Echographic analysis of the alterations musculoskeletal associated with endothelial dysfunction and obesity

Background: Modern imaging plays a central role in the care of obese patients, with an integral focus on its use and accessibility in individuals with 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 into two groups: control group (C) fed a standard diet and the obese group (Ob), fed with a hyper caloric diet of high fructose-fat for 4 mounts. Body weight, cholesterol, triglycerides, glucose, inflammatory cytokines and adhesion molecules (ICAM-1, VCAM-1) were measured. Additionally, two-dimensional echocardiography and abdominal ultrasound and musculoskeletal system studies in the lower extremities were performed. Results . Body weight in the Ob group was increased compared to the control group, (p <0.001); in addition, increased glucose, cholesterol and triglycerides of 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 were observed in obese rodents.

Some mechanisms that are triggered by obesity involve the inflammatory process, with the production of proinflammatory cytokines such as interleukine-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α). In addition, the endothelial dysfunction that is induced by the activation of adhesion molecules (ICAM-1, VCAM-1) is the cause of several vascular pathologies [2][3][4][5]. To understand the pathophysiological mechanisms, experimental models are used, as murine models (rats) are a valuable tool to understand these processes and their histological characteristics as well as the mechanisms linked to the comorbidities of these metabolic diseases [6]. In obesity, the accumulated storage of lipids in different organs, such as the liver, and heart, increases the macrophages of adipocyte origin, altering the essential lysosomal catabolism program for triglyceride hydrolysis and generating hepatic steatosis [7,8].
Within the enormous complexity of the individual, family and social management of the obese patient, imaging does not escape the inherent difficulties that come with the care of this type of population. The possibility of using widely tested imaging techniques in humans, such as ultrasound or echocardiography, has resulted in studying the structure and function of organs in small rodents [9].
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 difficult access with the one-dimensional echocardiogram [9].
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. 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 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.

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. 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.

Results
In the Ob group, an increase in these adhesion molecules and in proinflammatory cytokines such as TNF-α was demonstrated, with a significant difference compared with the C group (320% ± 3 vs 100% ± 0.05, p<0.001), and of IL-6. 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). However, in Figure 3A, the analysis of the two-dimensional ultrasound being more complete, fat filtration was observed in the obese group, with the presence of fatty liver (Table 3); on the other hand, a homogeneous hepatic parenchyma was found to be 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 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).

Discussion
In the present work, the results of this experimental model of obesity revealed alterations in metabolic parameters, such as lipids and HOMA-IR index, as mentioned by other studies where the use of hypercaloric diets similar to that used in this study, is associated with the early onset of lipid-like metabolic alterations [13], besides the endothelial damage that is had with the intake of hypercaloric diets, showing increase in TNF-α and adhesion molecules (ICAM-1 y VCAM-1). Together, with the ultrasound evaluation, the alterations of joint cysts and liver damage are highlighted. In individuals with metabolic alterations, it is difficult to diagnose and reverse this damage and also have the complications of cardiovascular diseases.
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 specific in our animals because the rats presented a relative parietal thickness below that limit. In addition, ultrasound changes in the liver were found as fatty liver in 75% of the rats (6 exemplary).
It is known that one of the main organs affected by long-term obesity is the liver, where the increase in lipogenesis and the decrease in mitochondrial β-oxidation of nonesterified 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 [12,14]. In addition, in a prospective longitudinal study, 86% of patients with non-alcoholic fatty liver (HGNA) and progressive fibrosis were obese, and only 27% of those with fibrosis remained stable [15].
At the renal level, changes such as pelvic dilation were found in 50% of the specimens. 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, which predominated in the females; therefore, certain joint alterations can be characterized according to gender.
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 inflammatory and degenerative processes, which could decrease physical functioning due to associations with mobility and pain [16].
An increase in proinflammatory cytokines, such as TNF-α, was observed in this study. It is known that in both humans and mice, an imbalance between TNF-α and adiponectin (a hormone of adipose tissue) seems to play an important role in the progression of steatosis to steatohepatitis. [16,17].
Adipose tissue is now recognized as a multifunctional organ. It plays an important role as an energy storage organ but also releases inflammatory mediators, proinflammatory molecules TNF-α and IL-6, and some other mediators such leptin, adiponectin, and resistin. In addition, endothelial damage increases molecules such as monocyte attractant chemoprotein 1 (MCP-1) and plasminogen activator inhibitor (PAI-1). IL-6, TNF-α and leptin act on immune cells and cause local and systemic inflammation [18,19,20]. Adhesion molecules were increased in the Ob group, which confirms endothelial damage, which has an impact on the joints. In addition, ICAM-1 and VCAM-1 are known to be 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, and can be modulated by body composition and eating pattern [21].
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 [15]. Changes in both static and dynamic alignment of the lower extremities could alter balance and gait and trigger pain throughout the lower limbs [22].
In this work was found the presence of Baker's cyst (QB), or popliteal cyst, which was first described in 1840 by Adams [23,24] and later by Baker [25]. In 1877, Baker published his experience on this entity, which gave rise to his name going to designate this type of cyst.
Baker's cyst is defined as an abnormal cluster of synovial fluid in the gastrocnemiussemimembranosus bursa or, failing that, a herniation of the posterior joint capsule with synovial fluid tension [25,26,27].

Conclusion
The results obtained from this study suggested 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 and better monitoring and treatment in support of reducing complications and improving the quality of life of patients.
In this study, also we showed a relationship between 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 were observed in obese rodents.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that there is no conflict of interest regarding the publication of this article.

Consent for publication
Not applicable.   Table 2 Effects on cytokines and CAMs in groups of study.

Supplementary Files
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