Nikola Bijelić

Trefoil factor family peptides TFF1 and TFF3 in the nervous tissues of developing mouse embryo.

Trefoil factor family peptides (TFF1, TFF2, and TFF3) are predominantly found in mucous epithelia of various organs. However, they have also been reported in the nervous tissue, particularly mouse, rat, porcine, and human brain. The aim of this research was to determine the presence of TFF1 and TFF3 in the nervous system of developing mouse embryo. Mouse embryos, at the stages E15 to E17 were isolated, fixed in 4% paraformaldehyde and embedded in paraffin blocks. Sagittal 6µm sections were made, processed for immunohistochemistry, and incubated with anti-TFF1 or anti-TFF3 primary polyclonal rabbit antibodies. Labeled streptavidin-biotin method was used for TFF detection. TFF1 and 3 were found in the cytoplasm of ganglion cell somata, while TFF3 staining was also visible in the cytoplasm of neurons in different areas and nuclei of brain and medulla oblongata. Neurons in the gray matter of spinal cord were also TFF1 and TFF3 positive, and signal for both peptides was found in the choroid plexus. TFF peptides might be involved in the complex processes of nervous system development and differentiation and brain plasticity.

High-fat diet induced changes in lumbar vertebra of the male rat offsprings

In obesity, bone marrow adiposity increases and proinflammatory cytokines excretion activates RANK/RANKL/OPG system, which leads to increased bone resorption. The aim of this study was to analyze trabecular and cortical bone parameters in animals exposed to the high-fat diet in utero and after lactation. Skeletal organ of interest was the fifth lumbar vertebra, which is not exposed to biomechanical loading in rats. Further aims were to determine TNF-α and IL-6 serum concentrations, and the intensity of the TNF-α immunohistochemical staining in the bone marrow. Ten female Sprague Dawley rats, nine weeks old, were randomly divided in two groups and fed either standard laboratory chow or food rich in saturated fatty acids during five weeks, and then mated with genetically similar male subjects. After birth and lactation male offsprings from both groups were divided in four subgroups depending on the diet they were fed until twenty-two weeks of age. The highest cholesterol and triglyceride concentration were found in both groups of offsprings fed with high-fat diet. The lowest trabecular bone volume, lowest trabecular number and highest trabecular separation were found in offsprings fed with high-fat diet of mothers on standard laboratory chow. The same group of offsprings was also characterized by the highest intensity of TNF-α immunostaining in the bone marrow and the highest TNF-α serum concentration, which suggest that this proinflammatory cytokine has interfered with bone metabolism, possibly by stimulation of bone resorption, which led to inadequate trabecular bone development and bone modeling of the fifth lumbar vertebra.

Trefoil factor family protein 3 (TFF3) is present in cartilage during endochondral ossification in the developing mouse fetus

Trefoil factor family protein 3 (TFF3) is found in cartilage affected by osteoarthritis and septic arthritis, whereas no TFF3 presence is observed in healthy cartilage. During endochondral ossification, bone tissue replaces degenerating cartilage. There is no data about the role of TFF3 in this process. Our aim was to study the localization of TFF3 in cartilage during endochondral ossification in the mouse fetus. CD1 mouse fetuses, days 14-17, were isolated, fixed, and paraffin embedded. Fetuses were cut into 6μm sections, and processed for immunohistochemical staining with affinity purified polyclonal rabbit anti-TFF3 antibody. TFF3 was present in cartilage chondrocytes undergoing endochondral ossification, particularly in zone of proliferation, hypertrophy and calcification as well as in zone of cartilage degeneration during the monitored fetal period. Resting cartilage showed no presence of TFF3, while during endochondral ossification TFF3 localization showed an analogous pattern to that reported in cartilage affected by osteoarthritis and septic arthritis. Our data indicate that the role of TFF3 in these pathological conditions is similar to its role in the physiological process of endochondral ossification.

Effect of different combination of maternal and postnatal diet on adipose tissue morphology in male rat offspring

Abstract Purpose: Adipose tissue expansion can occur through several different ways and, under certain conditions, can be connected with chronic inflammation. TNF-α is one of the important cytokines involved in this process. Prolonged inflammation in obesity can lead to obesity-related insulin resistance and tissue dysfunction. The aim of our study was to investigate how different combination of maternal and postnatal diet affects offspring adipose tissue morphology and adipose tissue TNF-α expression. Methods: Ten female Sprague Dawley rats, 9 weeks old, were randomly divided into two groups and fed either standard laboratory chow or food rich in saturated fatty acids during 6 weeks and then mated with the same male rat. After birth and lactation male rat offspring from both groups were divided into four subgroups depending on the diet they were fed until 22 weeks old. Samples of white adipose tissue were taken from the subcutaneous, epididymal, and perirenal fat pad. On tissue sections, histomorphometric analysis was conducted using CellProfiler program v 2.1.1, and immunohistochemical staining for TNF-α was performed. Results: Greater mean surface area of subcutaneous and epididymal adipocytes was found in groups of male rat offspring with altered diet. In perirenal adipose tissue, the highest number of adipocytes was measured in the group where both mother and offspring were fed a high-fat diet. Adipocyte staining intensity for TNF-α did not differ significantly between the groups. Conclusions: Together with our previously published data, our results lead to the conclusion that alteration of postnatal diet can lead to TNF-α and adipocyte morphology changes.

Raspodjela rizičnih čimbenika za moždani udar u istočnoj Hrvatskoj

SUMMARY – The aim of this study was to determine the distribution of risk factors according to age, gender, subtypes and recurrence of stroke in eastern Croatia. The study included 250 acute stroke patients admitted to University Department of Neurology, Osijek University Hospital Centre in 2011. Patients were grouped according to age, gender, subtypes and recurrence of stroke. The study showed significant differences in the distribution of cigarette smoking, diabetes, cardiomyopathy and hyperuricemia according to patient age. According to gender, male patients had a significantly higher prevalence of smoking and alcohol abuse, whereas in female patients the prevalence of arterial hypertension, atrial fibrillation and hyperuricemia was significantly higher. Regarding stroke subtypes, significant differences were noticed in the prevalence of arterial hypertension, atrial fibrillation, cardiomyopathy and cerebral blood vessel stenosis. Atrial fibrillation was significantly more common in first-ever than in recurrent stroke. Study results identified the groups of patients requiring special attention regarding particular risk factors in eastern Croatia and emphasized the need of developing regional strategies of screening, prevention and holistic care for stroke patients.

Pojavnost intramiokardijalnih masnih stanica u stijenci desne pretklijetke i desne klijetke – postmortalna humana analiza

SUMMARY – Histologic and radiologic studies describe intramyocardial fat tissue as a normal finding or as part of cardiac pathology. The role of fat cells within the myocardium is not fully understood. The aim of this study was to assess fat tissue distribution in the myocardium of right atrium (RA) and right ventricle (RV) and age differences in subjects free from cardiac disease. The study included 10 males without cardiac disease divided into two groups according to age (below/above 50 years). Three cross sections were performed (RV free wall and apex and RA free wall) with histomorphological analysis on digital photographs. The shares of total myocardial fat (TMF), perivascular fat (PVF) and non-perivascular (nPVF) fat were calculated. Samples from the older group had larger amounts of fat in the epicardium and myocardium, without statistically significant difference (TMF p=0.847, PVF p=0.4 and nPVF p=0.4). The largest quantities of fat tissue were found in the RV apex samples (14.9%), followed by RV free wall (7.5%) and RA (4.5%), where total apical RV fat share was significantly larger than in RA sample (p=0.044). Intramyocardial fat cells were present within the non-diseased RA and RV in all samples, mostly in the apex. Further investigations on age difference, effect of visceral obesity and sex differences are needed.

Histomorphometric Parameters of the Growth Plate and Trabecular Bone in Wild-Type and Trefoil Factor Family 3 (Tff3)-Deficient Mice Analyzed by Free and Open-Source Image Processing Software

Trefoil factor family 3 (Tff3) peptide is present during intrauterine endochondral ossification in mice, and its deficiency affects cancellous bone quality in secondary ossification centers of mouse tibiae. The aim of this study was to quantitatively analyze parameters describing the growth plate and primary ossification centers in tibiae of 1-month-old wild-type and Tff3 knock-out mice (n=5 per genotype) by using free and open-source software. Digital photographs of the growth plates and trabecular bone were processed by open-source computer programs GIMP and FIJI. Histomorphometric parameters were calculated using measurements made with FIJI. Tff3 knock-out mice had significantly smaller trabecular number and significantly larger trabecular separation. Trabecular bone volume, trabecular bone surface, and trabecular thickness showed no significant difference between the two groups. Although such histomorphological differences were found in the cancellous bone structure, no significant differences were found in the epiphyseal plate histomorphology. Tff3 peptide probably has an effect on the formation and quality of the cancellous bone in the primary ossification centers, but not through disrupting the epiphyseal plate morphology. This work emphasizes the benefits of using free and open-source programs for morphological studies in life sciences.

Different combinations of maternal and postnatal diet are reflected in changes of hepatic parenchyma and hepatic TNF-alpha expression in male rat offspring

Obesity is related to increased TNF-alpha production in different tissues. TNF-alpha is connected to mitochondrial dysfunction in the liver and also development of fatty infiltration of the liver. Also, postnatal change from normal to high-fat diet causes a significant increase in TNF-alpha serum levels. The aim of this research was to determine how maternal diet and switching male offspring to a different dietary regime after lactation influences rat liver. Ten female Sprague Dawley rats at nine weeks of age were randomly divided in two groups and fed either standard laboratory chow or high-fat diet during six weeks, and then mated with the same male subject. After birth and lactation male offspring from both groups were further divided into four subgroups depending on their subsequent diet. At 22 weeks of age, the animals were weighted, sacrificed and major organs were collected and weighted. Immunohistochemistry for TNF-alpha was performed on liver, and liver samples were analyzed for pathohistological changes. The group in which mothers were fed standard chow and offspring high-fat diet had the most pronounced changes: heaviest liver, poorest histopathological findings and strongest TNF-alpha immunohistochemical staining of liver parenchyma. High-fat diet during pregnancy and lactation and switching to high-fat diet postnatally affects liver weight, histological structure and TNF-alpha expression in male offspring.