Carlos Alberto Mandarim-de-Lacerda

Stereological tools in biomedical research

Stereological studies are more and more frequent in literature, particularly in the development/evolution, pathology, and neurosciences areas. The stereology challenge is to understand the structural inner three-dimensional arrangement based on the analysis of the structure slices only showing two-dimensional information. Cavalieri and Scherles methods to estimate volume, and Buffons needle problem, are commented in the stereological context. A group of actions is needed to appropriately quantify morphological structures (unbiased and reproducibly), e.g. sampling, isotropic and uniform randomly sections (Delesses principle), and updated stereological tools (disector, fractionator, nucleator, etc). Through the correct stereology use, a quantitative study with little effort could be performed: efficiency in stereology means a minimum slices sample counting (little work), low cost (slices preparation), but good accuracy. In the present text, a short review of the main stereological tools is done as a background basis to non-expert scientists.

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.

Protein restriction during gestation and/or lactation causes adverse transgenerational effects on biometry and glucose metabolism in F1 and F2 progenies of rats

Substantial evidence suggests that poor intrauterine milieu elicited by maternal nutritional disturbance may programme susceptibility in the fetus to later development of chronic diseases, such as obesity, hypertension, cardiovascular disease and diabetes. One of the most interesting features of fetal programming is the evidence from several studies that the consequences may not be limited to the first-generation offspring and that it can be passed transgenerationally. In the present study, female rats (F0) were fed either a normal-protein diet [control diet (C); 19 g of protein/100 g of diet] or a low-protein diet [restricted diet (R); 5 g of protein/100 g of diet]. The offspring were termed according to the period and the types of diet the dams were fed, i.e. CC, RC, CR and RR (first letter indicates the diet during gestation and the second the diet during lactation). At 3 months of age, F1 females were bred to proven males, outside the experiment, to produce F2 offspring. At weaning, F2 offspring were divided by gender. RC1 offspring (with the number indicating the filial generation) were born with low birthweight, but afterwards they had catch-up growth, reaching the weight of the CC1 offspring. The increased glycaemia in RC1 offspring was associated with insulin resistance. CR1 and RR1 offspring had impaired growth with no changes in glucose metabolism. RC2 offspring had high BM (body mass) at birth, which was sustained over the whole experiment in male offspring. The F2 generation had more alteration in glucose metabolism than the F1 generation. CR2 and RC2 offspring had hyperglycaemia accompanied by hyperinsulinaemia and insulin resistance in both genders. CR2 offspring had an increase in body adiposity with hyperleptinaemia. In conclusion, low protein during gestation improves BM, fat mass and growth rate in F1 rats, but has adverse effects on glucose and leptin metabolism, resulting in insulin resistance in adult F1 and F2 offspring. Low protein during lactation has adverse effects on glucose, insulin and leptin metabolism, resulting in insulin resistance in adult F2 offspring. These findings suggest that low protein during gestation and/or lactation can be passed transgenerationally to the second generation.

Comparative effects of telmisartan, sitagliptin and metformin alone or in combination on obesity, insulin resistance, and liver and pancreas remodelling in C57BL/6 mice fed on a very high-fat diet.

The aim of the present study was to evaluate the effects of monotherapies and combinations of drugs on insulin sensitivity, adipose tissue morphology, and pancreatic and hepatic remodelling in C57BL/6 mice fed on a very HF (high-fat) diet. Male C57BL/6 mice were fed on an HF (60% lipids) diet or SC (standard chow; 10% lipids) diet for 10 weeks, after which time the following drug treatments began: HF-T (HF diet treated with telmisartan; 5.2 mg x kg-1 of body weight x day-1), HF-S (HF diet treated with sitagliptin; 1.08 g x kg-1 of body weight.day-1), HF-M (HF diet treated with metformin; 310.0 mg x kg-1 of body weight x day-1), HF-TM (HF diet treated with telmisartan+metformin), HF-TS (HF diet treated with telmisartan+sitagliptin) and HF-SM (HF diet treated with sitagliptin+metformin). Treated groups also had free access to the HF diet, and treatments lasted for 6 weeks. Morphometry, stereological tools, immunostaining, ELISA, Western blot analysis and electron microscopy were used. The HF diet yielded an overweight phenotype, an increase in oral glucose intolerance, hyperinsulinaemia, hypertrophied islets and adipocytes, stage 2 steatosis (>33%), and reduced liver PPAR-alpha (peroxisome-proliferator-activated receptor-alpha) and GLUT-2 (glucose transporter-2) levels, concomitant with enhanced SREBP-1 (sterol-regulatory-element-binding protein-1) expression (P<0.0001). Conversely, all drug treatments resulted in significant weight loss, a reversal of insulin resistance, islet and adipocyte hypertrophy, and alleviated hepatic steatosis. Only the HF-T and HF-TS groups had body weights similar to the SC group at the end of the experiment, and the latter treatment reversed hepatic steatosis. Increased PPAR-alpha immunostaining in parallel with higher GLUT-2 and reduced SREBP-1 expression may explain the favourable hepatic outcomes. Restoration of adipocyte size was consistent with higher adiponectin levels and lower TNF-alpha (tumour necrosis factor-alpha) levels (P<0.0001) in the drug-treated groups. In conclusion, all of the drug treatments were effective in controlling the metabolic syndrome. The best results were achieved using telmisartan and sitagliptin as monotherapies or as a dual treatment, combining partial PPAR-gamma agonism and PPAR-alpha activation in the liver with extended incretin action.

Vascularization pattern in hypertrophic scars and keloids: a stereological analysis.

Wound healing is a complex process that does not always occur harmoniously and may lead to pathological scar development, such as hypertrophic scars and keloids. Considering that vascularization can play a role in the development of these scars, and that the literature is controversial, we performed a stereological analysis of dermal for vessels of normal skin, normal scars, hypertrophic scars, and keloids. The parameters studied concerned vessels: surface density, length density; for vessels and myofibroblasts: volume density, in papillary and reticular dermis. The pattern of dermal vascularization in normal skin and normal scar showed no differences. In papillary demis, the number of vessels was higher in hypertrophic scars and keloids than in normal skin (p < 0.05). Vessels of hypertrophic scars were more dilated than those of normal skin (p < 0.01). In reticular dermis, vessels were present in higher amount in hypertrophic scars and keloids than in normal skin (p < 0.025; p < 0.001, respectively). The pattern of vascularization did not show any differences between hypertrophic scars and keloids. Our results show that hypertrophic scars and keloids have a distinct pattern of vascularization compared to normal skin and normal scars. This indicates that abnormal vascularization can be involved in the development of hypertrophic scars and keloids.

Maternal high-fat intake predisposes nonalcoholic fatty liver disease in C57BL/6 offspring

OBJECTIVE This work aimed to verify the hypothesis that maternal intake of high-fat diet in critical periods of pregnancy and/or suckling period predisposes nonalcoholic fatty liver disease in adult C57BL/6 mice offspring. STUDY DESIGN Male pups were divided into 5 groups: (1) SC, from standard chow-fed dams; (2) G, from high-fat chow (HF)-fed dams during the gestation (G) period; (3) L, from HF-fed dams during the lactation (L) period; (4) GL, from HF-fed dams during the gestation and lactation (GL) periods; and (5) GL/HF, from HF-fed dams during GL, maintaining an HF diet from postweaning to adulthood. We analyzed body mass, plasma blood, and liver structure. RESULTS The G offspring showed insulin resistance and lower glucose transporter-2 expression. Hepatic steatosis was present in the G, L, GL, and mainly in GL/HF offspring. Sterol regulatory element-binding protein-1c expression was higher in G, GL, and GL/HF offspring. CONCLUSION Programming by HF chow predisposes hepatic adverse remodeling in the liver of adult offspring.

A critical analysis of three quantitative methods of assessment of hepatic steatosis in liver biopsies

The issue of adequately quantitatively evaluating hepatic steatosis is still unresolved. Therefore, we compared three methods of quantitative assessment. Two groups of mice (n = 10 each) were fed standard chow (10% fat, SC group) or a high-fat diet (60% fat, HF group) for 16 weeks, and hepatic triglyceride (HT) and liver tissue were then studied. Paraplast-embedded tissues stained by hematoxylin and eosin (H-E) were compared to frozen sections stained by Oil Red-O (ORO). In addition, the volume density of steatosis (Vv[steatosis, liver]) was measured by point counting (P-C, sections H-E or ORO) or by image analysis (I-A, sections ORO). HT was significantly higher in the HF group (104% greater, P = 0.0004) than in the SC group. With P-C and H-E, Vv[steatosis, liver] was 4.80 ± 0.90% in the SC group and 33.50 ± 3.17% in the HF group (600% greater, P < 0.0001). With P-C and ORO, Vv[steatosis, liver] was 4.86 ± 0.89% in the SC group and 25.21 ± 1.27% in the HF group (420% greater, P < 0.0001). With I-A and ORO, Vv[steatosis, liver] was 4.17 ± 0.85% in the SC group and 23.35 ± 1.58% in the HF group (460% greater, P < 0.0001). Correlations between Vv[steatosis, liver] and HT were strong and significant in all methods. In conclusion, all methods were appropriate and reproducible. In P-C and H-E, there is a slight overestimation of steatosis in the HF animals in comparison to frozen sections and ORO; in frozen sections, differences between P-C and I-A are insignificant.

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.

Fenofibrate (PPARalpha agonist) induces beige cell formation in subcutaneous white adipose tissue from diet-induced male obese mice

Browning is characterized by the formation of beige/brite fat depots in subcutaneous white adipose tissue (sWAT). This study aimed to examine whether the chronic activation of PPARalpha by fenofibrate could induce beige cell depots in the sWAT of diet-induced obese mice. High-fat fed animals presented overweight, insulin resistance and displayed adverse sWAT remodeling. Fenofibrate significantly attenuated these parameters. Treated groups demonstrated active UCP-1 beige cell clusters within sWAT, confirmed through higher gene expression of PPARalpha, PPARbeta, PGC1alpha, BMP8B, UCP-1, PRDM16 and irisin in treated groups. PPARalpha activation seems to be pivotal to trigger browning through irisin induction and UCP-1 transcription, indicating that fenofibrate increased the expression of genes typical of brown adipose tissue (BAT) in the sWAT, characterizing the formation of beige cells. These findings put forward a possible role of PPARalpha as a promising therapeutic for metabolic diseases via beige cell induction.

Beneficial effects of exercise training (treadmill) on insulin resistance and nonalcoholic fatty liver disease in high-fat fed C57BL/6 mice

C57BL/6 mice develop signs and symptoms comparable, in part, to the human metabolic syndrome. The objective of the present study was to evaluate the effects of exercise training on carbohydrate metabolism, lipid profile, visceral adiposity, pancreatic islet alterations, and nonalcoholic fatty liver disease in C57BL/6 mice. Animals were fed one of two diets during an 8-week period: standard (SC, N = 12) or very high-fat (HF, N = 24) chow. An exercise training protocol (treadmill) was then established and mice were divided into SC and HF sedentary (SC-Sed, HF-Sed), exercised groups (SC-Ex, HF-Ex), or switched from HF to SC (HF/SC-Sed and HF/SC-Ex). HF/HF-Sed mice had the greatest body mass (65% more than SC/SC-Sed; P < 0.0001), and exercise reduced it by 23% (P < 0.0001). Hepatic enzymes ALP (+80%), ALT (+100%) and AST (+70%) were higher in HF/HF mice than in matched SC/SC. Plasma insulin was higher in both the HF/HF-Sed and HF/SC-Sed groups than in the matched exercised groups (+85%; P < 0.001). Pancreatic islets, adipocytes and liver structure were greatly affected by HF, ultimately resulting in islet beta-cell hypertrophy and severe liver steatosis. The HF group had larger islets than the SC/SC group (+220%; P < 0.0001), and exercise significantly reduced liver steatosis and islet size in HF. Exercise attenuated all the changes due to HF, and the effects were more pronounced in exercised mice switched from an HF to an SC diet. Exercise improved the lipid profile by reducing body weight gain, visceral adiposity, insulin resistance, islet alterations, and fatty liver, contributing to obesity and steatohepatitis control.