Echography analysis altered in musculoskeletal, heart and liver associated with endothelial dysfunction of obese rat

Background: Modern imaging plays a central role in the care of obese patients, with an integral focus on its use and accessibility in individuals into this condition with alterations of various organs. Objective. To perform an echographical analysis of musculoskeletal system disorders, endothelial dysfunction and the left ventricle in obese rats. Methods. Sprague Dawley rats (250±5 g) were used and divided in two groups: control group (C) fed with a standard diet, and the obese group (Ob) fed with a hyper caloric diet of high fructose-fat for 4 months. Body weight, cholesterol, triglycerides, glucose, inammatory cytokines and adhesion molecules (ICAM-1, VCAM-1) were measured. Additionally, two-dimensional echocardiography, abdominal ultrasound and musculoskeletal system studies were performed in the lower extremities. Results. Body weight in the Ob group was increased compared to the control group, (p <0.001); in addition, increased glucose, cholesterol and triglycerides were found in the Ob group vs the C group, (p<0.05), and as well as increased adhesion molecules ICAM-1 and, VCAM-1 (p<0.01). On ultrasound, 75% of the Ob group presented, showed 75% fatty liver and distal joint abnormalities. Conclusion. Endothelial dysfunction and changes at the level of the musculoskeletal system with the presence of joint cysts in the posterior region of the distal joint of the lower extremities and fat liver were observed in obese rodents. Lipoprotein; MCP-1: Monocyte NAFLD: Non-Alcoholic Fatty Liver Ob: hypercaloric diet group; PAI-1: Plasminogen Activator Inhibitor; QB: Baker's cyst; SD: standard deviation; TG: Triglycerides; TNF-a: Tumor Necrosis Factor-a; VCAM-1: Vascular Adhesion Molecule-1; VI: left ventricle; VLDL: Very Low Density Lipoprotein.


Background
Overweight and obesity are de ned as an abnormal or excessive accumulation of fat that is harmful to health and it is an important risk factor for non-communicable diseases, such as cardiovascular diseases (mainly heart disease and strokes), associated to metabolic disorders (glucose intolerance, insulin resistance, hyperlipidemia, diabetes and hypertension), musculoskeletal disorders (especially osteoarthritis) and some cancers (endometrial, breast, ovarian, prostate, liver, gallbladder, kidney and colon) [1].
Some mechanisms that are triggered by obesity involve the in ammatory process, with the production of proin ammatory cytokines such as interleukine-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α). Thus, transendothelial migration of in ammatory cells (leucocytes) and production of cytokines are an early step in endothelial dysfunction, which continues with the activation of adhesion molecules (ICAM-1, VCAM-1); this contributed, from early endothelial dysfunction to the atherosclerotic plaques, causing vascular complications [2][3][4][5][6]. To understand these pathophysiological mechanisms, experimental models are used, as murine models (rats) which are a valuable tool to understand these mechanisms linked to the comorbidities of the metabolic disease [7].
In obese rat have been demonstrated excess lipid accumulates in other tissues including the liver, the skeletal muscle and the heart, also, these are associated with an increase in adipose mass and free fatty acids. Likewise, in liver together with other intrahepatic signals leading to derangement in glucostatic and lipidostatic functions, are generating to greater vulnerability to hepatic steatosis [8,9].
While echography is usually used in the clinical setting for the diagnosis and follow-up of patients with nonalcoholic fatty liver disease (NAFLD); also, this analysis is a good method which allows to examine arteries, cardiopathies and fatty tissue [10]. However, in obese murine models no study has assessed sonographic ndings of several organs in obesity, this could be identi ed to integration and relationship of dysfunctions of mechanism different in other organs or comorbidities in same organism.
Thus, ultrasound is an economical, accessible, fast, precise, simple, comfortable, and noninvasive procedure. It does not cause pain, radiation is not used, it has high sensitivity and accuracy to obtain images and it is essential in the study of a variety of organs such as the liver and the musculoskeletal system. Two-dimensional echocardiography is the study of the heart in two dimensions; it allows us to analyze the organ as a whole and the relationships that the cardiac structures maintain with each other. Two-dimensional echocardiography is very useful in the study of congenital anomalies, in the differentiation between thrombi and intracardiac masses and in the analysis of regions of di cult access with the one-dimensional echocardiogram [11].
Although there are ultrasound studies in rats where the various morphological and functional aspects are both analyzed at the cardiac level, mainly in the left ventricle, none of them has been considered important for a joint analysis of both, the left ventricle and the hepatic, renal and musculoskeletal systems represented in humans [12]. Therefore, we report here in this study the echographic analysis of the left ventricle, liver, musculoskeletal disorders and endothelial dysfunction in obesity.

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 weighing 200-250 g, were used. Animals were randomly allocated and were divided in two groups: the control group C (n=12) was fed with the standard diet (Formulab 5008 Diet; PMI Nutrition International, Brentwood, MO, USA) and the Ob group (n=8) was fed with a high fat-fructose diet (standard diet supplemented with 10% lard and 30% fructose mixed and dissolved in drinking water) by 16 weeks ad libitum, both groups had this diet until six months of age. Food and water intake, as well as, the animals weight, were recorded daily during these weeks. We have worked with this experimental model in other projects [5]. This animal model employed in this work with the ingestion of a high fat-fructose diet is clearly associated with insulin resistance development, disturbed glucose homeostasis and endothelial dysfunction in rodents [4][5][6]. The hypercaloric diet was used to induce obesity in (Ob group) experimental group.
All cages contained wood shavings, bedding and a cardboard tube for environmental enrichment. All rats of each group had ad libitum access to their 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. In this work the obese group had a hypercaloric diet during 4 months, and both groups; C and Ob were studied at 6 months of age. However, their feeding continued of both groups until 12 months of age for others of tissues and organs analysis.

Metabolic parameters and Body weight
Body weight. The weight of both groups: group C (n=12) and Ob (n=8) was recorded from the begging to the end of the study. Food and water intake were recorded per week.
Blood samples were collected at 8 am (during a 7 hours fasting at 6 month of age) from the tail vein in order to measure the biochemical blood parameters and in ammatory cytokines. The blood samples were transferred into tubes containing anticoagulant for measurement of biochemical blood parameters and in ammatory 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 glucose level was 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). In each animal was twice recorded.
Determinations of levels of total cholesterol, triglycerides (TG), high density lipoproteins (HDL) and lowdensity 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.

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 A niti 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 [11,12].
The measurement of the left ventricle in diastole was performed from the left septal endocardium to the posterior wall endocardium, measuring 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 signi cance was considered with a value of p <0.05. For each study variable: glucose, insulin, lipid pro le, HOMA-IR index, proin ammatory 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 signi cant. Figure 1 shows the differences in food consumption in the study groups, in Obese group with hypercaloric diet had a rise compared with control (p<0.001). Also, an increase in body weight was observed in the Ob group vs the C group (22.5%; 472 ± 25 vs 385 ± 20, p<0.001). In addition, the Ob group showed an increase in fasting glucose (p<0.05), lipid pro le and HOMA-IR index (300%; 4±1 vs 16±3, p<0.0001); therefore, also because of lipids analyzed, these rats were considered obese rats with metabolic alterations (Table 1). cytokines TNF-α, also adhesion molecules that are indicators of endothelial damage, such as ICAM-1 (87.6%, 122± 9 vs 65±5, p <0.0001) and VCAM-1 (29.4% 110±10 vs 85±2, p <0.05) ( Table 2). ICAM-1 and VCAM-1 are relevant in chronic in ammatory process, with highlights to an increased risk for type 2 diabetes (T2DM) and cardiovascular diseases (CVD) [6,7,13]  In the Ob group, an increase in these adhesion molecules and in proin ammatory cytokines such as TNF-α was demonstrated, with a signi cant difference compared with the C group (220%, 16±2 vs 5±0.05, p<0.001), and similar was for IL-6 (155%, 23±5 vs 9±1, p<0.001). According to the measurements of the metabolic parameters and cytokines, the Ob group, with the consumption of the hypercaloric diet, presented endothelial damage. In addition, modern imaging allowed us to evaluate an integral approach of the control group, where the aorta and left ventricle (VI) were observed. For VI, an increase in the septum and wall was found in the Ob group (Table 3, Figure 2).

Results
However, in Figure 3A, the analysis of the two-dimensional ultrasound being more complete, in the obese group was observed fat ltration, with the presence of fatty liver (Table 3); on the other hand, a homogeneous hepatic parenchyma was found being isoechoic in the control group ( Figure 3B).
Another analysis of the ultrasound dimensions, that was obtained in the Ob group showed alterations in pelvic dilatation in the kidney compared to the control group where the kidney was echogenic, with no evidence of dilation ( Figure 3C, D).
In the ultrasound images at the musculoskeletal level, we can observe alterations in the Ob group, where these changes were found due to fat deposits, demonstrating the presence of cysts at the articular level, in the posterior region of the distal joint, where it is possible to view the damage due to accumulated fat (Table 3, Figure 3E). Table 3. Descriptions of two-dimensional echocardiography of kidney and skeletal muscle (lower extremities) and left ventricles (LV), partition IV y wall of LV express in mm

Discussion
In this study, we showed relation among obesity, endothelial dysfunction and alterations in several organs as musculoskeletal system, kidney, liver and heart through a noninvasive method as is echography. Thus, our result showed that high fat-fructose diet increased intake food, with marked weight gain due the caloric contribution provided by the fat and carbohydrate in obese group. We also found metabolic alterations, such as rise in glucose, triglycerides, cholesterol and HOMA-IR index, similar as other studies where were used hypercaloric diets [14,15]. Hence, with the obesity is triggered dysmetabolism, in our results observed hyperglycemia and hypertriglyceridemia, also, an increase of the levels of in ammatory cytokines (TNF-a, IL-6). Likewise, as a consequence of obesity can be manifested alterations in other organs that could be analyzed in ultrasound. In this work was relevant the results in muscle-skeletal and articular levels to show the presence of cysts at the articular level, in the posterior region of the distal joint, where it was possible to view the damage due accumulated of fat. Other alterations were presented in heart, kidney, musculoskeletal and liver analyzed by echography.
In this study we used a noninvasive and fast method, in the evaluation of the ventricular anatomy, although the cut-off point for normal relative parietal thickness in humans is 0.42 mm, this criterion was highly speci c in our animals because the rats presented a relative parietal thickness below that limit [11,12]. Also, in this work was found the presence of Baker's cyst (QB), or popliteal cyst, which was rst described in 1840 by Adams [16] and later by Baker [17]. In 1877, Baker published his experience on this entity, which gave him a rise name going to designate this type of cyst. Baker's cyst is de ned as an abnormal cluster of synovial uid in the gastrocnemius-semimembranosus bursa or, failing that, a herniation of the posterior joint capsule with synovial uid tension [17][18][19]. Changes in both static and dynamic alignment of the lower extremities could alter balance and gait, and trigger pain throughout the lower limbs [20]. Besides, ultrasound changes in the liver were found as fatty liver in 75% of the rats (6 exemplary) in obese rats. It is known that one of the main organs affected by obesity is the liver, where long-term the increase in lipogenesis and the decrease in mitochondrial β-oxidation of nonesteri ed fatty acids, as well as hepatic triglyceride secretion, can contribute to the accumulation of fat in the liver, leading to the appearance of liver steatosis. As well as, in a prospective longitudinal study, 86% of patients with NAFLD and progressive brosis were obese [13,15,21].
At the renal level, changes such as pelvic dilation were found in 50% of the obese group. On the other hand, changes were observed at the level of the musculoskeletal system with the presence of joint cysts in the posterior region of the distal joint of the lower extremities of the rodents. Thus, with an excessive body weight creates a greater load stress, which in the musculoskeletal system affects muscle-skeletal and articular levels, causing joint misalignment (deformities) in the lower extremities and in ammatory and degenerative processes. Which could decrease physical functioning due associations with mobility and pain [22].
In several studies, it is known that the mechanical stress caused by overload or repetitive use can trigger tendon pathology. There are also extrinsic factors (posture and activity) and intrinsic factors (genetics and metabolic characteristics) that can interfere with their development [23]. This condition and metabolic factors (hyperglycemia, dyslipidemia and endothelial dysfunction) affect quality life of subjects.
Regarding to the in ammatory process and endothelial dysfunction, we also found an increase in proin ammatory cytokines, such as TNF-α and IL-6, these molecules augment monocytes adhesion to endothelial cell. It is known that in both, humans and mice, an imbalance between TNF-α, and adiponectin (adipokine of adipose tissue) participate in the progression to steatohepatitis [24][25][26]. Thus, our results of adhesion molecules showed an increase in the Ob group, which con rms endothelial damage, which has an impact on the joints. In addition, ICAM-1 and VCAM-1 are known being activating molecules of endothelial dysfunction because they play a crucial role in the adhesion of cells to the endothelial surfaces and in the integrity of the vascular wall, generating an accumulation of cells and sparking oxidative stress, that can be modulated by body composition and eating pattern [4,6,29,30].

Conclusion
This study we observed a relationship between endothelial dysfunction and the changes observed at the level of the musculoskeletal system, liver and heart with the presence of joint cysts in the posterior region of the distal joint of the lower extremities in obese rodents. Thus, we suggest that ultrasound is an excellent diagnostic tool that is accessible and easy to use in the chronic diseases that are increasing in our population, and it allows the integration of alterations in different organs, joints and tissues, besides, better monitoring and support to reducing complications and improving the quality of life of patients.