The Role of Macrophage Inhibitory Factor in TAA-Induced Liver Fibrosis in Mice: Modulatory Effects of Betaine.

Macrophage inhibitory factor (MIF) is a multipotent cytokine, involved in the inflammatory response to infections or injuries. This study investigates the role of MIF in liver fibrosis and the modulating effect of betaine on MIF in thioacetamide (TAA)-induced liver fibrosis. The wild-type and knockout MIF-/- C57BL/6 mice were divided into the following groups: control; Bet group, which received betaine; MIF-/-; MIF-/-+Bet; TAA group, which received TAA; TAA+Bet; MIF-/-+TAA; and MIF-/-+TAA+Bet group. After eight weeks of treatment, liver tissue was collected for further analysis. The results revealed that TAA-treated MIF-deficient mice had elevated levels of hepatic TGF-ß1 and PDGF-BB, as well as MMP-2, MMP-9, and TIMP-1 compared to TAA-treated wild-type mice. However, the administration of betaine to TAA-treated MIF-deficient mice reduced hepatic TGF-ß1 and PDGF-BB levels and also the relative activities of MMP-2, MMP-9 and TIMP-1, albeit less effectively than in TAA-treated mice without MIF deficiency. Furthermore, the antifibrogenic effect of MIF was demonstrated by an increase in MMP2/TIMP1 and MMP9/TIMP1 ratios. The changes in the hepatic levels of fibrogenic factors were confirmed by a histological examination of liver tissue. Overall, the dual nature of MIF highlights its involvement in the progression of liver fibrosis. Its prooxidant and proinflammatory effects may exacerbate tissue damage and inflammation initially, but its antifibrogenic activity suggests a potential protective role against fibrosis development. The study showed that betaine modulates the antifibrogenic effects of MIF in TAA-induced liver fibrosis, by decreasing TGF-ß1, PDGF-BB, MMP-2, MMP-9, TIMP-1, and the deposition of ECM (Coll1 and Coll3) in the liver.

NMDA Receptor Antagonist Memantine Ameliorates Experimental Autoimmune Encephalomyelitis in Aged Rats.

Aging is closely related to the main aspects of multiple sclerosis (MS). The average age of the MS population is increasing and the number of elderly MS patients is expected to increase. In addition to neurons, N-methyl-D-aspartate receptors (NMDARs) are also expressed on non-neuronal cells, such as immune cells. The aim of this study was to investigate the role of NMDARs in experimental autoimmune encephalomyelitis (EAE) in young and aged rats. Memantine, a non-competitive NMDAR antagonist, was administered to young and aged Dark Agouti rats from day 7 after immunization. Antagonizing NMDARs had a more favourable effect on clinical disease, reactivation, and apoptosis of CD4+ T cells in the target organ of aged EAE rats. The expression of the fractalkine receptor CX3CR1 was increased in memantine-treated rats, but to a greater extent in aged rats. Additionally, memantine increased Nrf2 and Nrf2-regulated enzymes' mRNA expression in brain tissue. The concentrations of superoxide anion radicals, malondialdehyde, and advanced oxidation protein products in brain tissue were consistent with previous results. Overall, our results suggest that NMDARs play a more important role in the pathogenesis of EAE in aged than in young rats.

Therapeutical Approach to Arterial Hypertension - Current State of the Art.

Arterial hypertension (AH) is recognized as the most common illness within the group of cardiovascular diseases and the most massive chronic non-infectious disease in the world. The number of hypertensive patients worldwide has reached 1.28 billion, contributing to an increase in cardiovascular diseases and premature death globally. The high prevalence of hypertension emphasizes the importance of effectively treating this condition. Elevated blood pressure often leads to lethal complications (heart failure, stroke, renal disorders, etc.) if left untreated. Considering an increase in AH prevalence in the future, a successful therapeutical approach to this disease and its complications is essential. The goal of AH treatment is to maintain normotensive blood pressure through various approaches, including lifestyle changes, a well-balanced diet, increased physical activity, psychoeducation, and, when necessary, pharmacotherapy. The evolving pharmacotherapeutic landscape reflects the progress made in our understanding of hypertension and emphasizes the need for continuous innovation to meet the challenges posed by this prevalent global health concern. The journey toward more effective and tailored treatments for hypertension is ongoing, and the introduction of new medications plays a pivotal role in shaping the future of antihypertensive pharmacotherapy.

Betaine Modulating MIF-Mediated Oxidative Stress, Inflammation and Fibrogenesis in Thioacetamide-Induced Nephrotoxicity.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with chemokine properties released by various immune and non-immune cells. It contributes to the pathogenesis of many inflammatory, autoimmune diseases and malignant tumors. OBJECTIVE: Our study aimed to investigate the role of betaine in the modulation of MIF-mediated oxidative stress, inflammation, and fibrogenesis during toxic kidney damage induced by thioacetamide (TAA). METHODS: The experiment is performed on wild-type and knockout MIF-/- C57BL/6 mice. They are randomly divided into groups: Control; Bet-group, received betaine (2% wt/v dissolved in drinking water); MIF-/- mice group; MIF-/- + Bet; TAA-group, treated with TAA (200 mg/kg b.w.), intraperitoneally, 3x/week/8 weeks); TAA+Bet; MIF-/-+TAA, and MIF-/- + TAA+Bet group. After eight weeks of treatment, animals are sacrificed and kidney samples are taken to determine oxidative stress parameters, proinflammatory cytokines, profibrogenic factors, and histopathology of renal tissue. RESULTS: In MIF-/-mice, TAA decreases malondialdehyde (MDA) concentration, IL-6, tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta 1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB) and increases superoxide dismutases (SOD) and catalase (CAT) activities, as well as glutathione (GSH) content in kidneys, compared to TAA group. Betaine alleviates the mechanism of MIF-mediated effects in TAA-induced nephrotoxicity, reducing MDA, IL-6, TNF-α, TGF-ß1, and PDGF-BB, and increasing SOD and CAT activity, as well as GSH levels. CONCLUSION: MIF mediates TAA-induced nephrotoxicity by increasing oxidative stress, inflammation, and profibrogenic mediators. MIF-targeted therapy could potentially alleviate oxidative stress and inflammation in the kidney, as well as pathohistological changes in renal tissue, but the exact mechanism of its action is not completely clear. Betaine alleviates MIF nephrotoxic effects by increasing the antioxidative capacity of kidney cells, and decreasing lipid peroxidation and cytokine production in the renal tissue. It suggests that betaine can be used for the prevention of kidney damage.

Artificial intelligence approaches to the biochemistry of oxidative stress: Current state of the art.

Artificial intelligence (AI) and machine learning models are today frequently used for classification and prediction of various biochemical processes and phenomena. In recent years, numerous research efforts have been focused on developing such models for assessment, categorization, and prediction of oxidative stress. Supervised machine learning can successfully automate the process of evaluation and quantification of oxidative damage in biological samples, as well as extract useful data from the abundance of experimental results. In this concise review, we cover the possible applications of neural networks, decision trees and regression analysis as three common strategies in machine learning. We also review recent works on the various weaknesses and limitations of artificial intelligence in biochemistry and related scientific areas. Finally, we discuss future innovative approaches on the ways how AI can contribute to the automation of oxidative stress measurement and diagnosis of diseases associated with oxidative damage.

Effects of Iron Oxide Nanoparticles on Structural Organization of Hepatocyte Chromatin: Gray Level Co-occurrence Matrix Analysis.

Gray level co-occurrence matrix (GLCM) analysis is a contemporary and innovative computer-based algorithm that can be used for the quantification of subtle changes in a cellular structure. In this work, we use this method for the detection of discrete alterations in hepatocyte chromatin distribution after in vivo exposure to iron oxide nanoparticles (IONPs). The study was performed on 40 male, healthy C57BL/6 mice divided into four groups: three experimental groups that received different doses of IONPs and 1 control group. We describe the dose-dependent reduction of chromatin textural uniformity measured as GLCM angular second moment. Similar changes were detected for chromatin textural uniformity expressed as the value of inverse difference moment. To the best of our knowledge, this is the first study to investigate the impact of iron-based nanomaterials on hepatocyte GLCM parameters. Also, this is the first study to apply discrete wavelet transform analysis, as a supplementary method to GLCM, for the assessment of hepatocyte chromatin structure in these conditions. The results may present the useful basis for future research on the application of GLCM and DWT methods in pathology and other medical research areas.

The Role of MIF in Hepatic Function, Oxidative Stress, and Inflammation in Thioacetamide-induced Liver Injury in Mice: Protective Effects of Betaine.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a multipotent cytokine that contributes to the inflammatory response to chemical liver injury. This cytokine exhibits pro- and anti-inflammatory effects depending on the etiology and stage of liver disease. OBJECTIVE: Our study aimed to investigate the role of MIF in oxidative stress and inflammation in the liver, and modulatory effects of betaine on MIF in thioacetamide (TAA)-induced chronic hepatic damage in mice. METHODS: The experiment was performed on wild type and knockout MIF-/- C57BL/6 mice. They were divided into the following groups: control; Bet-group that received betaine (2% wt/v dissolved in drinking water); MIF-/- mice group; MIF-/-+Bet; TAA-group that received TAA (200 mg/kg b.w.), intraperitoneally, 3x/week/8 weeks); TAA+Bet; MIF-/-+TAA, and MIF-/-+TAA+Bet. In TAA- and Bet-treated groups, animals received the same doses. After eight weeks of treatment, blood samples were collected for biochemical analysis, and liver specimens were prepared for the assessment of parameters of oxidative stress and inflammation. RESULTS: In MIF-/-mice, TAA reduced transaminases, γ-glutamyltranspeptidase, bilirubin, malondialdehyde (MDA), oxidative protein products (AOPP), total oxidant status (TOS), C-reactive protein (CRP), IL-6, IFN-γ, and increased thiols and total antioxidant status (TAS). Betaine attenuated the mechanism of MIF and mediated effects in TAA-induced liver injury, reducing transaminases, γ-glutamyltranspeptidase, bilirubin, MDA, AOPP, TOS, CRP, IL-6, IFN-g, and increasing thiols. CONCLUSION: MIF is a mediator in hepatotoxic, pro-oxidative, and proinflammatoryeffects of TAA-induced liver injury. MIF-targeted therapy can potentially mitigate oxidative stress and inflammation in the liver, but the exact mechanism of its action requires further investigation. Betaine increases anti-oxidative defense and attenuates hepatotoxic effects of MIF, suggesting that betaine can be used for the prevention and treatment of liver damage.

The Effect of CB1 Antagonism on Hepatic Oxidative/Nitrosative Stress and Inflammation in Nonalcoholic Fatty Liver Disease.

Dysfunction of the endocannabinoid system (ES) has been identified in nonalcoholic fatty liver disease (NAFLD) and associated metabolic disorders. Cannabinoid receptor type 1 (CB1) expression is largely dependent on nutritional status. Thus, individuals suffering from NAFLD and metabolic syndrome (MS) have a significant increase in ES activity. Furthermore, oxidative/ nitrosative stress and inflammatory process modulation in the liver are highly influenced by the ES. Numerous experimental studies indicate that oxidative and nitrosative stress in the liver is associated with steatosis and portal inflammation during NAFLD. On the other hand, inflammation itself may also contribute to reactive oxygen species (ROS) production due to Kupffer cell activation and increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The pathways by which endocannabinoids and their lipid-related mediators modulate oxidative stress and lipid peroxidation represent a significant area of research that could yield novel pharmaceutical strategies for the treatment of NAFLD. Cumulative evidence suggested that the ES, particularly CB1 receptors, may also play a role in inflammation and disease progression toward steatohepatitis. Pharmacological inactivation of CB1 receptors in NAFLD exerts multiple beneficial effects, particularly due to the attenuation of hepatic oxidative/nitrosative stress parameters and significant reduction of proinflammatory cytokine production. However, further investigations regarding precise mechanisms by which CB1 blockade influences the reduction of hepatic oxidative/nitrosative stress and inflammation are required before moving toward the clinical phase of the investigation.

Effect of Betaine Supplementation on Liver Tissue and Ultrastructural Changes in Methionine-Choline-Deficient Diet-Induced NAFLD.

Nonalcoholic fatty liver disease (NAFLD) represents a hepatic manifestation of metabolic syndrome. The aim of this study was to examine the effect of betaine on ultrastructural changes in the mouse liver with methionine- and choline-deficient (MCD) diet-induced NAFLD. Male C57BL/6 mice were divided into groups: Control-fed with standard chow, BET-standard chow supplemented with betaine (1.5% w/v drinking water), MCD-fed with MCD diet, and MCD + BET-MCD diet with betaine supplementation for 6 weeks. Liver samples were taken for pathohistology and transmission electron microscopy. The MCD diet-induced steatosis, inflammation, and balloon-altered hepatocytes were alleviated by betaine. MCD diet induced an increase in mitochondrial size versus the control group (p < 0.01), which was decreased in the betaine-treated group. In the MCD diet-fed group, the total mitochondrial count decreased versus the control group (p < 0.01), while it increased in the MCD + BET group versus MCD (p < 0.01). Electron microscopy showed an increase in the number of autophagosomes in the MCD and MCD + BET group versus control, and a significant difference in autophagosomes number was detected in the MCD + BET group by comparison with the MCD diet-treated group (p < 0.05). Betaine decreases the number of enlarged mitochondria, alleviates steatosis, and increases the number of autophagosomes in the liver of mice with NAFLD.

Iron-based nanoparticles and their potential toxicity: Focus on oxidative stress and apoptosis.

Recently, there have been several studies indicating that iron-based nanomaterials may exhibit certain toxic properties. Compared to conventional iron and iron oxides, iron nanoparticles (FeNPs) have some unique physical and chemical traits which impact their absorption, biodistribution and elimination. Facilitated passage through biological barriers enables FeNPs to reach various tissues and cells, and interact with a variety of different compounds. Currently, most of the recent research is focused on the potential cytotoxicity of FeNPs, and its implications on cell viability and functions. Some studies suggested that, in certain cell types, FeNPs may increase levels of oxidative stress and induce generation of reactive oxygen species. Oxidative stress may be one of the most important mechanisms by which FeNPs exhibit cytotoxic effects. Some authors have also suggested that, in certain conditions, exposure to FeNPs, in combination with other factors, may lead to changes in intracellular signaling resulting in programmed cell death. In this short review, we focus on the recent research on potential cytotoxicity of iron-based nanomaterials, and the potential implications of this new knowledge in medicine, chemistry and biology.

The effects of dietary methionine restriction on the function and metabolic reprogramming in the liver and brain - implications for longevity.

Methionine is an essential sulphur-containing amino acid involved in protein synthesis, regulation of protein function and methylation reactions. Dietary methionine restriction (0.12-0.17% methionine in food) extends the life span of various animal species and delays the onset of aging-associated diseases and cancers. In the liver, methionine restriction attenuates steatosis and delays the development of non-alcoholic steatohepatitis due to antioxidative action and metabolic reprogramming. The limited intake of methionine stimulates the fatty acid oxidation in the liver and the export of lipoproteins as well as inhibits de novo lipogenesis. These effects are mediated by various signaling pathways and effector molecules, including sirtuins, growth hormone/insulin-like growth factor-1 axis, sterol regulatory element binding proteins, adenosine monophosphate-dependent kinase and general control nonderepressible 2 pathway. Additionally, methionine restriction stimulates the synthesis of fibroblast growth factor-21 in the liver, which increases the insulin sensitivity of peripheral tissues. In the brain, methionine restriction delays the onset of neurodegenerative diseases and increases the resistance to various forms of stress through antioxidative effects and alterations in lipid composition. This review aimed to summarize the morphological, functional and molecular changes in the liver and brain caused by the methionine restriction, with possible implications in the prolongation of maximal life span.

Betaine modulates oxidative stress, inflammation, apoptosis, autophagy, and Akt/mTOR signaling in methionine-choline deficiency-induced fatty liver disease.

We examined the effects of betaine, an endogenous and dietary methyl donor essential for the methionine-homocysteine cycle, on oxidative stress, inflammation, apoptosis, and autophagy in methionine-choline deficient diet (MCD)-induced non-alcoholic fatty liver disease (NAFLD). Male C57BL/6 mice received standard chow (control), standard chow and betaine (1.5% w/v in drinking water), MCD, or MCD and betaine. After six weeks, serum and liver samples were collected for analysis. Betaine reduced MCD-induced increase in liver transaminases and inflammatory infiltration, as well as hepatosteatosis and serum levels of low-density lipoprotein, while it increased that of high-density lipoprotein. MCD-induced hepatic production of reactive oxygen and nitrogen species was significantly reduced by betaine, which also improved liver antioxidative defense by increasing glutathione content and superoxide-dismutase, catalase, glutathione peroxidase, and paraoxonase activity. Betaine reduced the liver expression of proinflammatory cytokines tumor necrosis factor and interleukin-6, as well as that of proapoptotic mediator Bax, while increasing the levels of anti-inflammatory cytokine interleukin-10 and antiapoptotic Bcl-2 in MCD-fed mice. In addition, betaine increased the expression of autophagy activators beclin 1, autophagy-related (Atg)4 and Atg5, as well as the presence of autophagic vesicles and degradation of autophagic target sequestosome 1/p62 in the liver of NAFLD mice. The observed effects of betaine coincided with the increase in the hepatic phosphorylation of mammalian target of rapamycin (mTOR) and its activator Akt. In conclusion, the beneficial effect of betaine in MCD-induced NAFLD is associated with the reduction of liver oxidative stress, inflammation, and apoptosis, and the increase in cytoprotective Akt/mTOR signaling and autophagy.

The effect of cannabinoid receptor 1 blockade on adipokine and proinflammatory cytokine concentration in adipose and hepatic tissue in mice with nonalcoholic fatty liver disease.

In high-fat diet (HFD) induced nonalcoholic fatty liver disease (NAFLD), there is an increase in the endocannabinoid system activity, which significantly contributes to steatosis development. The aim of our study was to investigate the effects of cannabinoid receptor type 1 blockade on adipokine and proinflammatory cytokine content in adipose and hepatic tissue in mice with NAFLD. Male mice C57BL/6 were divided into a control group fed with a control diet for 20 weeks (C, n = 6) a group fed with a HFD for 20 weeks (HF, n = 6), a group fed with a control diet and treated with rimonabant after 18 weeks (R, n = 9), and a group fed with HFD and treated with rimonabant after 18 weeks (HFR, n = 10). Rimonabant significantly decreased leptin, resistin, apelin, visfatin, interleukin 6 (IL-6), and interferon-γ (IFN-γ) concentration in subcutaneous and visceral adipose tissue in the HFR group compared to the HF group (p < 0.01). Rimonabant reduced hepatic IL-6 and IFN-γ concentration as well as plasma glucose and insulin concentration and the homeostatic model assessment index in the HFR group compared to the HF group (p < 0.01). It can be concluded that the potential usefulness of CB1 blockade in the treatment of HFD-induced NAFLD is due to modulation of the adipokine profile and proinflammatory cytokines in both adipose tissues and liver as well as glucose metabolism.

Gray-level co-occurrence matrix analysis of chromatin architecture in periportal and perivenous hepatocytes.

Periportal hepatocytes (PPHs) and perivenous hepatocytes (PVHs) in standard optical microscopy appear to be morphologically identical. However, the functional properties of these two cell populations and their roles in liver lobules are not the same. Despite significant differences in gene expression between these two hepatocyte populations, it is still unclear whether the differences are present at the higher levels of chromatin organization. In this study, we present results, indicating that periportal and perivenous hepatocytes, when stained using toluidine blue histological dye, have different chromatin textural patterns quantified with gray-level co-occurrence matrix (GLCM) method. Hepatic tissue was obtained from ten male, healthy mice. Chromatin structures were analyzed using GLCM. For each structure, we measured the values of angular second moment, inverse difference moment, GLCM Contrast, GLCM Variance, and GLCM Sum Variance. The results indicate that there is a statistically significant difference in all GLCM mathematical parameters except the contrast. In addition, some chromatin GLCM features were in correlation with serum aminotransferase levels in perivenous, but not in periportal hepatocytes. To the best of our knowledge, this is the first study to test the nuclear morphological differences between hepatocytes using GLCM and to investigate the respective relation with serum liver enzymes.

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.

Postnatal Developmental Changes in Fractal Complexity of Giemsa-Stained Chromatin in Mice Spleen Follicular Cells.

Although there are numerous recent works focusing on fractal properties of DNA and chromatin, many issues regarding changes in chromatin fractality during physiological aging remain unclear. In this study, we present results indicating that in mice, there is an age-related reduction of chromatin fractal complexity in a population of spleen follicular cells (SFCs). Spleen tissue was obtained from 16 mice and fixated in Carnoy solution. The youngest animal was newborn, and each animal was exactly 1 month older than the previous. We performed fractal analysis of SFC chromatin structure, stained using Giemsa technique. Fractal analysis was done in a plugin algorithm of ImageJ software. We also performed gray-level co-occurrence matrix (GLCM) analysis of all chromatin structures with the calculation of parameters such as angular second moment and inverse difference moment. Giemsa-stained SFC chromatin exhibited an age-dependent reduction of fractal dimension with statistically significant (p<0.01) linear trend. Moreover, there was a statistically significant increase of SFC chromatin lacunarity. The chromatin GLCM parameters did not significantly change. To our knowledge, this is the first study to perform fractal and GLCM analyses of SFC chromatin and to investigate potential changes of fractal parameters during postnatal development.

The effect of cannabinoid receptor 1 blockade on hepatic free fatty acid profile in mice with nonalcoholic fatty liver disease.

We used rimonabant to investigate the role of CB1 receptor on hepatic FFAs profile during NAFLD. Male mice C57BL/6 were divided into: control group fed with control diet 20 weeks (C; n=6); group fed with HFD 20 weeks (HF; n=6); group fed with control diet and treated with rimonabant after 18 weeks (R; n=9); group fed with HFD and treated with rimonabant after 18 weeks (HFR; n=10). Rimonabant (10mg/kg) was administered daily to HFR and R group by oral gavage. Rimonabant decreased liver palmitic acid proportion in HFR group compared to HF group (p<0.05). Liver stearic and oleic acid proportions were decreased in R group compared to control (p<0.01 respectively). Rimonabant increased liver linoleic and arachidonic acid proportions in HFR group compared to HF group (p<0.01 respectively). CB1 blockade may be useful in the treatment of HFD-induced NAFLD due to modulation of plasma lipid and hepatic FFA profile.

Age-related reduction of chromatin fractal dimension in toluidine blue - stained hepatocytes.

In this study, we proposed a hypothesis that chromatin of mouse hepatocytes exhibits age-related reduction of fractal dimension. This hypothesis was based on previously published works demonstrating that complexity of biological systems such as tissues, decreases during the process of physiological aging. Liver tissue was obtained from 24 male mice divided into 3 age groups: 10-days-old (young, juvenile), 210-days-old (adult) and 390-days-old. The tissue was stained using a modification of toluidine blue (nucleic acid - specific) staining method. A total of 480 chromatin structures (20 for each animal) were analyzed. For each structure, the values of fractal dimension, lacunarity, textural angular second moment and inverse difference moment were calculated using ImageJ software and its plugins. The results indicated the age-related reduction in fractal dimension and increase in lacunarity (p<0.01). Fractal dimension is a potentially good indicator of age associated changes in chromatin structure. To our knowledge, this is the first study to show that fractal complexity of hepatocyte chromatin decreases during the process of physiological aging. Fractal analysis as a method could be useful in detection of small age-related changes in chromatin distribution not otherwise visible with naked eye on conventional tissue micrographs.

Methionine-choline deprivation alters liver and brain acetylcholinesterase activity in C57BL6 mice.

Choline and methionine are precursors of acetylcholine, whose hydrolysis is catalyzed by acetylcholinesterase (AChE). Considering the possibility of their common deficiency, we investigated the influence of methionine-choline deprivation on AChE activity in liver and various brain regions (hypothalamus, hippocampus, cerebral cortex and striatum) in mice fed with methionine-choline deficient (MCD) diet. Male C57BL/6 mice (n = 28) were randomly and equally divided into following groups: control group fed with standard diet for 6 weeks (C) and groups fed with MCD diet for 2 weeks (MCD2), 4 weeks (MCD4) and for 6 weeks (MCD6). After the diet, mice were sacrificied and AChE activity in liver and brain was determined spectrophotometrically. Hepatic AChE activity was higher in MCD2, MCD4 and MCD6 compared to control (p < 0.01), with most prominent increase in MCD6. AChE activity in hypothalamus was higher in MCD4 and MCD6 vs. control (p < 0.05 and p < 0.01, respectively), as well as in MCD6 compared to MCD4 (p < 0.01). In hippocampus, increase in AChE activity was shown in MCD6 compared to control (p < 0.01). In cortex and striatum, increase in AChE activity was noted in MCD6 compared to control (p < 0.05). Our findings indicate the increase of hepatic and brain AChE activity in mice caused by methionine-choline deprivation.

NIKOLA TESLA AND MEDICINE: 160TH ANNIVERSARY OF THE BIRTH OF THE GENIUS WHO GAVE LIGHT TO THE WORLD - PART I.

INTRODUCTION: The interest in Nikola Tesla, a scientist, physicist, engineer and inventor, is constantly growing. In the millennialong history of human civilization, it is almost impossible to find another person whose life and work has been under so much scrutiny of such a wide range of researchers, medical professionals included. Although Tesla was not primarily dedicated to biomedical research, his work significantly contributed to the development of radiology, and high frequency electrotherapy. This paper deals with the impact of Tesla's work on the development of a new medical branch - radiology. Nikola Tesla and the Discovery of X-ray radiation. Tesla pioneered the use of X-rays for medical purposes, practically laying the foundations of radiology. Namely, since 1887, Tesla periodically experimented with X-rays, at that time still unknown and unnamed, which he called "shadowgraphs". Moreover, at the end of 1894, lie conducted extensive research focusing on X-rays, but unfortunately it was inlerrupted after the fire burning down his laboratory in 1895. In 1896 and 1897, Tesla published ten papers on the biologic effects of X-ray radiation. All his studies on X-rays were experimental. During 1896 and 1897, Tesla continued improving X-ray devices. Apart from this, Tesla was the first to point out the harmful effects of exposure to X-ray radiation on human body. CONCLUSION: Nikola Tesla was a visionary genius of the future. Tesla's pioneer steps, made more than a century ago in the domain of radiology, are still being used today.