ABC | Volume 112, Nº1, January 2019

Original Article Jevjdovic et al Prenatal stress affects rat heart ADRB1 Arq Bras Cardiol. 2019; 112(1):67-75 In order to better understand molecular mechanisms by which prenatal stress may potentially contribute to the development of cardiovascular diseases in adulthood, the present study was designed to investigate region-specific gene expression of adrenergic receptor subtypes (ADRB1, ADRB2 and ADRB3) and MAO-A in the left ventricular myocardium of female and male offspring. Methods Animals Three-month-old virgin female Wistar rats (266 ± 11.9 g) were housed with free access to food and water under constant light-dark cycle (12 h) in temperature-controlled conditions (22 ± 1°C) in the animal facility of the Faculty of Biology, University of Belgrade. Sample size was determined by convenience, and each of six pairs of female rats was caged with a sexually experienced male during a whole oestrus cycle. Day 0 of pregnancy was marked by appearance of sperm in vaginal smear. One female remained non-pregnant. To avoid selection bias, pregnant females who were mated with the same male were randomly assigned to control (n = 5) or stressed (n = 6) group and housed individually. All procedures were conducted according to the rules for animal care proposed by the Federation of European Laboratory Animal Science Associations (FELASA), and approved by the Ethics Committee of the Faculty of Biology, University of Belgrade. Prenatal stress protocol During the third week of gestation (gestational day 13‑20, GD13-GD20) pregnant rats were exposed to a chronic unpredictable mild stress (CUMS) protocol that included random and intermittent exposure to a variety of stressors. Detailed CUMS protocol is shown in Table 1. Briefly, animals were exposed to the following stressors in random order twice a day for 1 h or overnight: damp bedding, restraint in a Plexiglas® tube, cold room (4°C), cage displacement and noise, overnight illumination, and cage tilt. Control mothers were left undisturbed for the duration of their pregnancies with the exception of general handling. During the entire pregnancy, water and food intake were recorded. Biochemical assays Before first and after last exposure to the stressor, blood was collected from dam's tail vein in EDTA-containing tubes. Adrenocorticotropic hormone (ACTH) plasma levels were measured with a CLIA kit and glucose levels were measured with an Exac-tech glucose analyzer using Dextrostix reagent strips, both according to the manufacturers’ instructions. Litters At birth pups were counted and weighed, and litters were adjusted to eight pups with an equal number of males and females to avoid effects of litter size and litter sex-distribution on development. All pups were raised by their biological mothers. The offspring were weaned at 28 days, separated by gender and housed in groups of two per cage, according to the experimental group (C- offspring from unstressed mothers, PS- offspring from stressed mothers). Offspring’s body weight and water and food consumption were recorded during both pre- and post-weaning periods. The offspring were sacrificed by decapitation at two months of age. To avoid oestrus cycle dependent fluctuations, female offspring were sacrificed in dioestrus, as confirmed by vaginal smears. RNA isolation Total RNA from the basal and apical portions of the left ventricles was isolated using TRI Reagent (Sigma, Germany) according to manufacturer instructions. Total RNA concentrations were quantified by absorbance measurements at 260 and 280 nm using a spectrophotometer (Ultrospec 2000, Pharmacia Biotech, USA) according to manufacturer instructions. RNA quality was analyzed on 1.5% agarose gel containing ethidium bromide and visualized by UV transillumination (ChemiDoc-It imager, UVP, Germany). cDNA synthesis and quantitative real-time PCR RNA samples (2 µg) were subjected to DNase I treatment, using rDNase I, according to manufacturer protocol (DNA-free kit, Ambion, USA). Ready-to-go You-Prime First-Strand beads transcription kit (GE Healthcare, USA) was used to generate cDNA for subsequent quantitative real-time PCR. Samples without reverse transcriptase were used to control for possible contamination of gDNA. All reactions were carried out in Table 1 – Stress regime 10:00-11:00 14:00-15:00 18:00-08:00 GD14 Restraint Damp bedding Cage tilt GD15 Cold room (4 ° C) Displacement and noise Continuous illumination GD16 Damp bedding Restraint Cage tilt GD17 Displacement and noise Cold room (4 ° C) Continuous illumination GD18 Restraint Damp bedding Cage tilt GD19 Cold room (4 ° C) Displacement and noise Continuous illumination GD20 Damp bedding Restraint Cage tilt * GD: gestational day. 68