Caroline Fernandes-Santos

A Mouse Model of Metabolic Syndrome: Insulin Resistance, Fatty Liver and Non-Alcoholic Fatty Pancreas Disease (NAFPD) in C57BL/6 Mice Fed a High Fat Diet

Diet-induced obesity in C57BL/6 mice triggers common features of human metabolic syndrome (MetS). The purpose is to assess the suitability of a diet-induced obesity model for investigating non-alcoholic fatty pancreatic disease (NAFPD), fatty liver and insulin resistance. Adult C57BL/6 mice were fed either high-fat chow (HFC, 60% fat) or standard chow (SC, 10% fat) during a 16-week period. We evaluated in both groups: hepatopancreatic injuries, pancreatic islets size, alpha and beta-cell immunodensities, intraperitoneal insulin tolerance test (IPITT) and oral glucose tolerance test (OGTT). The HFC mice displayed greater mass gain (p<0.0001) and total visceral fat pads (p<0.001). OGTT showed impairment of glucose clearance in HFC mice (p<0.0001). IPITT revealed insulin resistance in HFC mice (p<0.0001). The HFC mice showed larger pancreatic islet size and significantly greater alpha and beta-cell immunodensities than SC mice. Pancreas and liver from HFC were heavier and contained higher fat concentration. In conclusion, C57BL/6 mice fed a high-fat diet develop features of NAFPD. Insulin resistance and ectopic accumulation of hepatic fat are well known to occur in MetS. Additionally, the importance of fat accumulation in the pancreas has been recently highlighted. Therefore, this model could help to elucidate target organ alterations associated with metabolic syndrome.

Image Analysis and Quantitative Morphology

Quantitative studies are increasingly found in the literature, particularly in the fields of development/evolution, pathology, and neurosciences. Image digitalization converts tissue images into a numeric form by dividing them into very small regions termed picture elements or pixels. Image analysis allows automatic morphometry of digitalized images, and stereology aims to understand the structural inner three-dimensional arrangement based on the analysis of slices showing two-dimensional information. To quantify morphological structures in an unbiased and reproducible manner, appropriate isotropic and uniform random sampling of sections, and updated stereological tools are needed. Through the correct use of stereology, a quantitative study can be performed with little effort; efficiency in stereology means as little counting as possible (little work), low cost (section preparation), but still good accuracy. This short text provides a background guide for non-expert morphologists.

Pan-PPAR agonist beneficial effects in overweight mice fed a high-fat high-sucrose diet.

OBJECTIVE We analyzed the effect of peroxisome proliferator-activated receptor (PPAR) agonists on adipose tissue morphology, adiponectin expression, and its relation to glucose and insulin levels in C57BL/6 mice fed a high-fat high-sucrose (HFHS) diet. METHODS Male C57BL/6 mice received one of five diets: standard chow, HFHS chow, or HFHS plus rosiglitazone (PPAR-gamma agonist), fenofibrate (PPAR-alpha agonist), or bezafibrate (pan-PPAR agonist). Diets were administered for 11 wk and medications from week 6 to week 11. Glucose intolerance (GI) and insulin resistance were evaluated by oral glucose tolerance testing and homeostasis model assessment for insulin resistance, respectively. Adipocyte diameter was analyzed in epididymal, inguinal, and retroperitoneal fat pads and by adiponectin immunostain. RESULTS Mice fed the HFHS chow had hyperglycemia, GI, insulin resistance, increased fat pad weight, adipocyte hypertrophy, and decreased adiponectin immunostaining. Rosiglitazone improved GI, insulin sensitiveness, and adiponectin immunostaining, but it resulted in body weight gain, hyperphagia, and adipocyte and heart hypertrophy. Fenofibrate improved all parameters except for fasting glucose and GI. Bezafibrate was the most efficient in decreasing body weight and glucose intolerance. CONCLUSION Activation of PPAR-alpha, -delta, and -gamma together is better than the activation of PPAR-alpha or -gamma alone, because bezafibrate showed a wider range of action on metabolic, morphologic, and biometric alterations due to an HFHS diet in mice.

Rosiglitazone aggravates nonalcoholic Fatty pancreatic disease in C57BL/6 mice fed high-fat and high-sucrose diet.

Objectives: Evaluate the effect of fenofibrate, bezafibrate, and rosiglitazone on nonalcoholic fatty pancreatic disease and islet peroxisome proliferator-activated receptor-&agr; (PPAR-&agr;) and PPAR-&bgr; immunostain in mice fed high-fat high-sucrose (HFHS) diet. Methods: Two-month-old male mice were fed standard chow (n = 10) or HFHS chow (n = 40) for 6 weeks. Afterward, HFHS mice were grouped by treatment: untreated HFHS and HFHS treated with rosiglitazone (HFHS-Ro), fenofibrate (HFHS-Fe), or bezafibrate (HFHS-Bz). Medications were administered for 5 weeks. After treatment, the pancreas was removed and analyzed by morphometry, stereology, and immunohistochemistry. Results: The HFHS-fed mice showed altered fasting glucose (+33%) and insulin (+138%); increased body (+20%) and pancreas (+28%) masses, pancreatic fat (+700%), islet hypertrophy (+38%); and decreased GLUT2 immunostain (−60%). Rosiglitazone reduced fasting glucose and insulin but induced weight gain. Fibrates impeded weight gain, but only bezafibrate prevented islet hypertrophy. The GLUT2 stain was improved in all treatments, and there were no alterations in PPAR-&agr;. There were morphological signs of pancreatitis with fenofibrate, although there were no alterations in amylase and lipase. Rosiglitazone exacerbated pancreatic fat infiltration (+75% vs HFHS group), and bezafibrate increased PPAR-&bgr; expression in pancreatic islets. Conclusions: Rosiglitazone is shown for the first time to exacerbate pancreatic fat infiltration; therefore, precaution has to be taken when rosiglitazone is prescribed to obese patients.

Exercise training enhances elastin, fibrillin and nitric oxide in the aorta wall of spontaneously hypertensive rats.

This work aimed to analyze the effect of low-intensity exercise training on ultrastructural and molecular aortic remodeling. Male Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were allocated into four groups: sedentary WKY (SED-WKY), exercised WKY (EX-WKY, 1 h/day, 5 days/week treadmill exercise training), sedentary SHR (SED-SHR), and exercised SHR (EX-SHR). EX-SHR showed blood pressure reduction of 26% in comparison to SED-SHR after 1 month of exercise (P<0.05). At the 20th week, BP level was not different between EX-SHRs and WKYs. Circumferential wall tension (CWT) was higher by 77% in SED-SHRs than in SED-WKYs (P<0.001). Exercise training reduced CWT by 30% in EX- vs. SED-SHR (P<0.001). In SED-SHRs, endothelial cells showed large and numerous cytoplasmatic vacuoles, fragmented inner elastic lamina and scarce elastin and fibrillin, while exercise training ameliorated it in EX-SHR group. The highest eNOS immunodensity was observed in EX-SHR, which was 50% higher than EX-WKY (P<0.01) and 120% higher than SED-SHR (P<0.0001). In conclusion, present findings indicate beneficial effects of exercise training in hypertensive rats since it increased elastin, fibrillin and eNOS content in the aortic wall.

Favorable cardiac and aortic remodeling in olmesartan-treated spontaneously hypertensive rats

Cardiovascular remodeling contributes to the progression of cardiovascular disease. Thus, our aim was to evaluate the action of long-term treatment with olmesartan on cardiac and aortic adverse remodeling and its relationship with blood pressure (BP) and tensile forces acting on the aortic wall. Five-month-old male rats were divided in: WKY group (n = 6), SHR group (n = 6), and SHRs treated with hydralazine 30 mg/kg/day (SHR-H, n = 8) or olmesartan 10 mg/kg/day (SHR-O, n = 8). Medications were administered for 16 weeks. The SHR group showed hypertension (189 ± 4 mmHg), cardiomyocyte hypertrophy (+107%), interstitial fibrosis (5.7% vs 1.9% in WKY), and reduced intramyocardial vascularization (9.1% vs 22.8% in WKY). In aorta, the SHRs showed outward hypertrophic remodeling, increased elastic fibers content (+36%), and increased circumferential wall tension (CWT, 2.79 × 104 dyne/cm) and tensile stress (TS, 261.4 × 104 dyne/cm2). Hydralazine and olmesartan decreased BP (−45% approximately) and likewise CWT and TS (−45% and −35% approximately). Both medications prevented left ventricle remodeling, but olmesartan improved cardiomyocyte hypertrophy better than hydralazine. Hydralazine did not alter media hypertrophy, but it enlarged lumen diameter and increased elastic fibers. It is unlikely that olmesartan prevented all aortic alterations. Taken together, long-term control of BP alone is not sufficient to prevent aortic remodeling due to hypertension, but in myocardium it seems to be enough, except for cardiomyocyte hypertrophy. The differential action of olmesartan suggests that it is essential to block growth stimulation by angiotensin II in cardiomyocytes and vascular smooth muscle cells in order to better prevent cardiovascular adverse remodeling due to arterial hypertension.

Cardiac and aortic structural alterations due to surgically-induced menopause associated with renovascular hypertension in rats.

Menopause and hypertension independently alter cardiovascular remodelling, but little is known about their effect on left ventricular and aortic wall remodelling. Eight‐weeks‐old Wistar rats were divided into four groups of six animals each: Sham group, OVX group (ovariectomized rats), 2K1C (two‐kidneys, one‐clip rats) and OVX + 2K1C group and kept until 19 weeks. Blood pressure (BP) increased 12% in OVX group, 35% in 2K1C and OVX + 2K1C groups compared with sham group. Vaginal cytology showed Sham and 2K1C rats cycling normally, whereas OVX and OVX + 2K1C rats were persistently in dioestrus or proestrus. At euthanasia, left ventricle (LV) and thoracic aorta were removed and analysed (immunohistochemistry and stereology). LV mass/tibia length ratio and cross‐sectional area of cardiomyocytes increased in all groups except Sham. The intramyocardial vascularization reduced 30% in comparison with Sham group, with no difference among OVX, 2K1C and OVX + 2K1C groups. The cardiac interstitium increased more than 45% in both 2K1C and OVX + 2K1C groups compared with Sham, but there was no significant difference between Sham and OVX groups. Nuclei number of LV cardiomyocyte diminished in OVX group, followed by 2K1C group and OVX + 2K1C group, with no difference between the 2K1C and the OVX + 2K1C groups. There was positive immunostaining for angiotensin II AT1 receptor in smooth muscle cell layer of aortic tunica media in all groups. These results show that both ovariectomy and renovascular hypertension enhance BP as a single stimulus and therefore produce adverse cardiac remodelling. However, renovascular hypertension exerts a far greater influence than surgically‐induced menopause in this parameter.

Quantitative Morphology Update: Image Analysis

La morfologia cuantitativa es una herramienta confiable en la biologia del desarrollo, clinica y en el envejecimiento. Los innumerables datos cuantitativos de mejoras histologica y / o deterioro debido a las manipulaciones dieteticas o intervenciones farmacologicas atraen la atencion de los investigadores en todo el mundo. La morfometria permite realizar una amplia gama de analisis en dos dimensiones, mientras que la segmentacion de imagenes permite la medicion de una unica estructura o la determinacion de la intensidad de su color despues de una adecuada inmunotincion o incluso el area ocupada por un tipo especifico de celula. Cuando se trata de un analisis imparcial, debe considerarse la posibilidad de la perfusion tisular adecuada, fijacion e inclusion, evitando artefactos. Ademas, se debe considerar la contraccion del tejido. Con el fin de asegurar la reproducibilidad, uno de los principios mas importantes en la investigacion cientifica, y para permitir que diferentes grupos se comparen, la adquisicion y segmentacion de la imagen aparecen como pasos cruciales y deben ser estandarizados. En este trabajo se abordan algunos cuidados importantes para el procesamiento de tejidos y evaluacion apropiadas, y se muestran algunos ejemplos practicos de como la segmentacion morfometrica y la imagen se pueden aplicar a su experimento.

Beneficial Effects of Angiotensin II AT1 Blocker on Cardiovascular Adverse Remodeling Due to Nitric Oxide Synthesis Blockade

Se estudiaron con herramientas morfologicas, los efectos de diferentes dosis de Losartan sobre el remodelamiento cardiovascular, en ratas deficientes en oxido nitrico. 30 ratas Wistar, con 15 semanas de edad, fueron separadas en 6 grupos. control (C), L-NAME (LN), y 4 grupos en que administro LN junto con Losartan, en diferentes dosis (1, 5, 20 y 40 mg/kg/dia). El L-NAME fue administrado durante 9 semanas y la administracion de Losartan se inicio en la segunda semana de experimentacion. Se estudiaron el corazon, la parte toracica de la aorta y la arteria mesenterica craneal, con microscopia de luz y estereologia. La presion arterial (PA) aumento desde la primera semana de administracion de L-NAME. El tratamiento con Losartan, en las dosis de 20 y 40 mg/kg/dia, fue eficiente para reducir la PA despues de la septima semana de tratamiento. El remodelamiento cardiaco adverso en el grupo LN se caracterizo por intensa fibrosis intersticial, disminucion de la microvascularizacion miocardica e hipertrofia y consecuente perdida de cardiomiocitos. La estructura de la pared de la aorta (densidad por area de nucleos de celulas musculares lisas y densidad de superficie de lamelas), y la relacion media/luz de la arteria mesenterica craneal, tambien fueron muy alteradas por la administracion de L-NAME. Solo en una dosis igual o mayor que 20 mg/kg/dia, el Losartan tuvo efecto benefico tratando estas alteraciones. En conclusion, tanto el corazon como la pared arterial de ratas deficientes en oxido nitrico, presentan un proceso de remodelamiento acentuado, y este es eficientemente tratado con Losartan en diferentes dosis. La eficiencia del tratamiento con Losartan en el modelo de bloqueo de la sintesis de oxido nitrico se correlaciona con el efecto hipotensor de la droga, principalmente en las dosis mas elevadas