Animals and Model of obesity
This was an experimental, cross-sectional and analytical study. The population was a murine model of rats of the Sprague Dawley strain obtained and used from an inbred colony of bioterium of the Specialty Hospital of the National Medical Center, Mexican Social Security Institute (Mexico City, Mexico), a total of 20 males were used and weighing 200-250 g. Animals were randomly allocated and were divided into the control group C (n=12) fed the standard diet (Formulab 5008 Diet; PMI Nutrition International, Brentwood, MO, USA), the Ob group (n=8) was fed a hypercaloric diet (high in fructose 30%), until 6 months of age, the rats stayed with this diet. All cages contained wood shavings, bedding and a cardboard tube for environmental enrichment. All rats had ad libitum access to a standard pellet diet and water; they were housed in groups of 4 rats, in conventional cages at room temperature (22-25°C), under a light cycle of 12 hours’ light/12 hours’ dark since at the start of the trial and hygienically controlled room. We have worked with this experimental model in other projects [5]. The hypercaloric diet was used to induce obesity and insulin resistance in (Ob group) experimental rats.
Ethical statement
All experiments were performed in accordance with relevant guidelines and regulations of bioterium of the Specialty Hospital of National Center Medical, of the Mexican Social Security Institute (CMN SXXI-IMSS), in accordance with the Official Mexican Standard (NOM-062-ZOO-1999, revised 2001) for the care and use of laboratory animals. Also, we obtained written informed consent to use the animals in our study. This study was approved by the Ethical Committee and the Local Research and Health Committee of the Mexican Social Security Institute, with number registered 3601-2015-95. The animals were treated according to the Official Mexican Standard for the care, use and sacrificing laboratory animals. All rats were fed until 12 months of age were anesthetized and sacrificed by cardiac puncture (NOM-062-ZOO-1999, revised 2001).
Metabolic parameters and Body weight
Body weight. From the beginning to group C (n=12) and Ob (n=8) their weight was recorded until the end of the study. Food and water intake were recorded for week.
Blood samples were collected at 8 am (during a fasting 7 hours at 6 month of age) from the tail vein in order to measure the blood biochemical parameters and inflammatory cytokines. The blood samples were transferred into tubes containing anticoagulant for measurement of blood biochemical parameters and inflammatory cytokines, respectively. The samples were centrifuged at 5200 g for 15 minutes. Plasma was separated and stored at -70 °C until use for bioassay analyses.
Blood parameters were determined by One drop of blood was placed on the blood glucose test strips of the FreeStyle Optium Xceed glucometer (Abbot Diabetes Care Ltd, OYL, UK).
Determinations of levels of total cholesterol, triglycerides (TG), high density lipoproteins (HDL) and low-density lipoproteins (LDL) were made on a Cardiocheck apparatus after placing one drop of blood (see previous section on blood samples) on a reactive strip, according to the manufacturer’s instructions.
Quantification of insulin and index HOMA-IR, insulin was measured for chemioluminiscence using Insulin IMMULITE kit (LKIN1 insulin IMMULITE);
Insulin resistance was calculated the index HOMA-IR through the formula of homeostasis model assessment-insulin resistance (HOMA-IR) index: insulin (μU/mL) × glucose (mg/dL)/405.
TNF-α, IL-6, adhesion molecules ICAM-1 and VCAM-1 were analyzed using enzyme immunoassay kits by ELISA R&D kit (R&D Systems, Inc., Minneapolis, MN, USA).
After, all rats were fed until 12 months of age. For collection of blood samples by cardiac puncture, also analysis of cytokines in tissue, rats were sacrificed with deeply anesthetized and administered pentobarbital (25 mg/kg, i.p.) [10].
Ultrasound analysis.
To carry out the study, the rat was placed in the left lateral decubitus position, with a slight inclination of the head and placing the transducer in the different acoustic windows. The equipment used was a Philips Affiniti 70 with a linear transducer of 9 mHz, where the results were obtained in millimeters. During the echocardiographic examination, two cuts were used for the evaluation of the heart: the left longitudinal parasternal section and the right longitudinal parasternal section. In the left longitudinal sternal section, it is possible to visualize the right ventricle, aorta with aortic valve, left atrium, mitral valve and left ventricle [11].
The right longitudinal sternal section that appears on the left and the base (atria) on the right, with the rotation of the transducer in the right direction, shows the left ventricular outlet, aortic valve, aortic root and proximal ascending aorta [12].
The measurement of the left ventricle in diastole was performed from the left septal endocardium to the posterior wall endocardium, measured below the level of the mitral valve. The interventricular septum is between the mitral ring in its posterior part and the endocardial surface of the high septum in its anterior part. In the free wall of the VI, the diastolic thickness is measured; its value is approximately equal to the diameter of the IVT, and its birth is parallel to it.
Statistical Analysis
The data are presented as means ± standard deviation (SD) of each group. The study groups were statistically analyzed the difference in means between group C vs Ob with a Student t test, the level of statistical significance was considered with a value of p <0.05. For each study variable: glucose, insulin, lipid profile, HOMA-IR index, proinflammatory cytokines, TNF-α, IL-6, adhesion molecules ICAM-1 and VCAM-1 statistical analysis was carried out using Graph Pad Prism. (GraphPad Prism 8 for Windows, San Diego, CA); p <0.05 was considered significant.