and

gonadotropin levels and uterin and ovarian variables throughout 4-day

and 5-day estrous cycles in the rat. Biol Reprod 1979; 20: 659–70.Nickenig G, Baumer AT, Grohe C, Kahlert S, Strehlow K, Rosenkranz S,

et al. Estrogen modulates AT1 receptor gene expression in vitro and

in vivo. Circulation 1998; 97: 2197–201.

Nogawa N, Sumino H, Ichikawa S, Kumakura H, Takayama Y,

Nakamura T, et al. Effect of long-term hormone replacement therapy

on angiotensin-converting enzyme activity and bradykinin in postme-

nopausal women with essential hypertension and normotensive post-

menopausal women. Menopause 2001; 8: 210–5.

Ohtani H, Nomoto M, Douchi T. Chronic estrogen treatment replaces

striatal dopaminergic function in ovariectomized rats. Brain Res 2001;

900: 163–8.

Ojeda NB, Royals TP, Black JT, Dasinger JH, Johnson JM, Alexander BT.

Enhanced sensitivity to acute angiotensin II is testosterone dependent

in adult male growth-restricted offspring. Am J Physiol Regul Integr

Comp Physiol 2010; 298: R1421–7.

Okumura M, Iwai M, Ide A, Mogi M, Ito M, Horiuchi M. Sex difference

in vascular injury and the vasoprotective effect of valsartan are related

to differential AT2 receptor expression. Hypertension 2005; 46:

577–83.Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New

York: Academic Press; 1985.

Przedborski S, Levivier M, Jiang H, Ferreira M, Jackson-Lewis V,

Donaldson D, et al. Dose-dependent lesions of the dopaminergic

nigrostriatal pathway induced by intrastriatal injection of

6-hydroxydopamine. Neuroscience 1995; 67: 631–47.Qin L, Liu Y, Wang T, Wei SJ, Block ML, Wilson B, et al. NADPH oxidase

mediates lipopolysaccharide-induced neurotoxicity and proinflamma-

tory gene expression in activated microglia. J Biol Chem 2004; 279:

1415–21.Ragonese P, D’Amelio M, Callari G, Salemi G, Morgante L, Savettieri G.

Age at menopause predicts age at onset of Parkinson’s disease. Mov

Disord 2006a; 21: 2211–4.Ragonese P, D’Amelio M, Savettieri G. Implications for estrogens in

Parkinson’s disease: an epidemiological approach. Ann NY Acad Sci

2006b; 1089: 373–82.Rey P, Lopez-Real A, Sanchez-Iglesias, Munoz A, Soto-Otero R,

Labandeira-Garcia JL. Angiotensin type-1-receptor antagonists reduce

6-hydroxydopamine toxicity for dopaminergic neurons. Neurobiol

Aging 2007; 28: 555–67.

Rodriguez-Pallares J, Parga JA, Munoz A, Rey P, Guerra MJ, Labandeira-

Garcia JL. Mechanism of 6-hydroxydopamine neurotoxicity: the role of

NADPH oxidase and microglial activation in 6-hydroxydopamine-

induced degeneration of dopaminergic neurons. J Neurochem 2007;

103: 145–56.

Rodriguez-Pallares J, Rey P, Parga JA, Munoz A, Guerra MJ, Labandeira-

Garcia JL. Brain angiotensin enhances dopaminergic cell death via

microglial activation and NADPH-derived ROS. Neurobiol Dis 2008;

31: 58–73.

Rodriguez-Perez AI, Valenzuela R, Villar-Cheda B, Guerra MJ,

Lanciego JL, Labandeira-Garcia JL. Estrogen and angiotensin inter-

action in the substantia nigra. Relevance to postmenopausal

Parkinson’s diseas. Exp Neurol 2010; 224: 517–26.

Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C,

Stefanick ML, et al. Risks and benefits of estrogen plus progestin in

healthy postmenopausal women: principal results From the Women’s

Health Initiative randomized controlled trial. JAMA 2002; 288:

321–33.Rueckschloss U, Quinn MT, Holtz J, Morawietz H. Dose-dependent

regulation of NAD(P)H oxidase expression by angiotensin II in

human endothelial cells: protective effect of angiotensin II type 1 re-

ceptor blockade in patients with coronary artery disease. Arterioscler

Thromb Vasc Biol 2002; 22: 1845–51.

Saavedra JM. Brain angiotensin II: new developments, unanswered ques-

tions and therapeutic opportunities. Cell Mol Neurobiol 2005; 25:

485–12.

Sakata A, Mogi M, Iwanami J, Tsukuda K, Min LJ, Fujita T, et al.

Sex-different effect of angiotensin II type 2 receptor on ischemic

brain injury and cognitive function. Brain Res 2009; 1300: 14–23.

Sandoval KE, Witt KA. Age and 17b-estradiol effects on blood–brain

barrier tight junction and estrogen receptor proteins in ovariectomized

rats. Microvascular Research 2011; 81: 198–205.


Dow



ovember 2021

Sauer H, Oertel WH. Progressive degeneration of nigrostriatal dopamineneurons following intrastriatal terminal lesions with

6-hydroxydopamine: a combined retrograde tracing and immunohis-

tochemical study in the rat. Neuroscience 1994; 59: 401–15.

Selvamani A, Sohrabji F. Reproductive age modulates the impact of focalischemia on the forebrain as well as the effects of estrogen treatment

in female rats. Neurobiol Aging 2010; 31: 1618–28.

Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK.

Angiotensin II stimulation of NAD(P)H oxidase activity: upstream me-diators. Circ Res 2002; 91: 406–13.

Shulman LM. Is there a connection between estrogen and Parkinson’s

disease? Parkinsonism Relat Disord 2002; 8: 289–95.Sohn HY, Raff U, Hoffmann A, Gloe T, Heermeier K, Galle J, et al.

Differential role of angiotensin II receptor subtypes on endothelial

superoxide formation. Br J Pharmacol 2000; 131: 667–72.

Sohrabji F, Bake S. Age-related changes in neuroprotection: is estrogenpro-inflammatory for the reproductive senescent brain? Endocrine

2006; 29: 191–7.

Sohrabji F. Estrogen: a neuroprotective or proinflammatory hormone?

Emerging evidence from reproductive aging models. Ann NY AcadSci 2005; 1052: 75–9.

Suarez C, Dıaz-Torga G, Gonzalez-Iglesias A, Cristina C, Becu-

Villalobos D. Upregulation of angiotensin II type 2 receptor expression

in estrogen-induced pituitary hyperplasia. Am J Physiol EndocrinolMetab 2004; 286: E786–94.

Suzuki S, Brown CM, Dela Cruz CD, Yang E, Bridwell DA, Wise PM.

Timing of estrogen therapy after ovariectomy dictates the efficacy ofits neuroprotective and antiinflammatory actions. Proc Natl Acad Sci

USA 2007; 104: 6013–8.

Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, et al. Expression

of a functionally active gp91phox-containing neutrophil-type NAD(P)Hoxidase in smooth muscle cells from human resistance arteries, regu-

lation by angiotensin II. Circ Res 2002; 14: 1205–13.

Tripanichkul W, Sripanichkulchai K, Finkelstein DI. Estrogen

down-regulates glial activation in male mice following1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication. Brain Res

2006; 1084: 28–37.

Tsuda M, Iwai M, Li JM, Li HS, Min LJ, Ide A, et al. Inhibitory effects ofAT1 receptor blocker, olmesartan, and estrogen on atherosclerosis via

anti-oxidative stress. Hypertension 2005; 45: 545–51.

Turgeon JL, Carr MC, Maki PM, Mendelsohn ME, Wise PM. Complex

actions of sex steroids in adipose tissue, the cardiovascular system, andbrain: insights from basic science and clinical studies. Endocr Rev 2006;

27: 575–605.

Turgeon JL, McDonnell DP, Martin KA, Wise PM. Hormone therapy:physiological complexity belies therapeutic simplicity. Science 2004;

304: 1269–73.

Unger T. Inhibiting angiotensin receptors in the brain: possible thera-

peutic implications. Curr Med Res Opin 2003; 19: 449–51.Ungvari Z, Csiszar A, Kaley G. Vascular inflammation in aging. Herz

2004; 29: 733–40.

Vegeto E, Benedusi V, Maggi A. Estrogen anti-inflammatory activity in

brain: a therapeutic opportunity for menopause and neurodegenera-tive diseases. Front Neuroendocrinol 2008; 29: 507–19.

Villar-Cheda B, Rodrıguez-Pallares J, Muno A, Valenzuela R, Guerra MJ,

Baltatu OC, Labandeira-Garcia JL. Nigral and striatal regulation ofangiotensin receptor expression by dopamine and angiotensin in

rodents: implications for progression of Parkinson’s disease. Eur J

Neurosci 2010; 32: 1695–706.

Villar-Cheda B, Valenzuela R, Rodriguez-Perez AI, Guerra MJ,Labandeira-Garcia JL. Aging-related changes in the nigral angiotensin

system enhances proinflammatory and pro-oxidative markers and

6-hydroxydopamine-induced dopaminergic degeneration. Neurobiol

Aging 2012; 33: 204.e1–204.e11.Wu D, Teisman P, Tieu K, Vila M, Jackson-Lewis V, Ischiropoulos H,

et al. NADPH oxidase mediates oxidative stress in the

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s

disease. Proc Natl Acad Sci USA 2003a; 100: 6145–50.Wu DC, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, et al.

Blockade of microglial activation is neuroprotective in the

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model ofParkinson disease. J Neurosci 2002; 22: 1763–71.

Wu Z, Zheng W, Sandberg K. Estrogen regulates adrenal angiotensin

type 1 receptors by modulating adrenal angiotensin levels.

Endocrinology 2003b; 144: 1350–6.Xue B, Johnson AK, Hay M. Sex differences in angiotensin II- induced

hypertension. Braz J Med Biol Res 2007a; 40: 727–34.

Xue B, Pamidimukkala J, Lubahn DB, Hay M. Estrogen receptor-alpha

mediates estrogen protection from angiotensin II-induced hypertensionin conscious female mice. Am J Physiol Heart Circ Physiol 2007b; 292:

H1770–6.

Xue B, Zhao Y, Johnson AK, Hay M. Central estrogen inhibition of angio-tensin II-induced hypertension in male mice and the role of reactive

oxygen species. Am J Physiol Heart Circ Physiol 2008; 295: H1025–32.

Zorad S, Dou J, Benicky J, Hutanu D, Tybitanclova K, Zhou J,

Saavedra JM. Long-term angiotensin II AT1 receptor inhibition pro-duces adipose tissue hypotrophy accompanied by increased expression

of adiponectin and PPAR�. Eur J Pharmacol 2006; 552: 112–22.


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ovember 2021

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