ABC | Volume 113, Nº3, September 2019

Original Article Imani et al. Sympathetic nervous system and cardioprotection Arq Bras Cardiol. 2019; 113(3):401-408 Methods A total of 32 male Wistar rats (200-250g) were kept in an air-conditioned room on a 12 hours light-dark cycle, at 22 ± 2˚C, with free access to water and food. The experimental protocols followed in this study conformed to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were further approved by the institutional ethical committee of Tehran University of Medical Sciences (Tehran, Iran). Stress box apparatus was used for physical stress exposure. It contained stainless bars at the bottom, connected to electroshock device using a connecting cable. Physical stress was induced using electrical foot shock (1mA) for 10 seconds with 50 seconds intervals for one hour. After that, animals were anaesthetized with sodium thiopental (60 mg/kg, i.p), 13 put on a surgical board. The chest was opened and the surgical silk suture (6-0) placed under the root of the left anterior descending coronary artery (LAD). Finally, the heart was removed from the chest and connected to Langendorff- perfusion apparatus. Heart was perfused in a retrograde manner using Krebs–Henseleit bicarbonate buffer (in mmol/l): sodium bicarbonate = 25, sodium chloride = 118.5, potassium chloride = 4.7, magnesium sulfate = 1.2, glucose = 11, gassed with 95% O 2 and 5% CO 2 (pH = 7.3-7.4 at 37˚C). Thereafter, the ends of the suture were passed through a plastic tube to create a snare for ischemia induction. Reperfusion was performed by releasing the snare. Latex fluid- filled balloon was inserted inside the left ventricle and connected to a pressure transducer (Harvard, March-Hugsteten, Germany), the biolab apparatus was used for recording the ventricular pressures. During the surgical procedure, recording was done during three designated periods: 20-30 minutes of the baseline (a period without any manipulation), 30 minutes of the local ischemia and 120 minutes of the reperfusion. After reperfusion, LADwas occluded again; Evans Blue dye (3 mL of 1.5% solution) was administrated to discriminate ischemic zone (the area at risk; [AAR]) from non-ischemic zone. 14 After freezing (−20°C for 24 hours), heart tissue was sliced into 2mm transverse sections and kept in 1% 2, 3, 5 triphenyltetrazolium chloride (TTC in 0.1 M phosphate buffer, pH = 7.4 Sigma) solution for 15–20 min at 37°C to delineate ischemic from infarct zone. 15 At the end of the experiments, the ratio of AAR and infarcted size (IS) were calculated by the Photoshop program. Animals were allotted in 4 groups: 1. IR group (n = 8): Rats were kept in stress box device (without stress exposure) for 1 hour and then, hearts were removed from the chest and subjected to ischemia and reperfusion. 2. Acute stress (St+IR) group (n=8): Rats were exposed electrical feet shock in the stress box for 1 hour and then, hearts were removed from the chest and subjected to ischemia and reperfusion. 3. Sympathectomy (Symp+IR) group (n = 8): chemical sympathectomy was done by injection of a 6-hydroxydopamine (6-OHDA, 100 mg/kg, sc) 24 hours prior to I/R induction. 16 4. Sympathectomy- physical stress (Symp+St+IR) group (n = 8): chemical sympathectomy was done 24 hours prior to physical stress and I/R induction. We measured serum corticoesterone levels by ELISA method. Moreover, systolic blood pressure was measured via non-invasive technique (Tail Cuff and power lab) to confirm chemical sympathectomy (n = 4). Statistical analysis The sample size and group divisions were defined based on our previous studies. 17 All data are reported as means ± S.E.M. Normality was checked using Kolmogorov-Smirnov test, SPSS software version 20. One way ANOVA and Tukey post hoc test was done for comparison of parameters between different groups. Analysis of changes in mean values over three times was done using repeated measurement ANOVA within each group. Sample t-test was used to compare systolic blood pressure before and after sympathectomy. Significant changes were considered as p < 0.05. Results Effect of acute physical stress on coronary flow and heart rate Figure 1 shows coronary flow (CF) at the end of the baseline, ischemia and reperfusion periods. There are significant differences for CF at the end of ischemia and reperfusion when compared to the end of the baseline period within groups (p < 0.01). HR was significantly decreased at the end of both ischemia and reperfusion in comparison with the end of baseline period within groups (p < 0.01), but no significant change were observed between different groups (Figure 2). Effect of acute physical stress on cardiac hemodynamic parameters The left ventricular developed pressure (LVDP, the difference between intraventricular systolic and diastolic pressures), rate product pressure (RPP, LVDP multiplied by HR) and were diminished at the end of reperfusion in comparison to the end of baseline period among groups, The amount of RPP and LVDP in acute stress group were extremely increased in comparison to IR group (p < 0.05 in induction of chemical sympathectomy before physical stress were considerably decreased RPP and LVDP in comparison to physical stress group p < 0.05, but there is no marked difference between sympathectomy group when compared to IR group (Figure 3). Effect of acute physical stress on infarcts size (%IS/AAR) Figures 4 shows the size of the infarct (%IS/AAR) in different groups. Infarct size was greatly deacreased in acute stress group as compared to the IR group (p < 0.05), but there was no considerable change in chemical sympathectomy group as compared to the control group. Chemical sympathectomy prior to acute physical stress represented no extermly change 402