Animals
All experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (National Research Council, USA), and were approved by the Institutional Review Board for Ethics and Animal Welfare, and by the Ethics Committee of FONDECYT (Fondo Nacional de Desarrollo Científico y Tecnológico, Chile).
Virgin Pirbright white guinea-pigs of ~600 g were kept under controlled conditions of humidity and temperature (25°C), with a 12-h light–dark cycle. Females were examined daily for perforation of the vaginal closure membrane. Upon this occurrence, they were caged with fertile males, and the following day was defined as day 1. Between days 18–20, pregnancy was confirmed by ecography (Aloka Flexus SSD-1100; Hitachi Aloka Medical, Tokyo, Japan). On day 20, pregnant guinea-pigs were anesthetized with intraperitoneal ketamine (60 mg/kg) and xylazine (4 mg/kg) prior to subcutaneous implantation of Alzet 2ML2 osmotic pumps (Durect, Cupertino, CA) in the interscapular region.
Over 14 days, these pumps delivered either saline solution (control; n = 6), angiotensin II (Sigma) 200 μg/kg/day (AII; n = 7), Bradyzide (Sigma) 62.5 μg/kg/day (BDZ; n = 5), or angiotensin II plus Bradyzide (AII + BDZ; n = 6). The dose of AII was subpressor, as determined in a pilot study in non-pregnant guinea-pigs. To evaluate the direct and the persistent consequences of the interventions, systolic blood pressure was determined during the last hours of the infusion on gestational day 34, and at 6 and 26 days after withdrawal of the osmotic pumps. Blood pressure was measured under anesthesia in the pad of the right paw compressed by a neonatal blood pressure cuff (Critikon, General Electric Healthcare, Connecticut, EEUU) using a Power Lab 8 SP system (ADInstruments, Sidney, Australia). Blood pressure data were analyzed with the Labchart graphic software 6.1 Pro (ADInstruments). After the blood pressure measurement on gestational day 34, the osmotic pumps were extracted while the animals were still anesthetized.
The study also included two groups of non-pregnant cycling guinea-pigs. The first group included control non-pregnant females (C NP; n = 5); in them the systolic blood pressure was measured in random days of the estrous cycle. The second group received subcutaneous infusions of AII + BDZ for 14 days (AII + BDZ NP; n = 6), and systolic blood pressure was measured on the last day of infusion, and at 6 and 26 days after its discontinuation.
On gestational day 60 or 26 days after AII + BDZ infusion in non-pregnant females, with the animals under ketamine and xylazine, urine was withdrawn from the bladder for determination of protein (Bradford method) and creatinine (Beckman Autoanalyser, Fullerton, CA) and blood from the left ventricle for creatinine and uric acid determination (Beckman Autoanalyser). Then the animals were euthanized with an overdose of ketamine/xylazine, and the uterus, feto-placental units, and kidneys were removed. The fetuses, placentas, and kidneys were weighed after trimming off the umbilical cord, amniotic membranes, and perirenal fat. Fetal loss was attributed to demise if an atrophic or necrotic feto-placental unit was observed, or to abortion when a fetus observed by ultrasonography was absent at term, since miscarriages in guinea-pigs are frequently not accompanied by persistence of the implantation site (Elger, personal communication). Only units with a live fetus were included for measurements of fetal and placental weight and for immunohistochemical studies. A central cross-section through the placenta, subplacenta, implantation site, and underlying myometrium was fixed as a single block. The placenta, and the subplacental decidua and myometrium were also isolated in other feto-placental units; from these, the fetal/placental weight ratio was calculated as a marker of placental efficiency [
39]. Tissues were immediately fixed with phosphate-buffered 10% formalin for 24 h, then dehydrated in a graded series of ethanol and xylene dilutions, and embedded in Paraplast-Plus® (Sigma, St. Louis, MO). Sections (6 μm) were mounted on silanized slides.
Immunostaining procedure
Immunostaining was performed at room temperature. Deparaffinized sections were rehydrated using ethanol, rinsed three times for five minutes each in phosphate-buffered saline with 50 mM Tris–HCl, and submitted to heat-induced antigen retrieval using citrate buffer (pH 6.0). Endogenous peroxidases were blocked by incubation in 10% H2O2 for ten minutes. Sections were then incubated in a humid chamber for 30 min with protein block (Cas-Block®; Zymed, San Francisco, CA), followed by incubation for 18 h at 4°C with anti-pancytokeratin mouse monoclonal antibody (1:50, P2871, Sigma). Sections were immunostained using a biotin-streptavidin-peroxidase system (LSAB + ®, DakoCytomation). Finally, the samples were treated for 15 min with 0.1% (w/v) 3-3′-diaminobenzidine in buffer containing 0.05% H2O2. The slides were counterstained with Harris hematoxylin (Sigma).
Evaluation of uterine arterial trophoblast invasion
Spiral arteries were identified as lateral if localized in the periphery of the subplacenta, or myometrial if in the uterine smooth muscle below the placental bed. The extravillous trophoblast (EVT) was defined as intramural when located in the media of the spiral artery, and as endovascular when lining the lumen of the spiral artery and replacing endothelial cells. Photographic images were acquired with a Nikon CoolPix 4500 camera (Nikon Inc., Tokyo, Japan), coupled to a Zeiss AxioImager AX.10 microscope (Carl Zeiss, CA). The portion of the endovascular trophoblast replacing endothelial cells was calculated by measuring the arterial perimeter occupied by cytokeratin-positive cells, using Axiovision 4.8.2.0 LE (Carl Zeiss AG, Inc., Oberkochen, Germany). Groups were compared according to the percentage of intraluminal perimeter occupied by cytokeratin-positive cells in the lateral and myometrial spiral arteries.
Statistics
A Bayesian analysis [
40] was performed to test differences between systolic blood pressures in different groups and periods and the proportion between viable versus lost fetuses according to treatment. Systolic pressure was modeled as a multivariate normal distribution with different mean vectors and variance-covariance matrices for groups and periods. Data are expressed as mean and dot-plot of individual values. The logarithm of the non-viable fetuses/[1 – the predicted non-viable fetuses] was modeled as a normal distribution, with group-dependent median and variances. After proving that variances were similar among groups, a common variance was used. For each group, we calculated the proportion of viable fetuses, the differences between these proportions, and their credible intervals (CI). Data are expressed as proportion of live fetuses and CI. The analyses were performed with the open-source OpenBUGS program. Differences were considered significant when the CI did not cross zero.
A one-way ANOVA with Fisher’s LSD post-hoc test was performed to determine the effects of the interventions on fetal, placenta, and kidney weights. Student’s t-test was used to evaluate differences in the protein/creatinine index among the different groups of pregnant dams and the non-pregnant females that received AII + BDZ. Data are expressed as mean and dot-plot of individual values. Data were analyzed using Graphpad Prism 6.01 (GraphPad Inc., San Diego, CA) and were considered significantly different when P < 0.05.