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The effects of experimental gestational hypertension on maternal blood pressure and fluid intake and pre-weanling hypothalamic neuronal activity
Accepted Manuscript The effects of experimental gestational hypertension on maternal blood pressure and fluid intake and pre-weanling hypothalamic neuronal activity Juan Arguelles, Carmen Perillan, Terry G. Beltz, Baojian Xue, Daniel Badaue-Passos, Jr., Jose A. Vega, Alan Kim Johnson PII:
S0195-6663(16)30820-0
DOI:
10.1016/j.appet.2017.04.008
Reference:
APPET 3421
To appear in:
Appetite
Received Date: 22 November 2016 Revised Date:
7 April 2017
Accepted Date: 8 April 2017
Please cite this article as: Arguelles J., Perillan C., Beltz T.G., Xue B., Badaue-Passos D., Jr, Vega J.A. & Johnson A.K., The effects of experimental gestational hypertension on maternal blood pressure and fluid intake and pre-weanling hypothalamic neuronal activity, Appetite (2017), doi: 10.1016/ j.appet.2017.04.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The Effects of Experimental Gestational Hypertension on Maternal Blood Pressure and Fluid Intake and Pre-Weanling Hypothalamic Neuronal Activity
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Passos Jrc, Jose A. Vegad,e, Alan Kim Johnsonb
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Juan Arguellesa, Carmen Perillana, Terry G. Beltzb, Baojian Xueb, Daniel Badaue-
a
Departamento de Biología Funcional, Área de Fisiología, Facultad de Medicina y
Ciencias de la Salud, Universidad de Oviedo, Spain b
Department of Psychological and Brain Sciences, The University of Iowa, Iowa
City, IA, USA, 52242 c
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Currently at Departamento de Fisiologia, Centro C. Biológicas e Saúde,
Universidade Federal de Sergipe, Sau Cristovao-SE, Brazil d
Departamento de Morfologia y Biologia Celular, Universidad de Oviedo, Spain
e
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Chile, Chile
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Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Santiago de
Corresponding author: Juan Arguelles, Departamento Biología Funcional, Area de Fisiología, Universidad de Oviedo, Spain; email: [email protected]
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ABSTRACT To examine the fetal programming effects of maternal hypertension, natriophilia and
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hyperreninemia [experimentally induced in rats by partial inter-renal aortic ligature (PAL) prior to mating] fos immunoreactivity was studied in 6-day-old offspring of PAL and control mothers. The purposes of the present set of experiments were twofold. The first was to characterize the effects of PAL on the mother’s arterial blood pressure and intake of salt (1.8% NaCl solution) and
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water over the course of gestation. Second, was to study the pattern of neuronal activation in key brain areas of 6-day-old offspring treated with the dipsogen isoproterenol that were from PAL
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and control mothers. Beta-adrenergic receptor agonist-treated pups allowed the determination whether there were neuroanatomical correlates within the neural substrates controlling thirst and the enhanced water intake evidenced by the isoproterenol treated pups of PAL mothers. Hydromineral ingestive behavior along with blood pressure and heart rate of PAL (M-PAL) and control (M-sPAL) dams throughout gestation was studied. Higher salt and water intakes along with blood pressures and heart rates were found during gestation and lactation in the M-PAL
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group. Maternal PAL evoked significantly increased isoproterenol-elicited Fos staining in brain regions (e.g. subfornical organ, organum vasculosum of the lamina terminalis, supraoptic nucleus, hypothalamic paraventricular nucleus and median preoptic nucleus) of 6-day-old pups, which is the age of animals shown enhanced thirst responses in PAL offspring. These results indicate that
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PAL is compatible with pregnancy, producing a sustained increase in blood pressure and heart rate, along with increased water and salt intake. The present study demonstrates that the neural
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substrates involved in cardiovascular homeostasis and fluid balance in adult rats are responsive in six-day-old rats, and can be altered by fetal programming. Key Words:
Ontogeny, Thirst, Gestation, Isoproterenol, Hypertension, c-fos, renin-angiotensin-aldosterone system
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1. Introduction1 Many authors have proposed that perturbations in maternal physiology during gestation alters the offspring in ways to make them more vulnerable to disease in adulthood. Intrauterine
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fetal programming (16, 26) can be induced by physiological challenges or threats to homeostasis that occur to the mother, during critical or sensitive periods of neonatal development. Such challenges affect the structure, physiology or metabolism in a manner that alters function later in life (3, 17).
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Intrauterine programming has frequently been discussed in relation to cardiovascular diseases (3, 17), and it has been hypothesized that such developmental influences on adult vital
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functions may have influenced population survival in times of drought and famine (41). Both experimental and natural challenges to maternal and offspring water and sodium distribution and balance and the cardiovascular system during perinatal critical periods, have been shown to have later consequences on body fluid and cardiovascular homeostasis in rats (6-8, 28, 31, 32, 48), sheep (42) and humans (10, 11).
Most experiments investigating the effects of challenges administered during the perinatal
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period have been aimed at demonstrating changes in adult systemic and cellular functions. However, recently studies on the effects of manipulations made during fetal development on the responsiveness of behavioral and neural substrates within postnatal period have been investigated by Perillan and colleagues (36, 37). These studies made use of earlier work characterizing the
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ontogenetic calendar of the onset of responsiveness to dipsogenic stimuli in neonates (29, 50). Wirth and Epstein (50) first described the sequence of the development of water drinking in
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response to a battery of cellular- and extracellular-depletion-related stimuli that elicit thirst in mature animals. Normal rat pups start to respond to cellular dehydration at 2 days of age, to hypovolemia at 4 days, and to beta-adrenergic receptor activation at 6 days (50). Perillan and colleagues (37) found that pharmacological treatment of the mothers during pregnancy with the 1
ANG, angiotensin; AVP, arginine vasopressin; BP, blood pressure; CNS, central nervous system; DOCA, deoxycorticosterone; FLI, Fos-like immunoreactive; Fos-ir, Fos immunohistochemistry; HR, heart rate; LT, lamina terminalis; M-PAL, partial aortic ligated mothers; M-sPAL, sham operated mothers; O-PAL, offspring from PAL mothers; OVLT, organum vasculosum; PAL, partial ligation of the aorta; PRA, paraformaldehyde; PBS, phosphate buffered saline; PVN, paraventricular nuclei; SFO, subfornical organ; SON, supraoptic nucleus
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mineralocorticoid agonist deoxycorticosterone (DOCA) altered responses to dipsogenic stimuli in neonates. The water intake of the pups of DOCA-treated females at 2 days of age in response to cellular dehydration (hypertonic saline administration) was slightly attenuated. Responding to
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hypovolemia induced by polyethylene glycol that nevertheless occurs in normal pups at 4 days of age was delayed until 6 days of age in the offspring of treated mothers, but the dipsogenic response to the beta-adrenergic receptor agonist isoproterenol was normal and was not affected by the prenatal maternal DOCA treatment.
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Under conditions of mineralocorticoid agonist treatment the levels of circulating renin and angiotensin are suppressed. Perillan and colleagues (37) have used the maternal manipulation-
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postnatal testing strategy to assess the effects of a condition where the renin-angiotensin system is activated rather than inhibited. In these studies the investigators used partial ligation of the aorta (PAL), a treatment which in adult rat has been shown to elevate plasma renin activity (15, 36), hypertonic saline and water intake
(1, 9, 38) and found that the dipsogenic response to
isoproterenol was enhanced in 6-day-old pups.
Costales, Fitzsimons and Vijande (9) made periodic measurements of arterial blood
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pressure (BP) by a catheter inserted in the carotid artery after PAL in adult male and nonpregnant female rats over the course of 3 weeks after surgery. Arterial pressure increased rapidly after PAL, but no significant correlation between BP and the increased intake of water and hypertonic saline was found. Although saline and water intakes and renin activities are elevated
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in pregnant PAL rats, the question of whether BP is elevated over the course of gestation after PAL has not been addressed. This becomes a particularly relevant issue as it is well established
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that in normal pregnancies the prospective mothers are both volume expanded and remarkably resistant to the hypertensive actions of many pressor agents. The current capability to measure BP continuously over extended periods of time using commercial arterial pressure transducer/telemetered data acquisition systems allow for studying the effects of PAL on blood pressures throughout gestation, while also collecting reliable behavioral information. The purposes of the present set of experiments were twofold. The first was to characterize the effects of PAL on the mother’s BP using continuous monitoring and to characterize the associated intake of salt and water. Second, in the course of conducting these studies it was
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possible to collect the brains from isoproterenol treated 6-day old offspring of PAL and control mothers.
As noted above, the 6-day-old offspring of PAL mothers show enhanced water
ingestion. Thus, the material from the beta-adrenergic receptor agonist-treated pups allowed us to
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determine if there is evidence for a neuroanatomical correlate within key neural substrates [i.e. subfornical organ (SFO), organum vasculosum (OVLT), supraoptic (SON) and paraventricular (PVN) nuclei] controlling thirst and the enhanced water intake evidenced by the isoproterenol treated pups of PAL mothers.
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2. Materials and methods 2.1. Animals
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Adult Sprague-Dawley male and female rats (10-12 weeks old) were obtained from a commercial supplier (Harlan Sprague-Dawley, Indianapolis, IN). Rats were housed individually in temperature- and light-controlled animal quarters and were provided with rat chow (7013 National Institutes of Health-31 modified rat diet, 0.25% NaCl), tap water and 1.8% NaCl solution ad libitum, except as noted below.
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All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Iowa Animal Care and Use Committee.
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2.2. Experimental Protocol
The animals were divided into two groups: 1) Partial Aortic Ligated Mothers (M-PAL;
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n=6); and 2) Sham Operated Mothers (M-sPAL; n=4). One week after the surgery, the female rats were mated with adult males to obtain two groups of offspring: 1) Offspring from PAL mothers (O-PAL), and 2) Offspring from Sham Operated Mothers (O-sPAL). Daily water and saline intakes were taken the six days during a baseline period before surgery, four days after surgery, and throughout the pregnancy.
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2.3. Surgical Preparations Partial Ligature of the abdominal aorta. PAL was produced by following a previously described procedure (9). Briefly, the abdominal aorta was approached through a ventral incision. The aorta
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between the renal arteries was cleared and partially occluded by tying a silk thread (no. 4/0) around it just below the mesenteric artery. A stylus (0.6 mm) was included within the ligature. After the ligatures were tied, the stylus was removed. The abdominal incision was then closed in layers. The sham operation was identical except that the aorta was not ligated.
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2.4. Telemetry probe implantation.
In a separate group of female rats, implantable rat BP transmitters (TA11PA-C40, Data
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Sciences International, St. Paul, MN) were used to measure BP and heart rate (HR) over the course of pregnancy. The rats were anesthetized with a ketamine-xylazine mixture. The carotid artery of the rat was accessed with a ventral midline incision. The left carotid artery was isolated and two occlusion sutures were placed beneath the artery and the telemetry catheter was inserted into the vessel. The catheter tip was advanced 3 cm into the vessel, the sutures were then tied and secured with tissue adhesive. Through the same ventral incision, a subcutaneous tunnel was
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formed across the right pectoral area, and a region was enlarged to form a pocket along the right flank. The body of the transmitter was slipped into the pocket and secured with tissue adhesive. The ventral incision was then closed with suture.
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All rats were allowed 7 days to recover from the transmitter implantation surgery before any measurements were made. This time interval permitted the rats to regain their circadian BP and HR rhythms. Recording was then initiated by telemetrically to record and store BP and HR
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with a Dataquest ART data acquisition system (Data Sciences International, St. Paul, MN). BP HR were monitored throughout the remainder of the protocol. One week after telemetry implantation, female rats underwent sham or PAL surgery, and
one week later they were mated using the same protocol described below.
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2.5. Mating and Pregnancy. After PAL or sham surgery, a single male breeder was housed overnight in each virgin female’s cage (M-PAL and M-sPAL) for mating. Pregnancy was assessed by daily vaginal
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smears, with the presence of spermatozoids taken as the first day of pregnancy. Once it had been determined that the breeding was successful, the males were removed from the cage and the female rats were housed singly.
One-day post-partum, all offspring were removed from the dam and cross-fostered to
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lactating, recently parturient control dams. Pups remained with surrogate mothers until postnatal day 6, when experiments for Fos expression were carried out.
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Immediately after the intake test was conducted on 6-day-old pups, the mothers were euthanized by intraperitoneal pentobarbital overdose. Following euthanization, both kidneys were removed, rinsed with cold isotonic saline, and weighted.
2.6. Beta-adrenergic agonist challenge and collection of brains of the offspring
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The day of birth was designated as Day 0. The cross-fostered litter, usually 8-12 pups, was kept intact until the day of testing. One 6-day-old pup from each litter was subcutaneously injected with isoproterenol (SIGMA, St. Louis, MO; 50µg/kg; n=6). This dose previously has been reported to produce significant water ingestion responses in pups 6 days of age (50). Another pup
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from each litter was subcutaneously injected with 0.15 M NaCl (control; n=4). All subcutaneous injections were made between the scapulae in a volume of 1.25 mL/100 g body wt (approx. 0.1
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mL vol/pup). They were placed individually in a cage lined with a cloth above a heating pad and skin temperature was monitored with a thermometer and maintained at 33°C. Ninety minutes after the injections, pups were anesthetized with sodium pentobarbital (Oak Pharmaceuticals, Inc., Lake Forest, IL; 50 mg/kg) and perfused transcardially with 10 ml of 0.1 M phosphate buffered saline (PBS) followed with 10 ml of 4% paraformaldehyde (PFA) in PBS. The brains were removed and immersed in PFA overnight and subsequently immersed in 30% sucrose-PBS for at least 24 h.
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2.7. Immunohistochemistry Coronal 40 µm thick sections were cut on a freezing microtome and processed for Fos immunohistochemistry (Fos-ir) by the avidin-biotin-peroxidase technique. Free-floating sections
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were rinsed twice in PBS, placed in a methanol-hydrogen peroxide solution for 20 min, immersed in 3% goat serum for 60 min and incubated overnight in primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; SC-52, 1:2000 dilution in antibody buffer with 0.3% Triton X100) at room temperature. The next day, sections were washed three times with PBS and
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incubated for 1 h with a secondary antibody (Anti-rabbit IgG, Biotinylated Vector labs, 1:200 dilution in PBS with 0.3% Triton X). They were washed again and processed using the Vectastain
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ABC kit (Vector labs, Burlingame, CA). Finally, the sections were treated for 3 min in 1 mg/ml diaminobenzidine tetrahydrochloride dissolved in PBS with 0.02% hydrogen peroxide, mounted on gelatinized slides, dried overnight, dehydrated in alcohol, and covered with coverslips and Permount®.
The brain nuclei of interest were the hypothalamic SON and PVN, the SFO, the MnPO, and the OVLT. All Fos grains were counted within the borders of each region or nucleus in two
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sections representing the largest coronal cross sectional area. The sections for SFO and SON are represented. respectively, by the coronal plates -0.92 and -1.30 mm from bregma in the Paxinos & Watson atlas (35). Two sections for PVN were selected for counting where the magnocellular subnucleus appeared largest (about -0.80 mm from bregma) (35). All nuclei were photographed
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by using a light microscope with a digital camera. The images were analyzed using a computer system running Leica QWin processing software (Servicio de análisis de Imágenes, University of
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Oviedo). SON, SFO, MnPO, and OVLT nuclei areas were measured after tracing its outline with a drawing tool by the Leica Q Win processing software 3.0 (Leica Microsystem Imaging Solutions Ltd, Cambridge, UK). Areas selected for counting were comparable for each of the four experimental groups. The density of Fos-IR was then calculated by dividing the total number of Fos-positive cells by the area. These densities are reported as cells per 10,000 µm2 per nucleus for the SFO, MnPO, or OVLT or per side for the PVN.
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2.8. Statistical analysis MAP and HR data were collected for 5 baseline days and the throughout the entire
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protocol, including pregnancies. MAP and HR are presented as mean daily values averaged from daytime and night time measurements. All data are presented as mean ± standard error of the mean. For multiple comparisons the means were compared by a two-way repeated measure analysis of variance (ANOVA) followed by Bonferroni post-test to compare means. Unpaired Student t tests were used to compare the differences in the means of two groups for body weight,
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temperature, and the birth number per litter. A p value of 0.05 was considered to be statistically significant. The statistical analyses were performed using IBM SPSS software (version 19, Inc.,
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Chicago, IL, USA).
3. Results 3.1. Dams
3.1.1. Water and saline intakes over pregnancy.
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Daily water and 1.8% NaCl intakes from M-sPAL and M-PAL animals over the course of pregnancy are presented in Figure 1. Baseline values for water and salt intake were comparable (23.79 ± 1.40 mL and 23.05 ± 1.91 mL for water, and 7.70 ± 1.54 and 8.60 ± 2.74 mL for salt).
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Partial aortic ligature produced a significant increase in both 1.8% NaCl and water intakes during the entire period of gestation (44.50 ± 1.45 mL) as compared with that of M-sPAL (35.38 ± 1.30;
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p <0.001).
3.1.2. The effects of Partial aortic ligation on blood pressure and heart rate. Figure 2 presents the results of the daily BP (Fig. 2A) and HR (Fig. 2B) changes.
Beginning the first day after partial aortic ligation, BP was significantly higher than that on the preoperative day (113.13 ± 4.40 mmHg vs 131.55 ± 6.26 mmHg; p<0.05). The average BP and HR were always higher in M-PAL group compared to the M-sPAL animals (p<0.05) every day
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throughout pregnancy (average 164.63 ± 2.38 mmHg vs 107.49 ± 1.01 mmHg; BP p<0.001, and 373.28 ± 3.45 beats/min vs 349.42 ± 3.45 beats/min; HR p<0.001).
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3.2. Offspring 3.2.1. Body weights
At 6 days of age, pups from M-PAL dams had similar body weights to pups from M-
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sPAL dams when tested with an isoproterenol challenge. (Table 1). No differences were found
3.2.2. c-Fos immunohistochemistry
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between sexes.
Fos-IR expression in the OVLT, SFO, SON, MnPO and PVN for each group are presented in Figure 3. In control pups infused with the vehicle, there was little or no Fos-ir in the hypothalamic structures. However, isoproterenol induced strong nuclear expression of c-Fos in the forebrain of the O-sPAL and O-PAL 6-day-old pups. The OVLT (p<0.001, t=4.42, df=53);
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p<0.001, t=6.65, df=38.8), MnPO (p<0.001, t=3.60, df=53; p<0.001, t=6.12, df=36), SON (p<0.001, t=10.17, df=71.9; p<0.001, t=8.34, df=57.78), PVN (p<0.001, t=3.49, df=31.98; p<0.001, t=4.17, df=21) and SFO (p<0.01, t=3.12, df=16.0; p<0.001, t=9.13, df=9.01) showed
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increased Fos-ir in O-PAL.
Isoproterenol treatment produced a significantly higher density of Fos-immunoreactivity in nuclei of the pups from PAL Dams, in OVLT (p=0.001, t=3.31. df=74), MnPO (p<0.001,
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t=5.11, df=64), SON (p=0.001, t=3.49, df=65.6), and SFO (p<0.001, t=5.44, df=22). No differences were found between pups injected with saline (Fig 3). 4. Discussion
This study characterized 1) the effects of PAL on cardiovascular variables and the intake of saline and water over the course of pregnancy of Sprague-Dawley rats, and 2) phenotypic changes in 6-day-old rat pups occurring in central nervous system (CNS) regions implicated in the mediation of enhanced drinking responses (i.e., water intake), induced by the beta-adrenergic
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receptor agonist, isoproterenol. The main findings of this study are as follows: (1) partial aortic occlusion produced a significant increase in BP, HR, and the intake of 1.8% NaCl and water over the course of the entire period of gestation, and (2) the 6-day-old offspring of mothers with
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gestational hypertension showed enhanced neural activity within forebrain structures involved in body fluid homeostasis after treatment with isoproterenol. The results of these experiments are consistent with the hypothesis that fetal programming of the offspring of mothers with gestational hypertension results in changes in CNS activity that accompanies enhanced drinking responses in
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6-day-old pups from PAL mothers. Six-day-old pups from PAL mothers drink significantly more water than control offspring (36, 37).
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Over the course of pregnancy there are many physiological changes that occur in the gravid mother. These are particularly notable in the systems that control BP and body fluid balance and distribution. During a healthy pregnancy, such changes as the expansion of blood volume are considered necessary for optimal perfusion of the placenta to meet the needs of the fetus. In humans, a hyponatremic, hypervolemic state develops over the course of pregnancy leading to an increase in total body water of 6-8 liters and a decrease of plasma osmolality of 8 to 10 mosm/kg
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by gestational week 36-38 (25, 40). Multiple behavioral, endocrine and hormonal mechanisms have been implicated in mediating these changes in body fluid homeostasis during pregnancy. The thresholds for both thirst and vasopressin release decrease over the course of the first two trimesters of pregnancy producing body fluid regulation around a new set-point (4, 25).
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Within the first few weeks of pregnancy, circulating levels of 17ß-oestradiol and progesterone increase along with elevated blood volume, HR, stroke volume and cardiac output. The last
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trimester of pregnancy is characterized by a rapid rise in estrogens, which coincides with enhanced plasma volume and interstitial fluid (19). The progressive increase in extracellular volume results in increases of 70% and 50% in plasma renal flow and glomerular filtration rate, respectively (5, 47). In spite of this, there is sodium and water retention as a result of the renin– angiotensin–aldosterone system and of increased action arginine vasopresina (AVP) on the kidney. Enhanced AVP secretion present during pregnancy is paralleled by an increased drinking response to osmotic challenge which may account for the greater water intake present during pregnancy.
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As Costales et al. showed (9), partial inter-renal aortic occlusion caused male and female rats with access to sodium chloride and water to increase their BP along with urine flow, creating fluid and electrolyte deficit in the animals. Also, single intracarotid BP measurements were made
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in 8 unanesthetized animals at times ranging from 7 to 24 days after PAL. In the present study, BP was measured continuously over the course of pregnancy by the use of telemetry in freely moving and stress-free pregnant rats without the need for anesthesia or restraint. BP reached plateau after 7 days of pregnancy, with values around 160 mmHg. In contrast, the pregnant
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control group BP progressively decreased. HR values were also significantly greater in the PAL than in the sham operated mothers. These results are consistent with prior reports in humans showing reduced BP during pregnancy which occurs despite blood volume expansion and
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activation of the renin-angiotensin system (5, 47).
Renal ischemia is a potent stimulus to increase renin secretion (12, 30, 49) Enhanced renin secretion contributes to the elevated intakes of water and NaCl, and is the major determinant to the onset of hypertension. The elevation in BP reaches a peak on the 4th day after occlusion, and hypertension is maintained throughout pregnancy. Increases in water and salt intakes by PAL
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rats are due to renin released by the ischemic kidneys and the formation of angiotensin II (Ang II), as captopril abolishes this effect (9).
The observed increase in thirst and salt appetite is consistent with prior reports of enhanced increase in water and salt intakes in pregnant animals (2, 13). In the present study the increased
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intake of water and 1.8% NaCl reached maximal levels about one week after ligation. This was the time chosen to mate these females, and the fluid intakes remained high throughout pregnancy.
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The current study is consistent with our previous work (36, 37) demonstrating that PAL is a surgical intervention that does not interfere with conception or full term pregnancy and is accompanied by an increase in the consumption of both water and 1.8% saline. Therefore, this procedure provides an effective model for studying the effects of an early endogenously altered hormonal environment on the ingestive behavior and the nervous system of the offspring of hypertensive mothers (1, 29). Using the PAL model, Perillan et al. (36) demonstrated that at 6 days of age, the offspring from hypertensive dams showed enhanced water intake when injected with the beta-adrenergic
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receptor agonist, isoproterenol. The administration of isoproterenol stimulates the release of renin resulting in the subsequent increase in circulating Ang II (14, 18, 20, 21, 24, 45, 46). Rettig et al. (39) reported that circulating levels of angiotensin produced by isoproterenol administration
mobilize thirst in the face of an isoproterenol challenge.
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reached dipsogenic levels and described that a renal factor, renin, provides a first-line defense to
In adult animals, isoproterenol treatment results in activating brain structures critical in body fluid homeostasis (34).
Ang II acting through AT1 receptors localized in the
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circumventricular organs (CVOs) induces rapid responses, including the release of AVP. Oldfield and McKinley (34), using an angiotensin type 1 receptor antagonist, losartan,
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demonstrated that cells in the lamina terminalis were activated directly or indirectly by Ang II, whereas other neurons in the hypothalamus and brain stem were responsive as a consequence of the peripheral vasodilation caused by isoproterenol.
Rowland (44) characterized isoproterenol as inducing strong Fos-like immunoreactive (FLI) neurons in the magnocellular PVN and SON nuclei, and moderate staining along the structures of
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the rostral wall of the LT. Fos-positive neurons have been shown in several studies present in a range of specific forebrain and hindbrain regions, frequently associated with body fluid balance and salt appetite (23, 43, 53). Nuyt et al. (33) demonstrated in late gestation the presence of AT1a mRNA and AT1 binding sites in forebrain nuclei suggesting that Ang II regulation of fluid
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homeostasis may be already functional early in life (33). The work by Wirth and Epstein (50) showed that 6 day-old rat pups first consumed water
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to subcutaneous isoproterenol. In the present study we found that the PVN, SON, MnPO, SFO and OVLT are all activated in a pattern similar to that seen in adults. The Fos-IR response was significantly greater in these regions in the offspring of PAL mothers. The differential degree of Fos-ir expression between both groups is likely the result of fetal programming of brain development produced by maternal PAL, which results in enhanced sensitivity of the RAAS system and HPA axis. Although increased behavioral and neural responsivity to an exogenous challenge under same environmental circumstances, such changes might result inappropriate when salt and water are plentiful at times later in ontogeny. Subjecting the fetus to conditions
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associated with maternal hypertension may result in fetal programing that leads to hypertension and metabolic disorders in adult animals (16, 51, 52). The fact that maternal salt loading during pregnancy induces changes in angiotensinogen mRNA in fetal liver and brain demonstrated that
genes implicated in hydromineral homeostasis (27).
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hydromineral challenges in mothers during pregnancy may affect the expression of some fetal
Isoproterenol-elicited Fos induction in the SFO and OVLT can be significantly reduced by peripheral pretreatment with AT1R antagonists (34), and these findings suggest that increased
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circulating Ang II stimulates AT1Rs in the SFO or OVLT, which in turn, activates the central pathways mediating water intake (22). Our pups, whose maturation in utero was produced in a
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hyperreninemic, hypertensive environment of the mother, could have higher levels of AT1Rs in CNS regions involved in maintaining body fluid homeostasis and produce increased responsiveness to isoproterenol.
In summary, the present studies demonstrate that PAL is compatible with pregnancy in the rat, producing a sustained increase in blood pressure and heart rate, along with increased water
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and salt intake. The 6-day-old offspring from these mothers, when challenged with isoproterenol, show adult-like Fos expression before they would normally develop independent ingestive behavior, but at that time they readily ingest water delivered by infusion into the mouth. The present study demonstrates that the neural substrates activated by thirst stimuli mature early and
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can be altered by fetal programming.
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Funding: This work was supported by the Excellence Mobility Grants for lectures and researchers of the
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Health R01 HL14388 (Alan Kim Johnson).
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University of Oviedo, Spain, 2013 & 2016 (Juan Arguelles) and by the National Institutes of
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Table1. Body weight in pups from mothers with atrial Aortic Ligature (O-PAL) or Sham Partial
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Aortic Ligature (O-sPAL) at 6 days old (g).
Males (n)
O-sPAL
14.2 ± 1.07 (3)
16.07 ± 1.35 (6)
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14.55 ± 0.98 (6)
14.53 ± 2.76 (3)
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Females (n)
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Fig 1. Daily intake of saline (1.8%) (+S.E.M.) of dams subjected to Partial Aortic Ligature (solid circles) or Sham Operation (open circles).
Fig. 3.
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Fig 2. Augmented pressor effects induced by aortic ligature during pregnancy in female rats
Quantification of c-Fos immunoreactivity cells per 10,000 µm2 in the organum
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vasculosum of the lamina terminals (OVLT), subfornical organ (SFO), supraoptic nucleus (SON), median preoptic nucleus (MnPO) and hypothalamic paraventricular nucleus (PVN) of pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL) 90 min after the
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administration of isoproterenol or vehicle subcutaneously injection. Values are presented as means ± SEMs. * p<0.05.
Fig. 4. c-Fos expression 90 min after the administration of isoproterenol or vehicle subcutaneously in pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL).
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Representative photomicrographs of c-Fos expression on coronal sections in the organum vasculosum of the lamina terminalis (OVLT).
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Fig. 5. c-Fos expression 90 min after the administration of isoproterenol or vehicle subcutaneously in pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL). Representative photomicrographs of c-Fos expression on coronal sections in the Median Pre-
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Optic nucleus (MnPO).
Fig. 6. c-Fos expression 90 min after the administration of isoproterenol or vehicle subcutaneously in pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL). Representative photomicrographs of c-Fos expression on coronal sections in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei.
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Fig. 7. c-Fos expression 90 min after the administration of isoproterenol or vehicle subcutaneously in pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL). Representative photomicrographs of c-Fos expression on coronal sections in the subfornical
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organ (SFO).
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S0195-6663(16)30820-0
DOI:
10.1016/j.appet.2017.04.008
Reference:
APPET 3421
To appear in:
Appetite
Received Date: 22 November 2016 Revised Date:
7 April 2017
Accepted Date: 8 April 2017
Please cite this article as: Arguelles J., Perillan C., Beltz T.G., Xue B., Badaue-Passos D., Jr, Vega J.A. & Johnson A.K., The effects of experimental gestational hypertension on maternal blood pressure and fluid intake and pre-weanling hypothalamic neuronal activity, Appetite (2017), doi: 10.1016/ j.appet.2017.04.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The Effects of Experimental Gestational Hypertension on Maternal Blood Pressure and Fluid Intake and Pre-Weanling Hypothalamic Neuronal Activity
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Passos Jrc, Jose A. Vegad,e, Alan Kim Johnsonb
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Juan Arguellesa, Carmen Perillana, Terry G. Beltzb, Baojian Xueb, Daniel Badaue-
a
Departamento de Biología Funcional, Área de Fisiología, Facultad de Medicina y
Ciencias de la Salud, Universidad de Oviedo, Spain b
Department of Psychological and Brain Sciences, The University of Iowa, Iowa
City, IA, USA, 52242 c
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Currently at Departamento de Fisiologia, Centro C. Biológicas e Saúde,
Universidade Federal de Sergipe, Sau Cristovao-SE, Brazil d
Departamento de Morfologia y Biologia Celular, Universidad de Oviedo, Spain
e
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Chile, Chile
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Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Santiago de
Corresponding author: Juan Arguelles, Departamento Biología Funcional, Area de Fisiología, Universidad de Oviedo, Spain; email: [email protected]
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ABSTRACT To examine the fetal programming effects of maternal hypertension, natriophilia and
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hyperreninemia [experimentally induced in rats by partial inter-renal aortic ligature (PAL) prior to mating] fos immunoreactivity was studied in 6-day-old offspring of PAL and control mothers. The purposes of the present set of experiments were twofold. The first was to characterize the effects of PAL on the mother’s arterial blood pressure and intake of salt (1.8% NaCl solution) and
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water over the course of gestation. Second, was to study the pattern of neuronal activation in key brain areas of 6-day-old offspring treated with the dipsogen isoproterenol that were from PAL
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and control mothers. Beta-adrenergic receptor agonist-treated pups allowed the determination whether there were neuroanatomical correlates within the neural substrates controlling thirst and the enhanced water intake evidenced by the isoproterenol treated pups of PAL mothers. Hydromineral ingestive behavior along with blood pressure and heart rate of PAL (M-PAL) and control (M-sPAL) dams throughout gestation was studied. Higher salt and water intakes along with blood pressures and heart rates were found during gestation and lactation in the M-PAL
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group. Maternal PAL evoked significantly increased isoproterenol-elicited Fos staining in brain regions (e.g. subfornical organ, organum vasculosum of the lamina terminalis, supraoptic nucleus, hypothalamic paraventricular nucleus and median preoptic nucleus) of 6-day-old pups, which is the age of animals shown enhanced thirst responses in PAL offspring. These results indicate that
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PAL is compatible with pregnancy, producing a sustained increase in blood pressure and heart rate, along with increased water and salt intake. The present study demonstrates that the neural
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substrates involved in cardiovascular homeostasis and fluid balance in adult rats are responsive in six-day-old rats, and can be altered by fetal programming. Key Words:
Ontogeny, Thirst, Gestation, Isoproterenol, Hypertension, c-fos, renin-angiotensin-aldosterone system
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1. Introduction1 Many authors have proposed that perturbations in maternal physiology during gestation alters the offspring in ways to make them more vulnerable to disease in adulthood. Intrauterine
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fetal programming (16, 26) can be induced by physiological challenges or threats to homeostasis that occur to the mother, during critical or sensitive periods of neonatal development. Such challenges affect the structure, physiology or metabolism in a manner that alters function later in life (3, 17).
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Intrauterine programming has frequently been discussed in relation to cardiovascular diseases (3, 17), and it has been hypothesized that such developmental influences on adult vital
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functions may have influenced population survival in times of drought and famine (41). Both experimental and natural challenges to maternal and offspring water and sodium distribution and balance and the cardiovascular system during perinatal critical periods, have been shown to have later consequences on body fluid and cardiovascular homeostasis in rats (6-8, 28, 31, 32, 48), sheep (42) and humans (10, 11).
Most experiments investigating the effects of challenges administered during the perinatal
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period have been aimed at demonstrating changes in adult systemic and cellular functions. However, recently studies on the effects of manipulations made during fetal development on the responsiveness of behavioral and neural substrates within postnatal period have been investigated by Perillan and colleagues (36, 37). These studies made use of earlier work characterizing the
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ontogenetic calendar of the onset of responsiveness to dipsogenic stimuli in neonates (29, 50). Wirth and Epstein (50) first described the sequence of the development of water drinking in
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response to a battery of cellular- and extracellular-depletion-related stimuli that elicit thirst in mature animals. Normal rat pups start to respond to cellular dehydration at 2 days of age, to hypovolemia at 4 days, and to beta-adrenergic receptor activation at 6 days (50). Perillan and colleagues (37) found that pharmacological treatment of the mothers during pregnancy with the 1
ANG, angiotensin; AVP, arginine vasopressin; BP, blood pressure; CNS, central nervous system; DOCA, deoxycorticosterone; FLI, Fos-like immunoreactive; Fos-ir, Fos immunohistochemistry; HR, heart rate; LT, lamina terminalis; M-PAL, partial aortic ligated mothers; M-sPAL, sham operated mothers; O-PAL, offspring from PAL mothers; OVLT, organum vasculosum; PAL, partial ligation of the aorta; PRA, paraformaldehyde; PBS, phosphate buffered saline; PVN, paraventricular nuclei; SFO, subfornical organ; SON, supraoptic nucleus
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mineralocorticoid agonist deoxycorticosterone (DOCA) altered responses to dipsogenic stimuli in neonates. The water intake of the pups of DOCA-treated females at 2 days of age in response to cellular dehydration (hypertonic saline administration) was slightly attenuated. Responding to
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hypovolemia induced by polyethylene glycol that nevertheless occurs in normal pups at 4 days of age was delayed until 6 days of age in the offspring of treated mothers, but the dipsogenic response to the beta-adrenergic receptor agonist isoproterenol was normal and was not affected by the prenatal maternal DOCA treatment.
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Under conditions of mineralocorticoid agonist treatment the levels of circulating renin and angiotensin are suppressed. Perillan and colleagues (37) have used the maternal manipulation-
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postnatal testing strategy to assess the effects of a condition where the renin-angiotensin system is activated rather than inhibited. In these studies the investigators used partial ligation of the aorta (PAL), a treatment which in adult rat has been shown to elevate plasma renin activity (15, 36), hypertonic saline and water intake
(1, 9, 38) and found that the dipsogenic response to
isoproterenol was enhanced in 6-day-old pups.
Costales, Fitzsimons and Vijande (9) made periodic measurements of arterial blood
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pressure (BP) by a catheter inserted in the carotid artery after PAL in adult male and nonpregnant female rats over the course of 3 weeks after surgery. Arterial pressure increased rapidly after PAL, but no significant correlation between BP and the increased intake of water and hypertonic saline was found. Although saline and water intakes and renin activities are elevated
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in pregnant PAL rats, the question of whether BP is elevated over the course of gestation after PAL has not been addressed. This becomes a particularly relevant issue as it is well established
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that in normal pregnancies the prospective mothers are both volume expanded and remarkably resistant to the hypertensive actions of many pressor agents. The current capability to measure BP continuously over extended periods of time using commercial arterial pressure transducer/telemetered data acquisition systems allow for studying the effects of PAL on blood pressures throughout gestation, while also collecting reliable behavioral information. The purposes of the present set of experiments were twofold. The first was to characterize the effects of PAL on the mother’s BP using continuous monitoring and to characterize the associated intake of salt and water. Second, in the course of conducting these studies it was
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possible to collect the brains from isoproterenol treated 6-day old offspring of PAL and control mothers.
As noted above, the 6-day-old offspring of PAL mothers show enhanced water
ingestion. Thus, the material from the beta-adrenergic receptor agonist-treated pups allowed us to
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determine if there is evidence for a neuroanatomical correlate within key neural substrates [i.e. subfornical organ (SFO), organum vasculosum (OVLT), supraoptic (SON) and paraventricular (PVN) nuclei] controlling thirst and the enhanced water intake evidenced by the isoproterenol treated pups of PAL mothers.
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2. Materials and methods 2.1. Animals
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Adult Sprague-Dawley male and female rats (10-12 weeks old) were obtained from a commercial supplier (Harlan Sprague-Dawley, Indianapolis, IN). Rats were housed individually in temperature- and light-controlled animal quarters and were provided with rat chow (7013 National Institutes of Health-31 modified rat diet, 0.25% NaCl), tap water and 1.8% NaCl solution ad libitum, except as noted below.
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All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Iowa Animal Care and Use Committee.
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2.2. Experimental Protocol
The animals were divided into two groups: 1) Partial Aortic Ligated Mothers (M-PAL;
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n=6); and 2) Sham Operated Mothers (M-sPAL; n=4). One week after the surgery, the female rats were mated with adult males to obtain two groups of offspring: 1) Offspring from PAL mothers (O-PAL), and 2) Offspring from Sham Operated Mothers (O-sPAL). Daily water and saline intakes were taken the six days during a baseline period before surgery, four days after surgery, and throughout the pregnancy.
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2.3. Surgical Preparations Partial Ligature of the abdominal aorta. PAL was produced by following a previously described procedure (9). Briefly, the abdominal aorta was approached through a ventral incision. The aorta
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between the renal arteries was cleared and partially occluded by tying a silk thread (no. 4/0) around it just below the mesenteric artery. A stylus (0.6 mm) was included within the ligature. After the ligatures were tied, the stylus was removed. The abdominal incision was then closed in layers. The sham operation was identical except that the aorta was not ligated.
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2.4. Telemetry probe implantation.
In a separate group of female rats, implantable rat BP transmitters (TA11PA-C40, Data
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Sciences International, St. Paul, MN) were used to measure BP and heart rate (HR) over the course of pregnancy. The rats were anesthetized with a ketamine-xylazine mixture. The carotid artery of the rat was accessed with a ventral midline incision. The left carotid artery was isolated and two occlusion sutures were placed beneath the artery and the telemetry catheter was inserted into the vessel. The catheter tip was advanced 3 cm into the vessel, the sutures were then tied and secured with tissue adhesive. Through the same ventral incision, a subcutaneous tunnel was
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formed across the right pectoral area, and a region was enlarged to form a pocket along the right flank. The body of the transmitter was slipped into the pocket and secured with tissue adhesive. The ventral incision was then closed with suture.
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All rats were allowed 7 days to recover from the transmitter implantation surgery before any measurements were made. This time interval permitted the rats to regain their circadian BP and HR rhythms. Recording was then initiated by telemetrically to record and store BP and HR
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with a Dataquest ART data acquisition system (Data Sciences International, St. Paul, MN). BP HR were monitored throughout the remainder of the protocol. One week after telemetry implantation, female rats underwent sham or PAL surgery, and
one week later they were mated using the same protocol described below.
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2.5. Mating and Pregnancy. After PAL or sham surgery, a single male breeder was housed overnight in each virgin female’s cage (M-PAL and M-sPAL) for mating. Pregnancy was assessed by daily vaginal
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smears, with the presence of spermatozoids taken as the first day of pregnancy. Once it had been determined that the breeding was successful, the males were removed from the cage and the female rats were housed singly.
One-day post-partum, all offspring were removed from the dam and cross-fostered to
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lactating, recently parturient control dams. Pups remained with surrogate mothers until postnatal day 6, when experiments for Fos expression were carried out.
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Immediately after the intake test was conducted on 6-day-old pups, the mothers were euthanized by intraperitoneal pentobarbital overdose. Following euthanization, both kidneys were removed, rinsed with cold isotonic saline, and weighted.
2.6. Beta-adrenergic agonist challenge and collection of brains of the offspring
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The day of birth was designated as Day 0. The cross-fostered litter, usually 8-12 pups, was kept intact until the day of testing. One 6-day-old pup from each litter was subcutaneously injected with isoproterenol (SIGMA, St. Louis, MO; 50µg/kg; n=6). This dose previously has been reported to produce significant water ingestion responses in pups 6 days of age (50). Another pup
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from each litter was subcutaneously injected with 0.15 M NaCl (control; n=4). All subcutaneous injections were made between the scapulae in a volume of 1.25 mL/100 g body wt (approx. 0.1
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mL vol/pup). They were placed individually in a cage lined with a cloth above a heating pad and skin temperature was monitored with a thermometer and maintained at 33°C. Ninety minutes after the injections, pups were anesthetized with sodium pentobarbital (Oak Pharmaceuticals, Inc., Lake Forest, IL; 50 mg/kg) and perfused transcardially with 10 ml of 0.1 M phosphate buffered saline (PBS) followed with 10 ml of 4% paraformaldehyde (PFA) in PBS. The brains were removed and immersed in PFA overnight and subsequently immersed in 30% sucrose-PBS for at least 24 h.
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2.7. Immunohistochemistry Coronal 40 µm thick sections were cut on a freezing microtome and processed for Fos immunohistochemistry (Fos-ir) by the avidin-biotin-peroxidase technique. Free-floating sections
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were rinsed twice in PBS, placed in a methanol-hydrogen peroxide solution for 20 min, immersed in 3% goat serum for 60 min and incubated overnight in primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; SC-52, 1:2000 dilution in antibody buffer with 0.3% Triton X100) at room temperature. The next day, sections were washed three times with PBS and
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incubated for 1 h with a secondary antibody (Anti-rabbit IgG, Biotinylated Vector labs, 1:200 dilution in PBS with 0.3% Triton X). They were washed again and processed using the Vectastain
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ABC kit (Vector labs, Burlingame, CA). Finally, the sections were treated for 3 min in 1 mg/ml diaminobenzidine tetrahydrochloride dissolved in PBS with 0.02% hydrogen peroxide, mounted on gelatinized slides, dried overnight, dehydrated in alcohol, and covered with coverslips and Permount®.
The brain nuclei of interest were the hypothalamic SON and PVN, the SFO, the MnPO, and the OVLT. All Fos grains were counted within the borders of each region or nucleus in two
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sections representing the largest coronal cross sectional area. The sections for SFO and SON are represented. respectively, by the coronal plates -0.92 and -1.30 mm from bregma in the Paxinos & Watson atlas (35). Two sections for PVN were selected for counting where the magnocellular subnucleus appeared largest (about -0.80 mm from bregma) (35). All nuclei were photographed
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by using a light microscope with a digital camera. The images were analyzed using a computer system running Leica QWin processing software (Servicio de análisis de Imágenes, University of
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Oviedo). SON, SFO, MnPO, and OVLT nuclei areas were measured after tracing its outline with a drawing tool by the Leica Q Win processing software 3.0 (Leica Microsystem Imaging Solutions Ltd, Cambridge, UK). Areas selected for counting were comparable for each of the four experimental groups. The density of Fos-IR was then calculated by dividing the total number of Fos-positive cells by the area. These densities are reported as cells per 10,000 µm2 per nucleus for the SFO, MnPO, or OVLT or per side for the PVN.
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2.8. Statistical analysis MAP and HR data were collected for 5 baseline days and the throughout the entire
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protocol, including pregnancies. MAP and HR are presented as mean daily values averaged from daytime and night time measurements. All data are presented as mean ± standard error of the mean. For multiple comparisons the means were compared by a two-way repeated measure analysis of variance (ANOVA) followed by Bonferroni post-test to compare means. Unpaired Student t tests were used to compare the differences in the means of two groups for body weight,
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temperature, and the birth number per litter. A p value of 0.05 was considered to be statistically significant. The statistical analyses were performed using IBM SPSS software (version 19, Inc.,
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Chicago, IL, USA).
3. Results 3.1. Dams
3.1.1. Water and saline intakes over pregnancy.
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Daily water and 1.8% NaCl intakes from M-sPAL and M-PAL animals over the course of pregnancy are presented in Figure 1. Baseline values for water and salt intake were comparable (23.79 ± 1.40 mL and 23.05 ± 1.91 mL for water, and 7.70 ± 1.54 and 8.60 ± 2.74 mL for salt).
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Partial aortic ligature produced a significant increase in both 1.8% NaCl and water intakes during the entire period of gestation (44.50 ± 1.45 mL) as compared with that of M-sPAL (35.38 ± 1.30;
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p <0.001).
3.1.2. The effects of Partial aortic ligation on blood pressure and heart rate. Figure 2 presents the results of the daily BP (Fig. 2A) and HR (Fig. 2B) changes.
Beginning the first day after partial aortic ligation, BP was significantly higher than that on the preoperative day (113.13 ± 4.40 mmHg vs 131.55 ± 6.26 mmHg; p<0.05). The average BP and HR were always higher in M-PAL group compared to the M-sPAL animals (p<0.05) every day
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throughout pregnancy (average 164.63 ± 2.38 mmHg vs 107.49 ± 1.01 mmHg; BP p<0.001, and 373.28 ± 3.45 beats/min vs 349.42 ± 3.45 beats/min; HR p<0.001).
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3.2. Offspring 3.2.1. Body weights
At 6 days of age, pups from M-PAL dams had similar body weights to pups from M-
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sPAL dams when tested with an isoproterenol challenge. (Table 1). No differences were found
3.2.2. c-Fos immunohistochemistry
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between sexes.
Fos-IR expression in the OVLT, SFO, SON, MnPO and PVN for each group are presented in Figure 3. In control pups infused with the vehicle, there was little or no Fos-ir in the hypothalamic structures. However, isoproterenol induced strong nuclear expression of c-Fos in the forebrain of the O-sPAL and O-PAL 6-day-old pups. The OVLT (p<0.001, t=4.42, df=53);
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p<0.001, t=6.65, df=38.8), MnPO (p<0.001, t=3.60, df=53; p<0.001, t=6.12, df=36), SON (p<0.001, t=10.17, df=71.9; p<0.001, t=8.34, df=57.78), PVN (p<0.001, t=3.49, df=31.98; p<0.001, t=4.17, df=21) and SFO (p<0.01, t=3.12, df=16.0; p<0.001, t=9.13, df=9.01) showed
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increased Fos-ir in O-PAL.
Isoproterenol treatment produced a significantly higher density of Fos-immunoreactivity in nuclei of the pups from PAL Dams, in OVLT (p=0.001, t=3.31. df=74), MnPO (p<0.001,
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t=5.11, df=64), SON (p=0.001, t=3.49, df=65.6), and SFO (p<0.001, t=5.44, df=22). No differences were found between pups injected with saline (Fig 3). 4. Discussion
This study characterized 1) the effects of PAL on cardiovascular variables and the intake of saline and water over the course of pregnancy of Sprague-Dawley rats, and 2) phenotypic changes in 6-day-old rat pups occurring in central nervous system (CNS) regions implicated in the mediation of enhanced drinking responses (i.e., water intake), induced by the beta-adrenergic
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receptor agonist, isoproterenol. The main findings of this study are as follows: (1) partial aortic occlusion produced a significant increase in BP, HR, and the intake of 1.8% NaCl and water over the course of the entire period of gestation, and (2) the 6-day-old offspring of mothers with
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gestational hypertension showed enhanced neural activity within forebrain structures involved in body fluid homeostasis after treatment with isoproterenol. The results of these experiments are consistent with the hypothesis that fetal programming of the offspring of mothers with gestational hypertension results in changes in CNS activity that accompanies enhanced drinking responses in
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6-day-old pups from PAL mothers. Six-day-old pups from PAL mothers drink significantly more water than control offspring (36, 37).
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Over the course of pregnancy there are many physiological changes that occur in the gravid mother. These are particularly notable in the systems that control BP and body fluid balance and distribution. During a healthy pregnancy, such changes as the expansion of blood volume are considered necessary for optimal perfusion of the placenta to meet the needs of the fetus. In humans, a hyponatremic, hypervolemic state develops over the course of pregnancy leading to an increase in total body water of 6-8 liters and a decrease of plasma osmolality of 8 to 10 mosm/kg
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by gestational week 36-38 (25, 40). Multiple behavioral, endocrine and hormonal mechanisms have been implicated in mediating these changes in body fluid homeostasis during pregnancy. The thresholds for both thirst and vasopressin release decrease over the course of the first two trimesters of pregnancy producing body fluid regulation around a new set-point (4, 25).
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Within the first few weeks of pregnancy, circulating levels of 17ß-oestradiol and progesterone increase along with elevated blood volume, HR, stroke volume and cardiac output. The last
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trimester of pregnancy is characterized by a rapid rise in estrogens, which coincides with enhanced plasma volume and interstitial fluid (19). The progressive increase in extracellular volume results in increases of 70% and 50% in plasma renal flow and glomerular filtration rate, respectively (5, 47). In spite of this, there is sodium and water retention as a result of the renin– angiotensin–aldosterone system and of increased action arginine vasopresina (AVP) on the kidney. Enhanced AVP secretion present during pregnancy is paralleled by an increased drinking response to osmotic challenge which may account for the greater water intake present during pregnancy.
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As Costales et al. showed (9), partial inter-renal aortic occlusion caused male and female rats with access to sodium chloride and water to increase their BP along with urine flow, creating fluid and electrolyte deficit in the animals. Also, single intracarotid BP measurements were made
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in 8 unanesthetized animals at times ranging from 7 to 24 days after PAL. In the present study, BP was measured continuously over the course of pregnancy by the use of telemetry in freely moving and stress-free pregnant rats without the need for anesthesia or restraint. BP reached plateau after 7 days of pregnancy, with values around 160 mmHg. In contrast, the pregnant
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control group BP progressively decreased. HR values were also significantly greater in the PAL than in the sham operated mothers. These results are consistent with prior reports in humans showing reduced BP during pregnancy which occurs despite blood volume expansion and
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activation of the renin-angiotensin system (5, 47).
Renal ischemia is a potent stimulus to increase renin secretion (12, 30, 49) Enhanced renin secretion contributes to the elevated intakes of water and NaCl, and is the major determinant to the onset of hypertension. The elevation in BP reaches a peak on the 4th day after occlusion, and hypertension is maintained throughout pregnancy. Increases in water and salt intakes by PAL
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rats are due to renin released by the ischemic kidneys and the formation of angiotensin II (Ang II), as captopril abolishes this effect (9).
The observed increase in thirst and salt appetite is consistent with prior reports of enhanced increase in water and salt intakes in pregnant animals (2, 13). In the present study the increased
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intake of water and 1.8% NaCl reached maximal levels about one week after ligation. This was the time chosen to mate these females, and the fluid intakes remained high throughout pregnancy.
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The current study is consistent with our previous work (36, 37) demonstrating that PAL is a surgical intervention that does not interfere with conception or full term pregnancy and is accompanied by an increase in the consumption of both water and 1.8% saline. Therefore, this procedure provides an effective model for studying the effects of an early endogenously altered hormonal environment on the ingestive behavior and the nervous system of the offspring of hypertensive mothers (1, 29). Using the PAL model, Perillan et al. (36) demonstrated that at 6 days of age, the offspring from hypertensive dams showed enhanced water intake when injected with the beta-adrenergic
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receptor agonist, isoproterenol. The administration of isoproterenol stimulates the release of renin resulting in the subsequent increase in circulating Ang II (14, 18, 20, 21, 24, 45, 46). Rettig et al. (39) reported that circulating levels of angiotensin produced by isoproterenol administration
mobilize thirst in the face of an isoproterenol challenge.
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reached dipsogenic levels and described that a renal factor, renin, provides a first-line defense to
In adult animals, isoproterenol treatment results in activating brain structures critical in body fluid homeostasis (34).
Ang II acting through AT1 receptors localized in the
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circumventricular organs (CVOs) induces rapid responses, including the release of AVP. Oldfield and McKinley (34), using an angiotensin type 1 receptor antagonist, losartan,
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demonstrated that cells in the lamina terminalis were activated directly or indirectly by Ang II, whereas other neurons in the hypothalamus and brain stem were responsive as a consequence of the peripheral vasodilation caused by isoproterenol.
Rowland (44) characterized isoproterenol as inducing strong Fos-like immunoreactive (FLI) neurons in the magnocellular PVN and SON nuclei, and moderate staining along the structures of
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the rostral wall of the LT. Fos-positive neurons have been shown in several studies present in a range of specific forebrain and hindbrain regions, frequently associated with body fluid balance and salt appetite (23, 43, 53). Nuyt et al. (33) demonstrated in late gestation the presence of AT1a mRNA and AT1 binding sites in forebrain nuclei suggesting that Ang II regulation of fluid
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homeostasis may be already functional early in life (33). The work by Wirth and Epstein (50) showed that 6 day-old rat pups first consumed water
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to subcutaneous isoproterenol. In the present study we found that the PVN, SON, MnPO, SFO and OVLT are all activated in a pattern similar to that seen in adults. The Fos-IR response was significantly greater in these regions in the offspring of PAL mothers. The differential degree of Fos-ir expression between both groups is likely the result of fetal programming of brain development produced by maternal PAL, which results in enhanced sensitivity of the RAAS system and HPA axis. Although increased behavioral and neural responsivity to an exogenous challenge under same environmental circumstances, such changes might result inappropriate when salt and water are plentiful at times later in ontogeny. Subjecting the fetus to conditions
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associated with maternal hypertension may result in fetal programing that leads to hypertension and metabolic disorders in adult animals (16, 51, 52). The fact that maternal salt loading during pregnancy induces changes in angiotensinogen mRNA in fetal liver and brain demonstrated that
genes implicated in hydromineral homeostasis (27).
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hydromineral challenges in mothers during pregnancy may affect the expression of some fetal
Isoproterenol-elicited Fos induction in the SFO and OVLT can be significantly reduced by peripheral pretreatment with AT1R antagonists (34), and these findings suggest that increased
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circulating Ang II stimulates AT1Rs in the SFO or OVLT, which in turn, activates the central pathways mediating water intake (22). Our pups, whose maturation in utero was produced in a
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hyperreninemic, hypertensive environment of the mother, could have higher levels of AT1Rs in CNS regions involved in maintaining body fluid homeostasis and produce increased responsiveness to isoproterenol.
In summary, the present studies demonstrate that PAL is compatible with pregnancy in the rat, producing a sustained increase in blood pressure and heart rate, along with increased water
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and salt intake. The 6-day-old offspring from these mothers, when challenged with isoproterenol, show adult-like Fos expression before they would normally develop independent ingestive behavior, but at that time they readily ingest water delivered by infusion into the mouth. The present study demonstrates that the neural substrates activated by thirst stimuli mature early and
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can be altered by fetal programming.
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Funding: This work was supported by the Excellence Mobility Grants for lectures and researchers of the
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Health R01 HL14388 (Alan Kim Johnson).
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Table1. Body weight in pups from mothers with atrial Aortic Ligature (O-PAL) or Sham Partial
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Aortic Ligature (O-sPAL) at 6 days old (g).
Males (n)
O-sPAL
14.2 ± 1.07 (3)
16.07 ± 1.35 (6)
O-PAL
14.55 ± 0.98 (6)
14.53 ± 2.76 (3)
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Females (n)
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Fig 1. Daily intake of saline (1.8%) (+S.E.M.) of dams subjected to Partial Aortic Ligature (solid circles) or Sham Operation (open circles).
Fig. 3.
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Fig 2. Augmented pressor effects induced by aortic ligature during pregnancy in female rats
Quantification of c-Fos immunoreactivity cells per 10,000 µm2 in the organum
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vasculosum of the lamina terminals (OVLT), subfornical organ (SFO), supraoptic nucleus (SON), median preoptic nucleus (MnPO) and hypothalamic paraventricular nucleus (PVN) of pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL) 90 min after the
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administration of isoproterenol or vehicle subcutaneously injection. Values are presented as means ± SEMs. * p<0.05.
Fig. 4. c-Fos expression 90 min after the administration of isoproterenol or vehicle subcutaneously in pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL).
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Representative photomicrographs of c-Fos expression on coronal sections in the organum vasculosum of the lamina terminalis (OVLT).
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Fig. 5. c-Fos expression 90 min after the administration of isoproterenol or vehicle subcutaneously in pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL). Representative photomicrographs of c-Fos expression on coronal sections in the Median Pre-
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Optic nucleus (MnPO).
Fig. 6. c-Fos expression 90 min after the administration of isoproterenol or vehicle subcutaneously in pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL). Representative photomicrographs of c-Fos expression on coronal sections in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei.
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Fig. 7. c-Fos expression 90 min after the administration of isoproterenol or vehicle subcutaneously in pups from PAL mothers (O-PAL) and from sham operated mothers (O-sPAL). Representative photomicrographs of c-Fos expression on coronal sections in the subfornical
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