Estradiol potentiates hypothalamic vasopressin and oxytocin neuron activation and hormonal secretion induced by hypovolemic shock.

Abstract

Estrogen receptors are located in important brain areas that integrate cardiovascular and hydroelectrolytic responses, including the subfornical organ (SFO) and supraoptic (SON) and paraventricular (PVN) nuclei. The aim of this study was to evaluate the influence of estradiol on cardiovascular and neuroendocrine changes induced by hemorrhagic shock in ovariectomized rats. Female Wistar rats (220-280 g) were ovariectomized and treated for 7 days with vehicle or estradiol cypionate (EC, 10 or 40 μg/kg, sc). On the 8th day, animals were subjected to hemorrhage (1.5 ml/100 g for 1 min). Hemorrhage induced acute hypotension and bradycardia in the ovariectomized-oil group, but EC treatment inhibited these responses. We observed increases in plasma angiotensin II concentrations and decreases in plasma atrial natriuretic peptide levels after hemorrhage; EC treatment produced no effects on these responses. There were also increases in plasma vasopressin (AVP), oxytocin (OT), and prolactin levels after the induction of hemorrhage in all groups, and these responses were potentiated by EC administration. SFO neurons and parvocellular and magnocellular AVP and OT neurons in the PVN and SON were activated by hemorrhagic shock. EC treatment enhanced the activation of SFO neurons and AVP and OT magnocellular neurons in the PVN and SON and AVP neurons in the medial parvocellular region of the PVN. These results suggest that estradiol modulates the cardiovascular responses induced by hemorrhage, and this effect is likely mediated by an enhancement of AVP and OT neuron activity in the SON and PVN.

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doi:10.1152/ajpregu.00800.2010
301:R905-R915, 2011. First published 1 June 2011;
Am J Physiol Regul Integr Comp Physiol
Lucila L. K. Elias and Jose Antunes-Rodrigues
Andre S. Mecawi, Tatiane Vilhena-Franco, Iracema G. Araujo, Luis C. Reis,
induced by hypovolemic shock
oxytocin neuron activation and hormonal secretion
Estradiol potentiates hypothalamic vasopressin and
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Estradiol potentiates hypothalamic vasopressin and oxytocin neuron activation
and hormonal secretion induced by hypovolemic shock
Andre S. Mecawi,1Tatiane Vilhena-Franco,1Iracema G. Araujo,1Luis C. Reis,2Lucila L. K. Elias,1
and Jose Antunes-Rodrigues1
1Faculty of Medicine of Ribeirao Preto, Department of Physiology, University of Sao Paulo, Sao Paulo; and2Department of
Physiological Sciences, Institute of Biology, Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil
Submitted 6 December 2010; accepted in final form 31 May 2011
Mecawi AS, Vilhena-Franco T, Araujo IG, Reis LC, Elias
LLK, Antunes-Rodrigues J. Estradiol potentiates hypothalamic
vasopressin and oxytocin neuron activation and hormonal secretion
induced by hypovolemic shock. Am J Physiol Regul Integr Comp
Physiol 301: R905–R915, 2011. First published June 1, 2011;
doi:10.1152/ajpregu.00800.2010.—Estrogen receptors are located in
important brain areas that integrate cardiovascular and hydroelectro-
lytic responses, including the subfornical organ (SFO) and supraoptic
(SON) and paraventricular (PVN) nuclei. The aim of this study was to
evaluate the influence of estradiol on cardiovascular and neuroendo-
crine changes induced by hemorrhagic shock in ovariectomized
rats. Female Wistar rats (220–280 g) were ovariectomized and
treated for 7 days with vehicle or estradiol cypionate (EC, 10 or 40
?g/kg, sc). On the 8th day, animals were subjected to hemorrhage
(1.5 ml/100 g for 1 min). Hemorrhage induced acute hypotension
and bradycardia in the ovariectomized-oil group, but EC treatment
inhibited these responses. We observed increases in plasma angio-
tensin II concentrations and decreases in plasma atrial natriuretic
peptide levels after hemorrhage; EC treatment produced no effects
on these responses. There were also increases in plasma vasopres-
sin (AVP), oxytocin (OT), and prolactin levels after the induction
of hemorrhage in all groups, and these responses were potentiated
by EC administration. SFO neurons and parvocellular and magno-
cellular AVP and OT neurons in the PVN and SON were activated
by hemorrhagic shock. EC treatment enhanced the activation of
SFO neurons and AVP and OT magnocellular neurons in the PVN
and SON and AVP neurons in the medial parvocellular region of
the PVN. These results suggest that estradiol modulates the car-
diovascular responses induced by hemorrhage, and this effect is
likely mediated by an enhancement of AVP and OT neuron activity
in the SON and PVN.
hemorrhage; female rats; supraoptic nuclei; paraventricular nuclei;
arterial pressure; heart rate
THE PRECISE REGULATION OF body fluids is essential for the
metabolic function of virtually all cells in the body. A variety
of mechanisms are activated to maintain plasma osmolality and
blood volume within a very narrow range of values (3). For
example, hypovolemia and/or hypotension induce vasopressin
(AVP) and oxytocin (OT) release from the magnocellular
neurons of the supraoptic (SON) and paraventricular (PVN)
nuclei in the hypothalamus (3, 56). It is estimated that a
decrease of 10–20% in total blood volume induces the release
of AVP in several species. This neurosecretory response is
modulated by peripheral baroreceptors in the aortic arch and
carotid sinus, cardiopulmonary volume receptors, and angio-
tensin II (ANG II) (50, 65).
The precise role of estrogen in maintaining body fluid
homeostasis is not yet fully understood (16). Our group has
previously reported that ANG II type 1 (AT1) receptors are
involved in the regulation of water and hypertonic saline intake
in ovariectomized (OVX) rats during the nocturnal period (37,
38). In addition, several reports have suggested that estrogen
has modulatory effects on cardiovascular function, as evi-
denced by the cardiovascular changes observed in postmeno-
pausal women, OVX rats, and, possibly, females of other
species during senescence (12, 46). These effects occur by
estrogen’s action on the renin-angiotensin, atrial natriuretic
peptide (ANP), and other peptidergic systems (33). Thus, it is
plausible to assume that investigating the association between
electrolyte and cardiovascular homeostasis and estrogen’s
mechanisms of action may help clarify the controversial car-
dioprotective effect of estrogen reported in experimental and
clinical trials (11).
In a recent review, Somponpun (61) described possible
mechanisms by which estrogen regulates electrolyte homeo-
stasis, focusing on AVP and OT. The fact that estrogen
receptors (ER) are expressed in key brain nuclei involved in body
fluid maintenance strongly suggests a role for estrogen in hydro-
electrolytic homeostasis; in rats, ER? are expressed in magnocel-
lular AVP and OT secretory neurons, and ER? are widely dis-
tributed in neurons in the basal forebrain nuclei (53, 55, 57, 58,
59, 60, 61).
Despite the apparently controversial results in the litera-
ture, estrogen’s effects on cardiovascular and hydroelectro-
lytic homeostasis seem to be at least partially due to its
influence on AVP secretion (55). In women, thirst and AVP
secretion induced by osmotic stimuli are diminished during
the luteal compared with the follicular phase of the men-
strual cycle (67). In postmenopausal women, Forsling et al.
(24) observed an increase in basal AVP plasma levels after
estrogen therapy. In animal models, Crofton et al. (14)
observed no changes in plasma AVP levels in OVX rats
treated with estrogen, whereas Skowsky et al. (54) observed
an increase in AVP secretion during proestrus compared
with diestrus in intact female rats and OVX rats treated with
estrogen. However, Crofton et al. (14) observed that estro-
gen plus progesterone reversed the OVX-induced increase
in AVP release, whereas Peysner and Forsling (45) observed
a reduction in plasma AVP concentrations after OVX and
either an increase (low dose) or reduction (high dose) in
AVP levels after estrogen treatment.
Although the influence of OT on reproductive function is
well established, knowledge of its effects on electrolyte bal-
Address for reprint requests and other correspondence: J. Antunes-
Rodrigues, Dept. of Physiology, Faculty of Medicine of Ribeirao Preto, Univ.
of Sao Paulo. Av. Bandeirantes, 3900, CEP 14049900. Ribeirao Preto, Sao
Paulo Brazil.
Am J Physiol Regul Integr Comp Physiol 301: R905–R915, 2011.
First published June 1, 2011; doi:10.1152/ajpregu.00800.2010.
0363-6119/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpregu.org R905
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ance and cardiovascular function is scarce. A positive correla-
tion between estrogen and plasma OT concentrations in young
women has been reported previously (2). Furthermore, gonadal
steroid hormones have been shown to be required for increases
in OT mRNA expression in magnocellular neurons of the SON
and PVN in rats subjected to osmotic stimulus (15).
Hemorrhagic hypovolemia is a common clinical condition
that occurs as a consequence of trauma, surgeries, gastrointes-
tinal diseases, and anticoagulant therapy (34). Understanding
the mechanisms involved in the control of homeostatic changes
after hemorrhage is extremely important for the development
of new therapeutic approaches. Because estrogen is closely
related to neuroendocrine systems, integrating cardiovascular
and hydroelectrolytic functions, the aim of this study was to
evaluate the influence of estrogen on cardiovascular and neu-
roendocrine changes induced by hemorrhagic hypovolemia in
OVX rats. Thus, we evaluated the effects of estradiol treatment
on ANG II, ANP, AVP, OT, and prolactin (PRL) secretion and
activation of vasopressinergic and oxytocinergic neurons in the
SON and PVN in OVX rats subjected to hemorrhage.
MATERIALS AND METHODS
Animals
Female Wistar rats (220–280 g) obtained from the Animal Facility
of the campus of Ribeirao Preto, University of Sao Paulo, Brazil, were
group-housed (4 animals/cage) under controlled temperature and light
conditions (23 ? 2°C, lights on between 0600 and 1800 h) with free
access to standard food pellets and tap water. All experiments were
performed in the morning (between 0700 and 1100 h).
This study was conducted according to the Guide for the Care and
Use of Laboratory Animals (NIH Publication No. 85–23, revised
1996), and the experimental protocols were approved by the Ethics
Committee on Animal Experiments of the Faculty of Medicine of
Ribeirao Preto at the University of Sao Paulo under protocol 041/
2009.
Surgeries
All surgical procedures were performed under anesthesia induced
by 2.5% 2,2,2-tribromoethanol (250 mg/kg, ip; Sigma) and were
followed by prophylactic doses of veterinary pentabiotic (Fort
Dodge).
Ovariectomy and estradiol treatment. Female rats were subjected
to bilateral OVX and randomly separated into groups treated with
corn oil (OVX-oil) or estradiol cypionate (EC; Pfizer) at 10 (OVX-EC
10) or 40 (OVX-EC 40) ?g/kg. Vehicle or EC administration (0.1
ml/rat, sc) started 24 h after OVX and was conducted one time per day
for eight consecutive days between 0700 and 1000 h. The efficiency
of the OVX procedure and estradiol treatment was confirmed by
estradiol plasma concentration and a uterine index (uterus weight/
body weight expressed as mg/100 g of body wt) after 8 days of
treatment.
We observed a significant increase in plasma estradiol concentra-
tions in a dose-dependent manner in the OVX-EC 10 and OVX-EC 40
groups compared with the OVX-oil group (72.2 ? 6.1 and 201.4 ?
30.4 vs. 21.6 ? 2.0 pg/ml, N ? 8, 10, and 7, respectively, P ? 0.05).
We also verified a significant increase in the uterine index in the
OVX-EC 10 and OVX-EC 40 groups when compared with the
OVX-oil group (216 ? 9.0 and 367.0 ? 23.5 vs. 53.5 ? 6.5 mg/100
g body wt, N ? 15, 14, and 15, respectively, P ? 0.001) (37, 38).
These results confirm the efficiency of estrogen treatment in reestab-
lishing estradiol plasma levels after ovariectomy and the trophic
uterine index. Additionally, published data in the literature support the
evidence that 10 ?g/kg of EC is sufficient for estrogen replacement as
a physiological dose (similar to estradiol concentrations found in
proestrus rats) and 40 ?g/kg as a supraphysiological dose (62).
However, because of different doses of estradiol used, several reports
have found diverse responses mediated by this steroid (24, 27, 45).
For this reason, we chose two different doses of estradiol in this study.
Femoral artery cannulation. For the hemorrhage procedure and
cardiovascular recordings, animals were subjected to right femoral
artery cannulation; a polyethylene cannula (PE-10 connected to PE-
50, Intramedic; Becton-Dickinson, Sparks, MD) was inserted and then
externalized in the dorsal cervical region. Soon after, the cannula was
flushed with isotonic saline containing heparin (100 IU/ml Liquemine;
Roche) to prevent obstruction. Animals were allowed to recover for
24 h before the experimental procedure.
Hemorrhage
Fifteen minutes before the hemorrhage procedure, isotonic sa-
line containing heparin (150 IU in 150 ?l) was administered
through the arterial catheter to prevent blood clotting during blood
removal. The hemorrhage procedure was achieved by blood re-
moval through the arterial catheter (15 ml/kg body wt, correspond-
ing to ?25% of total blood volume) for 1 min. False hemorrhage
was performed using the same protocols without blood withdrawal.
Cardiovascular Recordings
For mean arterial pressure (MAP, mmHg) and heart rate (HR,
beats/min) assessment, the arterial catheter was connected to a pres-
sure transducer (P23Gb; Statham Instruments, Hato Hey, Puerto Rico)
and data acquisition system (Windaq/200; Dataq Instruments). After
20 min of adaptation, baseline data were collected for 10 min, and the
blood withdrawal was then initiated, followed by monitoring of
hemodynamic changes for 30 min. The data represent several data
points from a continuous recording.
Plasma Hormone Determination
For plasma hormone measurements, animals were decapitated, and
the blood was collected from the trunk at 0 (baseline), 5, 15, and 30
min after hemorrhage induction in chilled tubes containing heparin
(for estradiol, AVP, OT, and PRL) or peptidase inhibitors (for ANG
II and ANP). Plasma was obtained after centrifugation (20 min, 3,000
rpm, 4°C) and stored at ?20°C until specific extraction and radioim-
munoassay procedures.
The radioimmunoassay for estradiol was performed with a com-
mercial kit from Diagnostic System Laboratories (DSL-4400; Web-
ster, TX). The specific antibodies for ANG II, AVP, and OT radio-
immunoassay were obtained from Peninsula (ANG II, T4007; AVP,
T4561; and OT, T4084; San Carlos, CA). PRL was obtained from the
National Institute of Diabetes and Digestive and Kidney Diseases
(PRL AFP131581570; Baltimore, MD), and ANP was donated by
Jolanta Gutkowska (Hotel Dieu, Univ. of Montreal, Montreal, Que-
bec, Canada). The radioimmunoassay sensitivity and intra- and inter-
assay coefficients of variation were 20 pg/ml, 1.4–27.1% for estradiol;
0.5 pg/ml, 10.9–17.1% for ANG II; 0.7 pg/ml, 4.8–10.0% for ANP;
0.8 pg/ml, 7.7–11.9% for AVP; 0.9 pg/ml, 7.0–12.6% to OT; and 0.9
ng/ml, 5.2–11.8% for PRL.
Perfusion, Tissue Preparation, and Immunohistochemistry
Ninety minutes after hemorrhage induction, the anesthetized ani-
mals were transcardially perfused with 200 ml of isotonic saline
containing heparin (50 IU/l) followed by 400 ml of 4% paraformal-
dehyde in 0.1 M phosphate buffer (PB), pH 7.2. The brains were
removed, fixed for 4 h in the perfusion solution, and stored at 4°C in
PB containing 30% sucrose.
Coronal sections of 30-?m thicknesses were obtained using a
cryostat (Micron) and collected in 0.01 M PB. Briefly, sections were
incubated with H2O2solution (0.03%) for 30 min and washed in 0.01
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PB. Sections were then incubated with 5% bovine albumin in 0.1 M
PB for 1 h. Sections were subsequently incubated at room temperature
for 12–14 h with the primary anti-Fos antibody (1:10,000, produced in
rabbits, Ab-5; Oncogene), washed, and then incubated with a biotin-
labeled antibody (1:200, produced in goats; Vector Laboratories,
Burlingame, CA) for 1 h. The avidin-biotin-peroxidase complex
(ABC) was used for staining (Vector Laboratories), followed by 0.5%
diaminobenzidine hydrochloride (DAB; Sigma Chemical, St. Louis,
MO) intensified with 5% cobalt chloride and 1% nickel ammonium
sulfate, which labels the cell nuclei black.
For double labeling, after concluding the Fos protocol described
above, sections were incubated with anti-OT or anti-AVP (1:10,000,
both produced in rabbits; Peninsula) for 48 h at 4°C. Thereafter,
sections were washed and subjected to the protocol described for Fos
labeling, using appropriate secondary biotinylated antibodies followed
by the ABC. The brown cytoplasmatic color was detected using a
nonintensified DAB solution. Finally, the sections were mounted on
gelatinized slides, air-dried overnight, dehydrated, cleared in xylene,
and placed under a cover slip with Ethelan.
Immunostained cells were quantified using a Leica microscope
equipped with a DC 200 Leica digital camera and coupled to a
computer using Leica Application Suite software. Visual counting
was performed unilaterally in one section per animal and was repeated
at least two times for each analyzed section.
The PVN and SON were identified and delimited according to the
Paxinos and Watson atlas (43). The subfornical organ (SFO) and SON
were examined on their medial portions (?0.84 and ?1.08 mm from
the bregma, respectively). The PVN was counted in the following four
different areas: medial magnocellular (PaMM, ?1.44 mm from the
bregma), lateral magnocellular (PaLM, ?1.72 mm from the bregma),
medial parvocellular (PaMP, ?1.72 mm from the bregma), and
posterior parvocellular (PaPO, ?2.04 mm from the bregma). The
schematic brain areas are shown in Fig. 1.
Statistical Analysis
Results are expressed as means ? SE. For statistical purposes,
estradiol results below the detection limit of the assay were assigned
the value of the detection limit. Statistical analyses were conducted
using one-way or two-way ANOVA (factors: time or hemorrhage and
treatment) followed by the Bonferroni posttest. The level of signifi-
cance was set at P ? 0.05.
Experimental Protocols
Protocol 1: Effects of estradiol on MAP and HR in OVX rats
submitted to hemorrhage. Animals were connected to the acquisition
system, and, after 20 min of adaptation, baseline parameters were
measured for 10 min. Next, blood withdrawal was performed, animals
were reconnected to the system, and recording of the cardiovascular
parameters resumed. Our results show the values of MAP and HR at
0 (baseline), 5, 15, and 30 min after hemorrhage induction.
Protocol 2: Effects of estradiol on hemorrhage-induced hormone
secretion in OVX rats. Blood collected during hemorrhage induction
(basal) and by decapitation (5, 15, or 30 min after hemorrhage) was
used for radioimmunoassay measurements of ANG II, ANP, AVP,
OT, and PRL plasma levels.
Protocol 3: Effects of estradiol on SFO, SON, and PVN neuronal
activation in OVX rats submitted to hemorrhage. Ninety minutes after
hemorrhage or false-hemorrhage induction, animals were anesthetized
and subjected to brain perfusion for subsequent immunohistochemical
procedures. Our results show the absolute number of Fos-positive
neurons in the SFO, SON, and PVN subdivisions and the percentage
of double-labeled neurons for Fos/AVP and Fos/OT in the SON and
PVN subdivisions.
RESULTS
Protocol 1: Effects of Estradiol on MAP and HR in OVX
Rats Subjected to Hemorrhage
Basal MAP was similar among the OVX-oil (110.2 ? 2.4
mmHg), OVX-EC 10 (107.0 ? 3.1 mmHg), and OVX-EC 40
(104.3 ? 3.1 mmHg) groups. However, we observed an estradiol
effect on HR in basal conditions [F(2,18) ? 4.3; P ? 0.05].
Estradiol treatment induced a significant reduction in basal HR in
the OVX-EC 10 (385.6 ? 20.5 beats/min, P ? 0.01) and
Fig. 1. Representative drawing (coronal sections) of the subfornical organ
(SFO), supraoptic nucleus (SON), and paraventricular nucleus (PVN) subnu-
clei. A1, SFO (bregma ?0.84 mm); A2, SFO representative photomicrograph;
B1, SON (bregma ?1.08 mm); B2, SON representative photomicrograph; C1,
PVN medial magnocellular region (PaMM) (bregma ?1.44 mm); C2, PaMM
representative photomicrograph; D1, PVN lateral magnocellular (PaLM) and
posterior magnocellular (PaMP) regions (bregma ?1.72 mm); D2, PaLM and
PaMP representative photomicrograph; E1, PVN posterior parvocellular region
(PaPO) (bregma ?2.04 mm); E2, PaPO representative photomicrograph.
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OVX-EC 40 (395.1 ? 12.5 beats/min, P ? 0.01) groups com-
pared with the OVX-oil group (452.3 ? 18.2 beats/min).
Figure 2 shows the difference (?) in basal MAP (Fig. 2A)
and HR (Fig. 2B) at 5, 15, and 30 min after hemorrhage in all
groups. There was a significant effect of time on ?MAP
[F(8,54) ? 5.2; P ? 0.01] and ?HR [F(8,54) ? 6.8; P ? 0.01].
We also observed an effect of treatment (oil, EC 10, and EC
40) on ?MAP [F(8,54) ? 7.2; P ? 0.01] and ?HR [F(8,54) ?
3.7; P ? 0.05]. Furthermore, there was an interaction between
treatment and time on ?MAP [F(8,54) ? 5.2; P ? 0.01] but
not ?HR.
In OVX-oil rats, hemorrhage induced a decrease in MAP (89.7 ?
3.9 vs. 110.2 ? 2.4 mmHg, P ? 0.01) and HR (400.4 ? 11.3 vs.
452.3 ? 18.2 beats/min, P ? 0.05) at 5 min compared with
basal values. However, estradiol treatment abolished the hy-
potension 5 min after hemorrhage in OVX-EC 10 (P ? 0.001)
and OVX-EC 40 (P ? 0.001) rats compared with the OVX-oil
group. Likewise, EC 10 and EC 40 treatment also abolished
(P ? 0.05) the HR decrease response induced by blood
withdrawal compared with the oil-treated rats simultaneously
with hemorrhage.
Protocol 2: Effects of Estradiol on Hemorrhage-Induced
Hormone Secretion in OVX Rats
Plasma concentrations of ANG II (Fig. 3A) and ANP (Fig.
3B) are shown in Fig. 3. We observed an effect of time on
plasma ANG II [F(11,99) ? 14.6; P ? 0.001] and ANP
[F(11,98) ? 12.5; P ? 0.001] concentrations. There was no
interaction between treatment and time after hemorrhage on
these parameters.
In the OVX-oil group, hemorrhage induced a significant
increase in plasma ANG II at 5 (P ? 0.01), 15 (P ? 0.01), and
30 (P ? 0.01) min after the procedure compared with basal
conditions. However, hemorrhage decreased plasma ANP con-
centrations at 5 (P ? 0.01), 15 (P ? 0.01), and 30 (P ? 0.01)
min in the OVX-oil rats compared with basal conditions.
Estradiol treatment (both doses) did not alter ANG II or ANP
secretion compared with oil treatment.
Plasma AVP (Fig. 4A), OT (Fig. 4B), and PRL (Fig. 4C)
concentrations are shown in Fig. 4. There was an effect of time
on plasma AVP [F(11,102) ? 29.3; P ? 0.001], OT
[F(11,102) ? 13.4; P ? 0.001], and PRL [F(11,106) ? 39.2;
P ? 0.001] concentrations. Plasma PRL basal levels were also
increased by estradiol treatment [F(11,106) ? 30.7; P ?
0.001]. Finally, we observed a significant interaction between
estradiol treatment and time after hemorrhage on AVP
[F(11,102) ? 3.2; P ? 0.01], OT [F(11,111) ? 3.2; P ? 0.01],
and PRL [F(11,106) ? 7.8; P ? 0.01] secretion.
In OVX-oil rats, hemorrhage induced an increase in plasma
AVP concentrations at 5 (P ? 0.001), 15 (P ? 0.001), and 30
(P ? 0.05) min postprocedure compared with basal conditions.
Five minutes after hemorrhage, EC 10 and 40 treatments
potentiated the AVP secretion induced by hemorrhage com-
pared with the OVX-oil group (P ? 0.001). Hemorrhage also
Fig. 3. The effects of EC treatment (10 or 40 ?g/kg) on plasma angiotensin II
(ANG II, A) and atrial natriuretic peptide (ANP, B) concentrations in response
to hemorrhage in ovariectomized (OVX) rats. The no. of animals/group is
shown in the respective bar. *P ? 0.05 between basal and hemorrhage.
Fig. 2. The effects of estradiol cypionate (EC) treatment (10 or 40 ?g/kg) on
mean arterial pressure (A) and heart rate (B) in response to hemorrhage in OVX
rats; N ? 7 animals/group. ?, Change. ?P ? 0.05 and ???P ? 0.001
between the OVX-oil and OVX-EC groups.
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induced an increase in plasma OT levels at 5 (P ? 0.05), 15
(P ? 0.05), and 30 (P ? 0.05) min in the OVX-oil rats
compared with baseline. However, only the EC 40 treatment
enhanced OT secretion 5 min after hemorrhage (P ? 0.05)
compared with the OVX-oil group.
We also observed an increase in plasma PRL concentrations
5 min after blood withdrawal (P ? 0.05) in OVX-oil rats
compared with baseline. At both 5 and 15 min after blood
withdrawal, PRL secretion was strongly potentiated by EC 10
(5 min, P ? 0.001; 15 min, P ? 0.05) and EC 40 (5 min, P ?
0.001; 15 min, P ? 0.001) compared with the OVX-oil group.
Protocol 3: Effects of Estradiol on SFO, SON, and PVN
Neuron Activation in OVX Rats Submitted to Hemorrhage
Estradiol effects on hemorrhage-induced Fos in SFO and
Fos/AVP or OT neuronal activation in the PVN and SON are
summarized in Table 1. Hemorrhage induced a significant
increase in the number of Fos-labeled neurons in the SFO,
SON, and PVN subdivisions and the percentage of Fos/AVP
and Fos/OT double-labeled neurons in the PVN subdivisions
and SON of the OVX-oil rats. The EC 10 and 40 treatments
induced a significant increase in Fos expression in the SFO
nucleus induced by hemorrhage in OVX rats. Additionally,
after hemorrhage, EC 10-treated rats showed a significant
increase in the number of Fos-positive neurons in the PaLM
and PaPo and in Fos/AVP double-labeled cells in the SON and
PaMM compared with the OVX-oil group. The results also
showed that hemorrhage increased the number of Fos-immu-
noreactive neurons in the SON and all PVN subdivisions in
OVX-EC 40 rats compared with the OVX-oil group. Further-
more, the higher EC dose potentiated the percentage of Fos/
AVP double-labeled neurons in the SON and PaMM, PaLM,
and PaMP subdivisions of the PVN after blood withdrawal
compared with oil treatment. EC 40 treatment also induced a
significant increase in the percentage of Fos/OT double-labeled
neurons in the SON and magnocellular PVN of hemorrhagic
rats compared with the OVX-oil group.
The representative photomicrographs from SFO showing
Fos-labeled neurons, SON showing Fos/AVP double-labeled
neurons, and the lateral magnocellular PVN showing Fos/OT
double-labeled neurons in sham or hemorrhage rats pretreated
with oil or EC are shown in Figs. 5, 6, and 7, respectively.
DISCUSSION
In the present report, we observed that blood withdrawal
reduced MAP and HR only at 5 min in the OVX-oil group.
Hypotension induced by mild to moderate hemorrhage is
usually accompanied by a tachycardic reflex response (29);
however, we used moderate to severe hemorrhage in the
present study, which is known to produce a bradycardic re-
sponse as a consequence of the vasovagal reflex, i.e., an acute
central hypovolemia with a consequent parasympathetic acti-
vation (1, 20, 40, 48). The bradycardic reflex induced by
parasympathetic activation is likely to involve activation of the
periaqueductal gray substance and serotoninergic system that
sends inhibitory projections to the rostral ventrolateral medulla
(RVLM), a crucial area implicated in the generation and
modulation of sympathetic activity to the cardiovascular sys-
tem (7, 9, 49, 51).
We observed an inhibition of the hypotension and brady-
cardic responses induced by hemorrhage in OVX-EC rats
compared with the OVX-oil group. The sex steroid hormones
are required for changes in baroreflex sensitivity, as shown by
the changes that occur in the cardiovagal baroreflex sensitivity
and renal sympathetic nerve activity during the estrous cycle of
female rats that are abolished by OVX (25). Additionally,
several studies have demonstrated the modulation of hypo-
tensive responses by estrogen, although the data are contro-
versial. Estrogen attenuated the hypotensive response after
the administration of a ?-adrenergic agonist (isoproterenol)
but did not influence the tachycardic reflex response (32).
However, the recovery of MAP and HR after hemorrhage in
Long Evans female rats was impaired by estrogen. This did
not occur in AVP-deficient animals (40). Our results con-
trast with those obtained by these authors, possibly because
of methodological differences in time and total blood vol-
Fig. 4. The effects of EC treatment (10 or 40 ?g/kg) on vasopressin (AVP, A),
oxytocin (OT, B), and prolactin (C) plasma concentrations in response to
hemorrhage in OVX rats. The no. of animals/group is shown in the respective
bar. *P ? 0.05, **P ? 0.01, and ***P ? 0.001 between basal and hemorrhage
levels. ?P ? 0.05, ??P ? 0.01, and ???P ? 0.001 between the OVX-oil
and OVX-EC groups.
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Table 1. Number of total Fos in SFO and total Fos and percentage of Fos/AVP and Fos/OT double-labeled neurons in SON and subdivisions of PVN of control
and hemorrhage OVX-oil-, OVX-EC 10-, and OVX-EC 40-treated animals
Control
Hemorrhage
Statistics
Oil
EC 10
EC 40
Oil
EC 10
EC 40
Hemorrhage
Treatment
Interaction
SFO
Fos
2.0 ? 0.5
2.3 ? 0.5
2.5 ? 1.0
34.9 ? 5.3a
63.7 ? 10a,b
79.1 ? 14.8a,b
F(5,29) ? 65.7, P ? 0.0001
F(5,29) ? 6.9, P ? 0.05
F(5,29) ? 6.6, P ? 0.05
SON
Fos
5.1 ? 1.4
4.4 ? 1.6
6.0 ? 1.2
57.3 ? 5.8a
86.8 ? 6.0a
126 ? 19.2a,b
F(5,29) ? 111, P ? 0.0001
F(5,29) ? 5.6, P ? 0.01
F(5,29) ? 6.5, P ? 0.01
Fos/AVP, %
2.5 ? 1.1
2.6 ? 1.6
1.3 ? 1.3
22.1 ? 3.4a
31.6 ? 4.1a,b
42.6 ? 6.6a,b
F(5,29) ? 641, P ? 0.0001
F(5,29) ? 5.8, P ? 0.01
F(5,29) ? 7.3, P ? 0.01
Fos/OT, %
2.5 ? 1.1
2.6 ? 1.5
1.3 ? 1.3
22.1 ? 3.4a
31.6 ? 4.0a
42.6 ? 6.5a,b
F(5,29) ? 100, P ? 0.0001
F(5,29) ? 3.5, P ? 0.05
F(5,29) ? 4.4, P ?0.05
PaMM
Fos
4.2 ? 1.5
2.9 ? 1.1
3.9 ? 2.5
34.9 ? 7.7a
58.4 ? 12.3a
90.6 ? 15.6a,b
F(5,29) ? 52,5, P ? 0.0001
F(5,29) ? 4.1, P ? 0.05
F(5,29) ? 4.1, P ? 0.05
Fos/AVP, %
3.6 ? 1.0
1.2 ? 0.7
0.9 ? 0.6
17.5 ? 2.4a
34.6 ? 6.6a,b
46.5 ? 5.4a,b
F(5,29) ? 110, P ? 0.0001
F(5,29) ? 6.7, P ? 0.01
F(5,29) ? 9.7, P ? 0.01
Fos/OT, %
0.7 ? 0.3
1.1 ? 0.6
1.1 ? 0.9
19.4 ? 2.5a
31.8 ? 3.3a
49.6 ? 7.8a,b
F(5,29) ? 112, P ? 0.0001
F(5,29) ? 8.2, P ? 0.01
F(5,29) ? 7.9, P ?0.01
PaLM
Fos
2.2 ? 1.0
3.1 ? 0.73
1.9 ? 1.1
36.6 ? 8.2a
66.0 ? 8.9a,b
97.4 ? 12.8a,b
F(5,29) ? 100, P ? 0.0001
F(5,29) ? 7.4, P ? 0.01
F(5,29) ? 7.5, P ?0.01
Fos/AVP, %
1.1 ? 0.3
0.7 ? 0,4
1.2 ? 0.6
33.7 ? 5.3a
45.3 ? 7.9a
59.9 ? 5.8a,b
F(5,29) ? 148, P ? 0.0001
F(5,29) ? 4.2, P ? 0.05
F(5,29) ? 4.1, P ?0.05
Fos/OT, %
0.4 ? 0.3
0.8 ? 0.4
0.8 ? 0.6
17.7 ? 3.7a
26,5 ? 5.1a
42.2 ? 5.9a,b
F(5,29) ? 88,7, P ? 0.0001
F(5,29) ? 5.9, P ? 0.01
F(5,29) ? 5.6, P ? 0.01
PaMP
Fos
3.8 ? 1.3
3.8 ? 1.4
5.9 ? 3.2
32.6 ? 5.0a
46.2 ? 10.9a
62.0 ? 5.9a,b
F(5,29) ? 77.4, P ? 0.0001
F(5,29) ? 3.6, P ? 0.05
P ? 0.05
Fos/AVP, %
2.0 ? 1.2
2.2 ? 1.0
5.5 ? 2.8
23.7 ? 4.7a
28.8 ? 5.9a
46.0 ? 5.7a,b
F(5,29) ? 68.4, P ? 0.0001
F(5,29) ? 4.6, P ? 0.05
P ? 0.05
Fos/OT, %
3.2 ? 2.1
1.1 ? 1.1
1.0 ? 1.0
24.5 ? 2.9a
29.3 ? 5.5a
33.4 ? 5.7a
F(5,29) ? 71,7, P ? 0.0001
P ? 0.05
P ? 0.05
PaPo
Fos
5.4 ? 1.1
5.4 ? 1.4
4.3 ? 0.7
23.7 ? 4.6a
42.6 ? 4.6a,b
44.0 ? 10.0a,b
F(5,29) ? 48.5, P ? 0.0001
P ? 0.05
P ? 0.05
Fos/AVP, %
3.8 ? 2.2
2.9 ? 1.0
1.7 ? 1.1
19.2 ? 5.8a
22.9 ? 4.9a
32.9 ? 6.1a
F(5,29) ? 49.4, P ? 0.0001
P ? 0.05
P ? 0.05
Fos/OT, %
2.6 ? 2.2
1.7 ? 0.8
1.0 ? 1.0
17.9 ? 3.4a
18.1 ? 3.6a
16.8 ? 3.5a
F(5,29) ? 48.1, P ? 0.0001
P ? 0.05
P ? 0.05
Data are expressed as means ? SE. Two-way ANOVA, followed by Newman-Keuls posttest, was used. OVX, ovariectomized; EC 10, 10 ?g/kg estradiol cypionate; EC 40, 40 ?g/kg estradiol cypionate;
SFO, subfornical organ; SON, supraoptic nucleus; PVN, paraventricular nucleus; PaMM, medial magnocellular; PaLM, lateral magnocellular; PaMP, medial parvocellular; PaPo, posterior parvocellular; AVP,
vasopressin; OT, oxytocin. P ? 0.05 vs. respective control group (a) and vs. respective oil group (b).
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ume removal. Women in the postmenopausal period and
during OVX, rats present an increase in sympathetic activity
with higher ?1-adrenergic activation and peripheral vaso-
constriction associated with a reduction in nitric oxide
synthesis, possibly because of estrogen deficiency (68). As
a result, we suggest that both the lower basal HR in
OVX-EC rats and attenuation of cardiovascular responses
induced by hemorrhage in the OVX-EC group found in the
present study could be a consequence of estrogen effects on
autonomic activity.
Fig. 5. Representative photomicrographs (coronal sections) of the SFO showing Fos immunoreactive neurons in sham or hemorrhage rats pretreated with oil or
EC (10 or 40 ?g/kg). In detail, the SFO is shown in a small magnification. Scale bar: 100 ?m.
Fig. 6. Representative photomicrographs (coronal sections) of the SON showing Fos/AVP immunoreactive neurons in sham or hemorrhage rats pretreated with
oil or EC (10 or 40 ?g/kg). In detail, the SON is shown in a small magnification. Scale bar: 100 ?m.
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Plasma ANP concentrations were reduced in all groups after
blood withdrawal in accordance with well-established data in
the literature demonstrating a decrease in its concentrations in
response to hypovolemia (69). However, despite previous dem-
onstrations of the colocalization of estrogen and ANP receptors
in cardiomyocytes and ANP secretion induced by estrogen
perfusion in atrial cardiac cells, we did not observe an influ-
ence of estrogen on ANP secretion under basal or hemorrhagic
conditions (4, 19). Although estrogen was shown to modulate
ANP secretion in response to osmolality changes and extracel-
lular volume expansion (66), it seems that the reduction of
ANP release during hemorrhage is not influenced by estrogen
treatment.
We also showed an important increase in plasma ANG II
concentrations in all groups after the hemorrhage procedure.
Systemic ANG II interacts with AT1receptors located in the
vascular smooth muscle, leading to its contraction and conse-
quently elevating arterial pressure, an important mechanism in
reestablishing tissue perfusion after blood withdrawal (17).
Furthermore, circulating ANG II also acts on the central
nervous system (CNS) structures devoid of blood-brain barrier,
such as the SFO, contributing to the arterial pressure mainte-
nance (17, 36). Estradiol had no effect on the ANG II increase
induced by hemorrhage.
It is possible that blood loss may not be the best experimen-
tal paradigm to evaluate the effects of estrogen on ANP
secretion and RAS activity because hemorrhage is a potent
stimulus for RAS and inhibits ANP secretion. Therefore, the
potent neuroendocrine stimulation or inhibition may overcome
the modulatory effects of estradiol on these systems.
Our results show an increase of Fos-immunoreactive neu-
rons in the SFO after hemorrhage in OVX-EC-treated animals.
Because there was no difference between plasma ANG II of
OVX-oil and OVX-EC groups after hemorrhage and because it
is well established that estrogen treatment reduces AT1mRNA
expression (18), we suggest that the increase in SFO neuronal
firing by estrogen is not related to the enhancement of ANG II
signaling. The SFO integrates the information from other areas
that participate in the control of cardiovascular function with
those from plasma ANG II concentrations and retransmits this
information to the PVN and SON, a possible pathway through
which plasma ANG II at least partially modulates AVP and OT
secretion (64). The SFO receives (and sends) important pro-
jections from (and to) areas involved with hydroelectrolytic
and cardiovascular balance, including the PVN, SON, RVLM,
and nucleus tractus solitarius (23). The influence of estrogen on
SFO neuronal activation could be exerted by direct action on
the ER? in the SFO, decreasing the threshold activation of
these neurons or acting through the above-mentioned areas to
increase stimulatory inputs to the SFO.
As expected, plasma AVP concentration was increased 5
min after blood withdrawal in all groups evaluated, and this
response is mediated by baroreceptors, volume receptors, and
increased plasma ANG II concentrations (3). Interestingly,
there was an increase in AVP secretion induced by hemorrhage
in the OVX-EC rats, which is in accordance with previous
studies observing a similar estrogen effect in rats and women
both in basal conditions and after an osmotic or hypovolemic
stimulus (24, 27, 45, 54).
Hypovolemia induced by hemorrhage increases AVP and
OT plasma levels and the number of Fos-positive magnocel-
lular neurons in the PVN and SON (8, 52). This response is
likely modulated by estrogen because it changes the number of
Fos-labeled cells in these nuclei observed in response to a
variety of stimuli (27, 31). Estrogen also attenuates hypoten-
sion and the number of Fos-positive cells in the area postrema
and lateral parabrachial nucleus, areas closely related to these
neuroendocrine responses (32).
Fig. 7. Representative photomicrographs (coronal sections) of the lateral magnocellular PVN showing Fos/OT immunoreactive neurons in sham or hemorrhage
rats pretreated with oil or EC (10 or 40 ?g/kg). In detail, the PVN is shown in a small magnification. Scale bar: 100 ?m.
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During hemorrhage, vasomotor brain nuclei relay peripheral
information to the PVN and SON, culminating with the marked
magnocellular vasopressinergic neuron activation seen in the
present study and subsequent enhancement of plasma AVP
levels (6). It has been demonstrated that exogenous adminis-
tration of AVP induces significant bradycardia in AVP-defi-
cient rats, suggesting that the endogenous hormone may con-
tribute to the vagally mediated bradycardia induced by hem-
orrhage (28). Peuler et al. (44) demonstrated that sympathetic
nerve activity and HR during hemorrhage were consistently
higher in diabetes insipidus rats compared with normal Long-
Evans or AVP-treated rats both before and after vagotomy. In
addition, these authors show that vagotomy attenuated the
inhibitory effect of AVP on the HR response but not renal
sympathetic nerve activity following hemorrhage in the same
experimental model of AVP-deficient animals. Taken together,
these results suggest that AVP may play a major role in the
cardiovascular adjustments following hemorrhage.
In the present report, we observed an increase in OT and
AVP magnocellular neuronal activation, assessed by the num-
ber of neurons double labeled for Fos/OT and Fos/AVP and
hormone plasma concentrations in response to hemorrhage.
Previous studies have demonstrated that AVP and OT could
present negative chronotropic effects mediated by cardiac V1
receptors (13, 22). However, the estrogen effect attenuating the
bradycardic response seems not to be related to OT and AVP
plasma concentrations because estrogen potentiated hormone
secretion after hemorrhage.
We also report a potentiation of AVP magnocellular neuro-
nal activation mediated by estrogen following hemorrhage.
Previous reports have already shown estrogen effects on AVP
secretion and SON neuronal activation in female rats subjected
to hemorrhage. However, they did not demonstrate the neuro-
nal phenotype involved in this response, which could be AVP-
or OT-producing cells (27). Because PVN and SON neurons
express ER?, the modulation exerted by estrogen on AVP
magnocellular activity and AVP secretion could be produced
by a direct effect on the hypothalamic neuroendocrine system
or mediated by the activation of other central areas controlling
plasma volume and arterial pressure, such as the SFO (53, 55,
57, 58, 59, 60, 61). Thus, the increase of AVP secretion
mediated by estrogen after blood withdrawal is consistent with
the attenuation of hypotension mediated by estrogen observed
5 min after the hemorrhage.
Additionally, we demonstrated that parvocellular PVN neu-
rons, mostly vasopressinergic neurons, were activated in re-
sponse to hemorrhage, as previously described (5). Hypoten-
sive hemorrhage culminates in the simultaneous activation of
AVP- and corticotropin-releasing hormone parvocellular neu-
rons in the PVN, and it increases adrenocorticotropin hormone
secretion (8). Parvocellular PVN neurons maintain reciprocal
communication with other CNS areas related to the integrated
control of cardiovascular and hydromineral balance. The in-
creased activation of vasopressinergic parvocellular PVN neu-
rons could indicate that AVP may act as a neurotransmitter
and/or neuromodulator in areas related to arterial pressure
control in hemorrhagic conditions (3, 8, 43, 56, 52). Further-
more, we also demonstrated that estrogen treatment potentiated
AVP neuronal activation in the medial parvocellular PVN.
In our study, we observed a significant increase in magno-
cellular oxytocinergic activation in the PVN and SON and a
concomitant increase in plasma OT concentrations following
blood withdrawal, as previously described in response to hy-
potension and/or hypovolemia (56). In addition, we demon-
strate an intense parvocellular oxytocinergic neuronal activation
induced by hemorrhage, which could indicate some physiological
role for OT release as a neurotransmitter in hypovolemia and/or
hypotension conditions. Treatment with estrogen potentiated the
effects of hemorrhage on the activation of OT magnocellular
neurons in the PVN, which corresponded with an enhancement of
OT secretion in OVX-EC rats. However, estrogen did not change
the response induced by hemorrhage on the number of Fos/OT
double-labeled cells in the parvocellular groups, suggesting that
estrogen acts selectively on AVP parvocellular neurons from the
PVN in hypovolemic hemorrhagic conditions.
The potent increase in AVP and OT secretion observed in
response to an acute hemorrhage induced a simultaneous re-
duction of hormone content in the SON and PVN under the
same experimental conditions in male rats (47). Previous
reports have also demonstrated the increased secretion of PRL
after hemorrhage in male rats (35).
Some studies have shown a positive correlation between
stress intensity, OT hypothalamic synthesis, and its release to
systemic circulation, which was not found for AVP (21).
Furthermore, PRL, a stress-related hormone in rodents, was
increased by hemorrhage in the present study. This evidence
also supports the hypothesis that the observed increase in OT
secretion following hemorrhage could be a nonspecific stress
response rather than an effect induced by hypotension or
decreased circulating volume (30). Our study also showed that
estrogen induces a further increase in OT magnocellular neu-
ronal activation and OT and PRL secretion in response to
hemorrhage, which corroborates previous findings showing
that estrogen can potentiate some effects demonstrated by
stress models (39).
Somponpun and Sladek (60) showed a clear influence of
hypovolemia on reducing ER? expression in the PVN and
SON. However, these authors did not perform acute challenges
because ER? expression was evaluated 8 h after polyethylene
glycol administration plus water deprivation or 20 and 26 h
after water deprivation. In our study, we found a potentiation of
AVP and OT secretion mediated by estrogen (5 min after
hemorrhage) and the double labeling of Fos/AVP and Fos/OT
90 min after hemorrhage. Thus, it is important for future
studies to verify the ability of an acute hypovolemic stimulus
and its time-dependent profile in altering the expression of
ER?. However, since hormonal evaluations were performed 5
min after hypovolemia in the present study, it is unlikely that
a change in ER? expression induced by hemorrhage is respon-
sible for these observed responses. Instead, this response could
be mediated by estrogen signaling through ER? in the PVN
and SON and/or ?- and ?-receptors located in other important
brain areas for cardiovascular maintenance.
Nomura et al. (41) reported a decrease in AVP transcript
levels induced by estrogen, which was abolished in ER?
knockout mice. Thus, we suggest that the potentiating effect on
AVP secretion and PVN and SON neuronal activation induced
by estrogen may not be related to ER? signaling. In a recent
report, Grassi et al. (26) demonstrated that the increase in AVP
immunoreactivity in the PVN and SON induced by estrogen
was not due to ER? signaling, but it could be mediated by
ER?. Because magnocellular neurons in the PVN and SON do
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not express ER?, this response (as in our experimental design)
could be mediated by afferent ER?-expressing neurons from
different areas, such as the SFO.
Estrogen has important genomic actions regulating the ex-
pression of several genes related to cellular excitability, includ-
ing the synthesis of neurotransmitters, expression of ion chan-
nels, and activation of intracellular cascades (10). Therefore,
we hypothesize that estrogen, through its genomic and non-
genomic actions, can change the excitability threshold of
circuits related to AVP and OT secretion. However, treatment
with estrogen has been shown to decrease the expression of
ER? in the PVN (42). Moreover, ER? is mainly expressed in
AVP neurons, whereas ?7–10% of ER? reside in magnocel-
lular OT cells in both male and female rats (58, 63). However,
we observed an estrogen-mediated potentiation of OT release
and neuronal activity after hemorrhage compared with the
oil-treated OVX group. In this context, Nomura et al. (41)
demonstrated an increase in OT mRNA levels in the PVN after
estrogen therapy that was abolished in ER? knockout mice,
suggesting a positive effect of ER? signaling in OT neurons.
Additionally, inhibitory afferents (i.e., activation of barorecep-
tors) also participate in the tonic control of OT and AVP
secretion (via the brain stem, which express ER? and ER?)
(53). Together with the potentiation of SFO neuronal activation
induced by estrogen, these data led us to again hypothesize that
estrogen could also act in other brain areas potentiating stim-
ulatory pathways and/or decreasing inhibitory pathways to the
PVN and SON, thus acting on the AVP and OT.
Significance and Perspectives
Taken together, our results suggest that estrogen modulates
some cardiovascular and neuroendocrine responses induced by
acute hemorrhage, and the data provide valuable evidence on
the influence of estrogen on body fluid homeostasis. Further-
more, hemorrhage is a very common condition in medical
clinics, and our results discriminate between several cardiovas-
cular and neuroendocrine responses in OVX rats receiving or
not receiving estrogen therapy after blood loss, which could
give new insights into hemorrhage management during the
postmenopausal and/or postpartum period. Further studies are
needed to investigate the role of ER? on AVP and OT
secretion and the importance of estrogen signaling in other
brain areas in the acute hypovolemia model performed in this
study.
ACKNOWLEDGMENTS
We thank Rubens Fazan, Jr., for kindly providing equipment for the
cardiovascular assessments and Maria Valci dos Santos Silva, Milene Man-
tovani, Rubens Fernando de Mello, and Carlos Alberto A. Silva for excellent
technical assistance.
GRANTS
This work was supported by the Fundação de Amparo a Pesquisa do Estado
de São Paulo, Brazil, and the Conselho Nacional de Desenvolvimento Cientí-
fico e Tecnológico.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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