Manual De Cardiologia Veterinaria Pdf Merge
The pathogenesis of nonalcoholic fatty liver disease (NAFLD) is not fully understood, and experimental models are an alternative to study this issue. We investigated the effects of a simple carbohydrate-rich diet on the development of obesity-related NAFLD and the impact of physical training on the metabolic abnormalities associated with this disorder.
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Sixty Wistar rats were randomly separated into experimental and control groups, which were fed with sucrose-enriched (18% simple carbohydrates) and standard diet, respectively. At the end of each experimental period (5, 10, 20, and 30 weeks), 6 animals from each group were sacrificed for blood tests and liver histology and immunohistochemistry. From weeks 25 to 30, 6 animals from each group underwent physical training. The experimental group animals developed obesity and NAFLD, characterized histopathologically by steatosis and hepatocellular ballooning, clinically by increased thoracic circumference and body mass index associated with hyperleptinemia, and metabolically by hyperglycemia, hyperinsulinemia, hypertriglyceridemia, increased levels of very low-density lipoprotein- (VLDL-) cholesterol, depletion of the antioxidants liver enzymes superoxide dismutase and catalase, and increased hepatic levels of malondialdehyde, an oxidative stress marker. Rats that underwent physical training showed increased high-density lipoprotein- (HDL-) cholesterol levels.
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In conclusion, a sucrose-rich diet induced obesity, insulin resistance, oxidative stress, and NAFLD in rats. Introduction Over the last decades, obesity has become a global epidemic and an important public health problem in many countries.
This condition is largely due to excessive consumption of saturated fats and simple sugars , , which, associated with sedentarism, represent the modern lifestyle. Obesity is recognized as a risk factor for many disorders including type-2 diabetes and nonalcoholic fatty liver disease (NAFLD). NAFLD encompasses a spectrum of increasingly severe clinicopathological conditions ranging from fatty liver to steatohepatitis (NASH) with or without hepatic fibrosis/cirrhosis. Recent evidence suggests that NAFLD is also associated with cardiovascular and chronic kidney disease and increased risk of hepatocellular carcinoma –. It has been considered that insulin resistance and hyperinsulinemia play a key role in the pathogenesis of NALFD (first causative step).
Excessive deposition of fat in adipocytes and muscles determines insulin resistance with subsequent accumulation of fat in the liver , which, in turn, increases the rate of mitochondrial beta-oxidation of fatty acids and ketogenesis that can promote lipid peroxidation and accumulation of reactive oxygen species (ROS) in the hepatocytes ,. These compounds generate a variety of cellular stimulations with subsequent inflammatory response, which has been recognized as the causal factor of NASH/fibrosis (second causative step) ,. In spite of growing knowledge, several aspects of NAFLD pathogenesis are still unknown. Considering the difficulty in developing human studies to evaluate the influence of nutrition in the development of NAFLD and associated metabolic abnormalities, experimental models constitute a reliable alternative way.
Manual De Cardiologia Veterinaria
Different animal models of NAFLD/NASH have been developed, but few of them replicate the entire human phenotype ,. These models may be classified into three basic categories: those caused by either spontaneous or induced genetic mutation; those produced by either dietary or pharmacological manipulation; and those involving genetic mutation and dietary or chemical challenges.
The dietary manipulations used in these last two types of models usually do not resemble human dietary pattern. In the present study, we developed a model of obesity and obesity-related NAFLD in nongenetically modified Wistar rats using a simple carbohydrate-rich diet, which resembles the current dietary pattern of humans, and followed the sequence of the pathophysiologic events and their clinical and metabolic consequences. In this context, it should be noted that, in the vast majority of studies on NAFLD in which animal models were employed, the description of the sequence of the pathophysiologic events and their consequences have not been addressed, as their key goal is usually the evaluation of a specific aspect such as a therapeutic intervention. Furthermore, we evaluated the impact of physical training on the metabolic abnormalities associated with this disorder.
Animals and Experimental Design Sixty male Wistar rats, approximately 28 days old (after weaning), were housed individually and had free access to water and rat diet. The animals were randomly separated into the following groups: experimental group (EG), fed with highly palatable diet (see below) during 5 (EG5, 6 rats), 10 (EG10, 6 rats), 20 (EG20, 6 rats), and 30 (EG30, 12 rats) weeks, and control group (CG), fed with standard rat chow during 5 (CG5, 6 rats), 10 (CG10, 6 rats), 20 (CG20, 6 rats), and 30 (CG30, 12 rats) weeks.
From week 25 to week 30, 12 animals belonging to the EG30 (6 rats) and CG30 (6 rats) were submitted to physical training (see below). At the end of each experimental period, after fasting for 10 hours, the animals were sacrificed. Blood samples were taken by cardiac puncture and stored at −20°C. The livers were immediately removed and fragments of about 1 mm thickness were fixed in 4% formaldehyde, dehydrated, immersed in xylene, and then embedded in paraffin for histology. Fresh tissue samples were collected to evaluate antioxidant enzymes activity. All experiments were approved by the Ethics Committee of the Universidade Federal de Minas Gerais for the Care and Use of Laboratory Animals (CETEA 53/2007) and were carried out in accordance with the regulations described in the Committee's Guiding Principles Manual. A rat belonging to the CG20 died and was excluded from all analyses.
Anthropometric Parameters and Physical Training On a weekly basis, the body weight, thoracic circumference (TC) (measured between the foreleg and hind leg), and nasoanal length were measured. Body mass index (BMI), that is, the ratio between body weight (g) and the square of body length (cm²), was calculated. All animals were acclimatized to exercise on the motor-driven treadmill (Gaustec, Brazil) by running at a speed of 10 mmin −1 at 5% inclination for 5 minutes/day, during 5 consecutive days. After exercise familiarization, trained rats were submitted to the physical training protocol, which consisted of running sessions with gradual increase in intensity across 5 weeks, 5 days/week. The speed and duration of the exercise bouts were increased until the rats were able to run at 25 mmin −1, 5% inclination, during 60 minutes/day.
The achievement of this exercise intensity ensures that a significant endurance training effect is produced. In order to ensure that all animals were subjected to the same handling stress, untrained group was submitted to running exercise on the same days of physical training, at the same speed, but for 2 minutes only. Analytical Procedures of Blood Parameters Measurement of glucose, total cholesterol, very low-density lipoprotein- (VLDL-) cholesterol, low-density lipoprotein (LDL-) cholesterol, high-density lipoprotein- (HDL-) cholesterol, and triglycerides was performed as recommended by the manufacturer (Bioclin, Quimbasa, Basic Chemistry Ltda, Brazil) using an autoanalyzer (StatPlus 2300, Yellow Spring Inst, USA).
Serum concentrations of leptin and insulin were determined by radioimmunoassay (Rat Leptin Ria Kit, Rat Insulin Ria Kit, LINCO Research, USA) using a gamma-ray counter (Mor-ABBOT, USA). The minimum detection value was 0.5 ng/mL. Evaluation of Antioxidant Enzyme Activity The determination of superoxide dismutase (SOD) activity was adapted from Dieterich et al. Briefly, fresh liver samples were homogenized in 50 mM sodium phosphate buffer (1 mL, pH 7.8, 37°C) and 1 mM of diethylenetriamine pentaacetic acid (DTPA), immediately after their removal. The reaction was initiated by addition of pyrogallol acid (0.2 mM/L, 37°C for 3 minutes) and the absorbance measured at 420 nm. SOD activity was calculated as U/mg protein, where 1 U of the enzyme was defined as the amount required to inhibit the oxidation of pyrogallol by 50%.
Catalase (CAT) activity was measured in the supernatant of liver homogenate as described by Nelson and Kiesow. Briefly, 0.04 mL of H 2O 2, 0.06 mL of liver homogenate, and 1.9 mL of potassium phosphate buffer (50 mM, pH 7.0) were mixed to give a final concentration of 6 mM of H 2O 2.
It took 1 minute for the reaction to occur at room temperature. The decomposition of H 2O 2 by CAT was evaluated by the change in absorbance at 240 nm. The experiments were performed in duplicate. CAT activity was expressed as mmol of H 2O 2 decomposed per minute per milligram of protein.
This procedure was adopted to avoid the possibility of interference in the activity of glutathione peroxidase, once the necessary cofactors were not present in the reaction medium. Histological and Immunohistochemistry Evaluations Histological sections were prepared from the material embedded in paraffin and stained with hematoxylin-eosin. The histological analysis was performed simultaneously by two examiners. The criteria established by Brunt et al. Were used to describe the histological lesions. According to these criteria, macrovesicular steatosis is quantified based on the percentage of involved hepatocytes (0 = absent; 1 66%), and its zonal distribution and the presence of microvesicular steatosis are noted; hepatocellular ballooning is evaluated for zonal location, and the estimate of its severity (mild, marked) is based on the numbers of hepatocytes showing this abnormality. Hepatic expression of malondialdehyde (MDA), leptin, and the leptin receptor Ob-R was evaluated by immunohistochemistry in the animals sacrificed at weeks 20 and 30.
From paraffin embedded tissues, sections on salinized slides (4 mm) were collected, deparaffinized, and hydrated. For immunohistochemistry, antigen reaction with ethylenediaminetetraacetic acid (EDTA) at pH 8.0, no steamer for 30 minutes at 98°C, was conducted, followed by Tris HCl pH 7.6 washing. The whole procedure was performed using Polymer Detection System kit (Novolink Polymer Detection System, Novocastra, USA). The primary antibodies used were anti-MDA monoclonal antibody (1F83) (Cosmo Bio Co., Ltd., Japan) diluted in 0.5 mL; anti-Ob (A-20) sc-84; and anti-Ob-R (H-300) sc-8325 (Santa Cruz Biotechnology Inc., USA) at a dilution of 1: 250 and 1: 100, respectively. Statistical Analysis Data are presented as frequencies and percentages, mean ± standard deviation (SD), and median and interquartile range (IQR).
For each quantitative response's variables, we developed linear regression models in which all variables with P value ≤0.25 at univariate analysis would be included initially. However, due to the high level of correlation between the explanatory covariates, we opted to adjust the final model with the following covariates: group, physical training, variation in BMI (ΔBMI), and variation in the amount of ingested calories (ΔKcal). The adequacy of the models was assessed by analysis of the residues. For the categorical variables, logistic regression models were developed, with inclusion of the variables that showed on the univariate analysis a P value ≤0.25, and also clinical significance.
The model fit was assessed by the Hosmer-Lemeshow test. Statistical analysis was performed using the R public domain software. Significance level was set at P value. Comparison of hormonal levels and enzyme activity between experimental and control groups. Liver histology was normal in the CG in all times of the experiment. Steatosis and hepatocellular ballooning (Figures and ) were observed only in the EG, from week 10. Steatosis was macro- and microvacuolar, located predominantly in zone 3 of the liver acinus.
The intensity of the macrovacuolar steatosis varied from mild (involvement of less than 33% of the hepatocytes) to severe (involvement of more than 66% of the hepatocytes) regardless of the time of the experiment. Ballooning was localized in zones 2 and 3 of the acinus, ranging from mild to marked and mismatched with the time of experiment. Neither inflammatory foci nor fibrosis was observed. Liver histology and immunohistochemistry. (a) Rats fed with standard diet (control group) at 30 weeks; normal histology, hematoxylin and eosin stain ×10.
(b, c, d, and e) Rats fed a sucrose-rich diet (experimental group) at 30 weeks; (b) macro. The reaction for identifying MDA was positive and intense, of cytoplasmic localization in zone 3 of the hepatic acinus, around the central vein, in EG20 and EG30.
No MDA was detected in CG rats. Leptin was identified in the cytoplasm especially in zone 3 of the acinus, in EG20 and EG30. In CG rats, the reaction was weakly positive, at the same location. Ob-R was expressed as a weak cytoplasmic reaction predominantly in zone 3 of the acinus, in the rats of both groups, at weeks 20 and 30. The comparison of the different variables between physical trained and untrained groups showed higher serum levels of HDL-cholesterol in the first group: medians 75 mg/dL and 52.2 mg/dL, respectively ( P = 0.007). No other clinical or metabolic variable was significantly different between the groups after the physical training.
Multivariate Analysis shows the results of the final linear and logistic regressions models. In summary, blood glucose levels were 49% higher in EG rats than in CG rats, and the rats studied for 10 and 30 weeks had an increase of 49% and 65%, respectively, in serum glucose compared to those studied for 5 weeks.
Total cholesterol was 19.2 mg/dL higher in the EG in comparison with the CG. Rats undergoing physical training showed an average of 27.1 mg/dL increase in HDL-cholesterol than those that did not exercise; and each increase of 1 unit in Δkcal intake caused an average reduction of 0.03 mg/dL in HDL-cholesterol levels.
Regarding LDL-cholesterol, there was an average increase of 60.2 mg/dL for each increase of 1 unit in ΔBMI. Linear and logistic regression models for the response variables. Two models were adjusted for the dependent variable insulin. The first, composed by the time (categorical) and groups of rats, showed that the EG20 and EG30 had, respectively, lower insulin values of 83% and 89% compared to EG5.
Furthermore, the animals of EG had an average insulin levels increased by 123% compared to the CG. The second model, including time (quantitative form), groups of rats, and Δkcal intake, showed that, for each increase of 1 unit in time, the average value of insulin decreased by 7% and, for each increase of 1 unit in Δkcal intake, the average value of insulin increased by 0.2%. The EG rats had an average insulin level increased by 100% compared to those of the CG. In EG20 and EG30, the leptin values were 33% and 40% higher, respectively, compared to the rats followed for 5 weeks. The EG had a mean value of leptin increased by 267% compared to the CG, and for every increase of 1 unit in ΔBMI the average value of leptin increased by 124.6%.
The amount of SOD was 24% lower in the animals followed for 30 weeks in relation to those studied for 5 weeks. In the EG, the mean values of SOD were 11% lower compared to the CG; and, for each increase of 1 unit in ΔBMI, the mean SOD values decreased by 54%. The rats studied for 20 weeks presented an average of 2.4 less CAT units than those studied for 5 weeks, and in the EG an average of 1.8 less units of CAT relative to the CG was observed. Concerning the histological findings, it was found that, for each increase of 1 unit in the ΔTC, the chance of expressing ballooning and steatosis increased by 50%.