All procedures with the animals were in accordance with the standards described in the Guidelines for Ethical Conduct in the Care and use of Animals, the Guideline of the Committee on Care and Use of Laboratory Animal Resources of the National Research Council of the United States of America and the authors complied with the ARRIVE guidelines. This project was approved by the Research Ethics Committee for Animals (CEUA-USJT) at São Judas University 038/2019.
21 female mice, 6 months old, ApoE-Ko, that are animals prone to the development of atherosclerosis14, were divided into 3 groups: control submitted during 9 months to normolipidic diet (C); ovariectomized submitted during 9 months to normal diet (Ovx); ovariectomized submitted during 9 months to high-fat diet (OvxHF). The animals came from the Lipid Laboratory of the Faculty of Medicine of the University of São Paulo, and were transported to the biotery of São Judas University, where they were kept in plastic boxes (5 animals per cage), in an environment with controlled temperature (22o – 24oC) and with controlled light, in a 12-hour cycle (light / dark) and fed with water and feed “ad libitum”.
At the beginning of the protocol, the mice were anesthetized (mixture of 0.5% -2% isoflurane and 98% O2 at a flow rate of 1.5 L / min) and were placed in the supine position, where a small incision (1 cm) was made in parallel with the body line on the skin and muscles in the lower third in the abdominal region. The ovaries were located and ligation of the oviducts, including blood vessels, was performed. The oviducts were cut, and the ovaries removed. The musculature and skin were sutured, analgesic were administered 11,15–17.
The administration of the diets began in the 6th month of life of the mice. “AIN - 93M” diet was administered in C and Ovx groups (REEVES; NIELSEN; FAHEY, 1993), whose caloric content is 3,802.8 Kcal/kg, of which 76% were provided by carbohydrates, 14% by proteins and 10% by lipids (soy oil). For OvxHF group, “AIN-93M” adapted diet, whose caloric content is 5,362.8 Kcal/kg, of which 60% was provided by lipids (lard and soybean oil), 30% by carbohydrates and 10% proteins, as previously explained and detailed (REEVES; NIELSEN; FAHEY, 1993).
Water and feed were offered unrestricted, with feed consumption measured 3 times a week, on alternate days. At the end of the study, the average weekly consumption of each box was calculated and divided by the number of animals in the box to obtain the average individual consumption value. In addition, the caloric consumption of each animal was calculated. For normolipidic feed, 3.8 kcal/gram was calculated, for high-fat diet, 5.6 kcal/gram.
The blood glucose measurement and the oral glucose tolerance test (OGTT) were performed in the 13th month of animal life and at the end of the protocol (15th month of animal life). Blood glucose concentrations were determined on a 12-hour fast using the Roche © Advantage® device and its reagent strips (IRIGOYEN et al., 2005a; SANCHES et al., 2012, 2014; SOUZA et al., 2007a). Then, a glucose solution (1.4g/kg of body weight of the animal) was gavaged and blood glucose measured after 15, 30, 60, 90 and 120 minutes and, subsequently, the area under the curve was calculated 11.
The echocardiographic examination was performed at the end of the protocol (15th month) with the anesthetized animals (mixture of 0.5% -2% isoflurane and 98% O2 at a flow rate of 1.5 L / min). SEQUOIA 512 equipment (ACUSON Corporation, Mountain View, CA) was used with a 15 MHz transducer. From the visualization of the left ventricle (cross section) at the level of the papillary muscles, diastolic (DDVE) and systolic (DSVE) diameters of the left ventricle and the thickness of the interventricular septum (IVS) and the left ventricular posterior wall (PP) in systole and diastole. After the measurements were taken, the left ventricular mass was calculated, according to guidance from the American Society of Echocardiography, which estimates the left ventricle mass using the following mathematical formula: LVM = [(LVDD + SIV + PP) 3- (DDVE) 3] x1.047, where 1.047 (mg/mm3) corresponds to myocardial density. In addition to the left ventricle mass, the left ventricular shortening force (D% = [(DDVE-DSVE) / DDVE] x100) was calculated. The absolute values of left ventricular mass were normalized by body weight. The cardiac hypertrophy index was calculated by dividing the left ventricular mass by body weight. The images obtained through Doppler were used to calculate the parameters of the left ventricular diastolic function. Peak E wave velocities, peak A wave velocities, isovolumetric relaxation time (TRIV) and deceleration time were measured, and the E/A wave ratio was also calculated. Also using the ejection time (TE) of the left ventricular outflow tract, the circumferential shortening speed of the myocardial fiber (Vcf = [(DDVE-DSVE) / DDVE] / TE) was calculated. Although VCF is sensitive to acute changes in arterial pressure in hemodynamic overload, at baseline conditions, in the absence of acute changes in blood pressure, the calculation of VCF provides information regarding myocardial contractility.
One day after echocardiographic evaluations, the animals were anesthetized (mixture of 0.5% - 2% isoflurane and 98% O2 at a flow rate of 1.5 L/min) and placed in the supine position. A small incision was made in the neck, through which polyethylene catheters (cannulas; tygon P50) filled with saline were implanted. These cannulas were positioned inside the carotid artery and jugular vein to record arterial pressure (AP), heart rate (HR) and drug administration, respectively. After the correct and firm implantation of the cannulas in the carotid artery and jugular vein, these were externalized on the animal's back in the cervical region and fixed with cotton thread on the skin. 11,16–21. Each animal was kept in a standard individual box during the systemic hemodynamic evaluations that started 24h after the cannulation.
With the animal awake, the arterial cannula was connected to an extension of 20 cm, allowing free movement of the animal through the box, during the entire period of the experiment. This extension was connected to an electromagnetic pressure transducer (Kent Instruments) which, in turn, was connected to a preamplifier (Stemtech). BP signals were recorded over a period of 30 minutes on a microcomputer equipped with a data acquisition system (WinDaq Recording and Playback Software), allowing analysis of pressure pulses, beat-to-beat, with a sampling frequency of 4KHz by channel to study the BP and HR values (DE ANGELIS et al., 2012; HEEREN et al., 2009).
After recording BP, an extension of approximately 20 cm (P10) was connected to the venous cannula for injection of vasoactive drugs. With animals at rest, baroreflex sensitivity was tested by infusing increasing doses of phenylephrine (100 ng/ml, 150 ng/ml, 250 ng/ml) and sodium nitroprusside (100 ng/ml, 150 ng/ml, 250 ng/ml). The drugs were injected randomly between the animals, beginning the session with one or the other drug. Phenylephrine (Sigma Chemical Company, St. Louis, MO, USA) is potent α1 stimulator, whose predominant action occurs in peripheral arterioles, causing vasoconstriction, was used to cause increased BP, which is followed by reflex bradycardia commanded by the preceptors. Sodium Nitroprusside (Sigma Chemical Company, St. Louis, MO, USA) is a potent vasodilator, both for arterioles and veins, whose action occurs through the activation of guanylate cyclase and increased synthesis of 3',5'- guanosine monophosphate (cyclic GMP) in the smooth muscles of vessels and other tissues, was used to cause a fall in BP, followed by a reflex tachycardic response commanded by the pressoreceptors. The α index was obtained from the division between the PI and SAP variability in the two main low frequency bands (LF) 22.
From the baseline record of the awake animals, it was possible to use the time-frequency analysis tool for pulse interval (IP) and systolic arterial pressure (SAP) variabilities. The parameters for analysis in the time domain consisted of calculating the mean values of SAP and IP, and their variability was quantified by calculating the mean of standard deviations. The variability of the pulse interval was obtained by analyzing the tachogram from the SAP record, where the frequency of beats was determined by the interval between two systolic peaks. For this analysis, stable records of at least 5 minutes and sampling frequency of 4,000 Hz were used 23.
The analysis in the frequency domain consisted of the decomposition of the sistogram by the Fast Fourier Transform. After this mathematical remodeling, the absolute powers of the very-low frequency band of the pulse interval (VLF-IP: 0.00 - 0.4 Hz), low frequency band of the pulse interval (LF-IP: 0.4 Hz -1.50 Hz), and high frequency band of the pulse interval (HF-IP: 1.5 - 5.0Hz) were obtained 24–27. The LF component was used as an indicator of sympathetic modulation. The HF component was used as an indicator of parasympathetic modulation. The LF/HF ratio indicated the sympathetic-vagal balance 24.
At the end of the study, the following tissues were collected and weighed: Heart, left ventricle, white adipose tissue, soleus and gastrocnemius muscle. Subsequently, the % of the body weight of the tissues was calculated for comparison between the groups.
Data analysis were performed using the Graph Pad Prisma software (version 8.0). The arithmetic mean and the standard error of the mean (SEM) were calculated for all variables. The Shapiro-Wilk test was used to verify the normality of the results. After the experimental period, the values obtained were analyzed by the two- way variance test (Two- way ANOVA), followed by Turkey's post hoc. The level of significance used in all analyzes was 5%.