Prenatal Androgenization Induces Anxiety-Like Behavior in Female Rats, Associated with Reduction of Inhibitory Interneurons and Increased BDNF in Hippocampus and Cortex.

Anxiety is one of the most frequent psychiatric disorders. Despite the fact that most studies describe an anxiolytic effect of testosterone, hyperandrogenemia in mothers is assumed to be related to an increased risk of mood disorders in their offspring. An increasing body of scientific evidence suggests that an altered expression of interneuronal markers of the hippocampus may be the cause of anxiety. The aim of this study was to examine the influence of maternal hyperandrogenemia on behavioral parameters of anxiety-like behavior, neuropeptide Y (NPY) and parvalbumin (PV) expression in the hippocampus, and the level of the brain-derived neurotrophic factor (BDNF) in the hippocampus and cerebral cortex. Pregnant female Wistar albino rats were treated with testosterone undecanoate on the 20th day of gestation. Anxiety-like behavior in adult female offspring was evaluated by the elevated plus maze test and the open field. The number of PV and NPY immunoreactive cells in the hippocampus was determined immunohistochemically. The level of BDNF expression in the hippocampus and cerebral cortex was analyzed with the Western blot test. Prenatal hyperandrogenization increased anxiety-like behavior in female offspring and decreased expression of NPY+ and PV+ in the CA1 region of the hippocampus as compared to the control group. BDNF expression in the hippocampus and cerebral cortex of prenatally androgenized female offspring was significantly increased in comparison with the controls. Prenatal hyperandrogenization may be the cause of anxiety-like behavior in female offspring. Decrease in NPY and PV expression in the hippocampus may explain the possible mechanism of hyperandrogenization induced anxiety.

Differences in Chromatin Texture and Nuclear Fractal Dimension Between Hashimoto's and Lymphocytic Thyroiditis Lymphocytes.

Previous evidence suggested that lymphocytic thyroiditis (LT) was a variant of Hashimoto's thyroiditis (HT), thus the aim of the current study is to quantify structural changes in histological specimens taken from HT and LT patients. A total of 600 images containing a single lymphocyte nucleus (300 nuclei per group) were obtained from 20 patients with HT and LT. In order to quantify changes in the nuclear architecture of investigated lymphocytes, the fractal dimension (FD) and some gray-level co-occurrence matrix texture parameters (angular second moment, inverse difference moment, contrast, entropy, and correlation) were calculated for each nucleus. A statistically significant difference in the FD of the "binary-outlined" nucleus and that of the corresponding "black-and-white" nucleus was detected between HT and LT lymphocyte nuclei. In addition, there was also a statistically significant difference in contrast and correlation between HT and LT lymphocyte nuclei. In conclusion, the results of this study suggested that there was a difference in structural complexity between investigated lymphocyte nuclei; additionally, LT lymphocytes possessed probably more complex texture and larger variations as well as more asymmetrical nuclei compared with HT lymphocytes. Accordingly, these findings indicate that LT is probably not a variant of HT; however, more complex studies are necessary to estimate differences between these types of thyroiditis.

Correlation of structural defects in the ascending aortic wall to ultrasound parameters: benefits for decision-making process in aortic valve surgery.

BACKGROUND: Histopathological changes in the ascending aorta wall in patients with severe tricuspid aortic valve (TAV) stenosis were graded and correlated to echocardiographic parameters. Objective was to associate threshold echocardiographic values with structural defects in the ascending aorta providing a tool to improve decision-making process in cases when simultaneous aortic valve replacement (AVR) and ascending aorta replacement is considered. METHODS: Biopsies from 108 TAV stenosis patients subjected to AVR were graded into three grades according to severity of aortic wall changes. Echocardiographic parameters obtained preoperatively and correlated to grade, age, gender and risk factors, were diameters of ventriculo-aortic junction (AA), sinus Valsalva (SV), sinotubular junction (STJ), the largest diameter of the visualized ascending aorta (AscA) as well as indexes: sinus Valsalva (SVI), sinotubular junction (STJI), AscA/AA and STJ/AA. RESULTS: Two echocardiographic parameters portrayed grades with statistical significance: STJ (F = 5.417; p = 0.006 (p < 0.05)) and AscA (F = 3.924; p = 0.023 (p < 0.05)). By using multiple predictors in the setting of Regression analysis, statistically significant differences among grades were reached for AA, SV, STJ, AscA and SVI. With further ROC curves analysis, threshold values for different grades were recognized. Grade 2 is identified in patients with AscA > 3.3 cm, while Grade 3 is identified in patients with values of AscA > 3.5 cm, STJ > 2.9 cm and STJI > 1. CONCLUSIONS: Hemodynamic stress induced by TAV stenosis leads to elastic lamellae disruption in the aortic wall. Those changes could be graded and correlated with echocardiographic parameters of the aortic root and ascending aorta, providing a tool for decision to replace ascending aorta concomitantly with AVR.

The Effects of Betaine on the Nuclear Fractal Dimension, Chromatin Texture, and Proliferative Activity in Hepatocytes in Mouse Model of Nonalcoholic Fatty Liver Disease.

The effects of betaine on hepatocytes chromatin architecture changes were examined by using fractal and gray-level co-occurrence matrix (GLCM) analysis in methionine/choline-deficient (MCD) diet-induced, nonalcoholic fatty liver disease (NAFLD). Male C57BL/6 mice were divided into groups: (1) Control: standard diet; (2) BET: standard diet and betaine supplementation through drinking water (solution 1.5%); (3) MCD group: MCD diet for 6 weeks; (4) MCD+BET: fed with MCD diet + betaine for 6 weeks. Liver tissue was collected for histopathology, immunohistochemistry, and determination of fractal dimension and GLCM parameters. MCD diet induced diffuse micro- and macrovesicular steatosis accompanied with increased Ki67-positive hepatocyte nuclei. Steatosis and Ki67 immunopositivity were less prominent in the MCD+BET group compared with the MCD group. Angular second moment (ASM) and inverse difference moment (IDM) (textural homogeneity markers) were significantly increased in the MCD+BET group versus the MCD group (p<0.001), even though no difference between the MCD and the control group was evident. Heterogeneity parameters, contrast, and correlation were significantly increased in the MCD group versus the control (p<0.001). On the other hand, betaine treatment significantly reduced correlation, contrast, and entropy compared with the MCD group (p<0.001). Betaine attenuated MCD diet-induced NAFLD by reducing fat accumulation and inhibiting hepatocyte proliferation. Betaine supplementation increased nuclear homogeneity and chromatin complexity with reduction of entropy, contrast, and correlation.

Open Fracture Management in Low-Resource Settings: A Medical Training Experience in Cambodian Hospitals.

INTRODUCTION: A prospective interventional study has been carried out on the teaching effect and sustainability of low-cost trauma training program in open tibia fracture management for health workers. MATERIALS AND METHODS: In 2007, an external fixator and a patella-bearing orthosis were developed at a rural workshop in Cambodia. From 2010 to 2016, a core group of nine Cambodian health workers was trained in open fracture management by Norwegian senior surgeons, using the locally made fixator and brace. The training outcome was also assessed by a questionnaire comprising of assertions regarding theoretical understanding, technical skills and self-confidence in understanding the biomechanical properties of locally made external fixator and its application; the use of handmade orthosis and principle in covering of soft-tissue defects. RESULTS: The students managed 23 cases with the new technique with a primary healing rate of 70% (95% CI 48.1-85.5). A significant increase in self-reported technical skills, understanding, and self-confidence was reported. CONCLUSION: This study demonstrates that the capacity building of reconstructive surgery in low-resource settings by local doctors and paramedics is clearly a reasonable option that may substantially reduce amputation of the limbs.

Combined effects of electromagnetic field and low-level laser increase proliferation and alter the morphology of human adipose tissue-derived mesenchymal stem cells.

In recent years, electromagnetic field (EMF) and low-level laser (LLL) have been found to affect various biological processes, the growth and proliferation of cells, and especially that of stem cells. The aim of this study was to investigate the effects of EMF and LLL on proliferation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and thus to examine the impact of these therapeutic physical modalities on stem cell engraftment. hAT-MSCs were isolated from subcutaneous adipose tissue of six persons ranging in age from 21 to 56 years. EMF was applied for a period of 7 days, once a day for 30 min, via a magnetic cushion surface at a frequency of 50 Hz and an intensity of 3 mT. LLL was applied also for 7 days, once a day for 5 min, at radiation energies of 3 J/cm2, with a wavelength of 808 nm, power output of 200 mW, and power density of 0.2 W/cm2. Nonexposed cells (control) were cultivated under the same culture conditions. Seven days after treatment, the cells were examined for cell viability, proliferation, and morphology. We found that after 7 days, the number of EMF-treated hAT-MSCs was significantly higher than the number of the untreated cells, LLL-treated hAT-MSCs were more numerous than EMF-treated cells, and hAT-MSCs that were treated with the combination of EMF and LLL were the most numerous. EMF and/or LLL treatment did not significantly affect hAT-MSC viability by itself. Changes in cell morphology were also observed, in terms of an increase in cell surface area and fractal dimension in hAT-MSCs treated with EMF and the combination of EMF and LLL. In conclusion, EMF and/or LLL treatment accelerated the proliferation of hAT-MSCs without compromising their viability, and therefore, they may be used in stem cell tissue engineering.

Effects of IL-33/ST2 pathway in acute inflammation on tissue damage, antioxidative parameters, magnesium concentration and cytokines profile.

AIM: The aim of this study was to examine the role of IL-33/ST2 pathway in a pathogenesis of acute inflammation and its effects on tissue damage, antioxidative capacity, magnesium concentration and cytokine profile in acutely inflamed tissue. MATERIAL AND METHODS: Male mice were randomly divided in four groups: wild-type control group (WT-C), ST2 knockout control group (KO-C), wild-type inflammatory group (WT-I), and ST2 knockout inflammatory group (KO-I). Acute inflammation was induced in WT-I and KO-I by intramuscular injection of turpentine oil, while mice in WT-C and KO-C were treated with saline. After 12h, animals were euthanized, and blood was collected for determination of creatine kinase (CK) and aspartate transaminase (AST) activity. The treated tissue was used for histopathological analysis, determination of volume density of inflammatory infiltrate (Vdii) and necrotic fiber (Vdnf), gene expression of interleukin (IL)-33, ST2, tumor necrosis factor alpha (TNF-alpha), IL-6, IL-12p35, and transforming growth factor beta (TGF-beta), concentration of magnesium (Mg), copper (Cu), selenium (Se), manganese (Mn) and reduced glutathione (GSH), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity. RESULTS: Presence of inflammatory infiltration and necrosis in the treated tissue was histopathologically confirmed in WT-I and KO-I. Vdii was significantly higher in WT-I when compared to KO-I, whereas Vdnf did not significantly differ between WT-I and KO-I. CK and AST significantly increased in both inflammatory groups when compared to corresponding control groups. However, the values of CK and AST were significantly higher in WT-I than in KO-I. Mg in the treated tissue was significantly lower in WT-I in comparison to WT-C and KO-I, while there was no significant difference between KO-C and KO-I. There was no significant difference in Cu, Se, and Mn in the treated tissue between WT-C, KO-C, WT-I and KO-I. Gene expression of IL-33 in the treated tissue increased in both inflammatory groups when compared to the corresponding control groups, but it was significantly higher in KO-I than in WT-I. Gene expression of ST2 in the treated tissue was significantly higher in WT-I than in WT-C. Gene expression of TNF-alpha, IL-6, and IL-12p35 in the treated tissue was significantly higher in WT-I and KO-I than in the corresponding control groups, and IL-6 was significantly higher in KO-C than in WT-C. TGF-beta gene expression in the treated tissue was significantly higher in KO-I when compared to WT-I, while there was no difference between WT-C and KO-C. SOD activity decreased at the site of acute inflammation in both inflammatory groups, while the GPx activity increased. GSH in the treated tissue was significantly higher in KO-I than in KO-C or WT-I. CONCLUSION: The results of our study have indicated, to our knowledge for the first time, that IL-33/ST2 pathway plays a role in enhancing inflammation and tissue damage at the site of acute inflammation by affecting the concentration of magnesium and GSH, important for antioxidative capacity, as well as gene expression of anti-inflammatory cytokine TGF-beta.

Expression of p300 and p300/CBP associated factor (PCAF) in actinic keratosis and squamous cell carcinoma of the skin.

p300 and p300/CBP-associated factor (PCAF) are histone modifiers and transcriptional co-factors involved in a number of cell processes. We investigated their expression patterns in 79 actinic keratoses (AK), 45 cases of Bowen's disease (BD), and 168 invasive squamous cell carcinomas of the skin (SCC). Using tissue microarray and immunohistochemistry, we evaluated p300 and PCAF expression in relation to the type of the lesion and SCC prognostic parameters (grade, diameter, thickness and level of invasion). High nuclear expression of p300 (>60% of positive cells) (p=0.001) and absent cytoplasmic expression (p=0.026) were more frequent in SCC compared to AK and BD, respectively. Cytoplasmic expression of p300 was associated with the SCC invasion of subcutaneous fat and deeper tissues (p=0.049). Diffuse distribution of cells with p300 nuclear expression was more commonly seen in BD and SCC compared to AK (p<0.001), in moderately- and poorly-differentiated SCC compared to well-differentiated SCC (p<0.001), in tumors thicker than 6mm (p<0.001), and in deeply invading tumors (p=0.001). More frequent loss of PCAF nuclear expression was observed in SCC than in AK and BD (p<0.001). Diffuse distribution of cells with PCAF cytoplasmic expression was more common in BD and SCC compared to AK (p<0.001), and in poorly-differentiated SCC compared to well- and moderately-differentiated SCC (p<0.001). Our results suggest that increase in nuclear expression of p300, as well as the presence of cytoplasmic but loss of nuclear expression of PCAF, could play an important role in the development and progression of cutaneous SCC.

Co-expression of vascular and lymphatic endothelial cell markers on early endothelial cells present in aspirated coronary thrombi from patients with ST-elevation myocardial infarction.

INTRODUCTION: Angiogenesis is the growth of both new vascular and lymphatic blood vessels from the existing vasculature. During this process, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) express specific markers, which help their discrimination and easier identification. Since the coronary thrombi material aspirated from patients with ST-elevation myocardial infarction (STEMI) proved as good angiogenesis model, we investigated the expression of CD34 and CD31 as BECs markers, and D2-40, LYVE-1 and VEGFR3 as LEC markers in this material. MATERIALS AND METHODS: Aspirated thrombi were stained immunohistochemically for CD34, CD31, D2-40, LYVE-1 and VEGFR3. Organizational patterns of immunopositive cells were graded as single cells, clusters or microvessels. Double immunofluorescence for CD31, D2-40, LYVE-1 and VEGRF3 was done. Thrombi were also graded as fresh (<1day old), lytic (1-5days old) and organized (>5days old). RESULTS: Serial sections of aspirated thrombi showed concordant BEC and LEC markers immunopositivity. Double immunoflorescence proved co-expression of CD31 and LEC markers on the same cells. Cells expressing LEC markers organized in clusters and microvessels were mainly present in lytic and organized thrombi. CONCLUSION: Co-expression of BEC and LEC markers on the same non-tumorous cell during thrombus neovascularization indicates existing in vivo plasticity of endothelial cells under non-tumorous pathological conditions. It also points that CD34 and CD31 on one hand, and D2-40, LYVE-1 and VEGFR3 immunostaining on the other hand, cannot solely be a reliable indicators whether vessel is lymphatic or not.

Effects of Il-33/St2 pathway on alteration of iron and hematological parameters in acute inflammation.

AIM: The aim of this study was to examine the role of the IL-33/ST2 pathway in pathogenesis of acute inflammation by investigating its possible role in alteration of iron and hematological parameters in experimental model of acute inflammation. MATERIAL AND METHODS: Wild-type and ST2 knockout BALB/c mice were divided into four groups: wild-type control group, ST2-/- control group, wild-type inflammatory group, and ST2-/- inflammatory group. Acute inflammation was induced by intramuscular injection of turpentine oil, while control groups were injected with saline. After 12h animals were anesthetized, and the treated tissue, blood and spleen were collected. Iron concentration in the treated tissue, hemoglobin blood concentration, mean corpuscular hemoglobin (MCH), hematocrit, erythrocyte, neutrophil and lymphocyte blood count, and erythrocytes percentage in spleen were determined. RESULTS: Iron concentration in the treated tissue was significantly higher in wild-type inflammatory group (WT-I) when compared to both, the wild-type control group (WT-C) and ST2-/- inflammatory group (KO-I). There was no significant difference in iron concentration between ST2-/- control group (KO-C) and the KO-I. MCH had significantly decreased in WT-I when compared to WT-C, while there was no significant difference between KO-C and KO-I. Hemoglobin blood concentration significantly increased in KO-I in comparison to KO-C, while it did not significantly differ between WT-I and KO-I. Erythrocyte count and hematocrit had significantly increased, while the percentage of erythrocytes in spleen decreased in both inflammatory groups when compared to their controls. Neutrophil count significantly decreased in WT-I, when compared to WT-C. Lymphocyte count decreased in both inflammatory groups when compared to their controls. CONCLUSION: Results of this study indicate that the IL-33/ST2 axis could have a role in the alteration of iron in acute inflammation, namely in an increase of iron concentration at the site of acute inflammation and a decrease of blood mean corpuscular hemoglobin.