Capítulo II MARCO TEÓRICO
DISCUSION DE LOS RESULTADOS
Dawley rats on high fat/sugar diet in a sex specific manner
Milorad Z1, Balog M1, Senka M2, Labak I2, Fenrich M1, Vari SG3, Gaspar R4, Gajovic S5,
Heffer M1
1 Faculty of Medicine, J.J. Strossmayer University of Osijek, Croatia 2Department of Biology, J.J. Strossmayer University of Osijek, Croatia
3 International Research and Innovationin Medicine Program, Cedars-Sinai Medical Center,
Los Angeles, CA, USA
4 Department of Pharmacodynamics and Biopharmacy, University of Szeged, Hungary 5 School of Medicine, University of Zagreb
Corresponding author: prof. Marija Heffer, [email protected]
Key words: high fat/sugar diet, aging, neurodegeneration, neuroinflammation, Metformin, Liraglutide
Introduction High content of sugars and fat in diet promote long term metabolic changes, which can lead to a variety of diseases starting with metabolic syndrome, specifically diabetes type 2 which has lately been connected to central diabetes and Alzheimers disease. Aging puts additional pressure on entire body, especially on maintenance of nervous system. Aim of this study was to compare sex specific neurodegeneration and neuroinflammation in Sprague Dawley rats on high fat/sugar diet (HFHSD) upon treatment with Metformin and Liraglutide. Methods Male and female rats were divided in two control groups: one fed with standard laboratory diet (SD) and second fed with standardized HFHSD. Two experimental groups were fed with HFHSD with addition of therapy – Metformin (MF) (50 mg/kg) or Liraglutide (LG) (0,3 mg/kg). Feeding with HFHSD or SD and two types of therapies had lasted for 6 months starting at the age of 9 months. Brains were collected at the age of 15 months and immunostained with inflammatory marker Iba1 and GFAP together with histological stainings for neurodegeneration (Congo Red and Bielschowski stainings). Images were photographed using Zeiss Axioskop 2 MOT microscope and analyzed in ImageJ software. Statistical analysis was performed in SPSS software.
Results Inflammation measured with Iba1 antibody detection is reduced in male and female groups treated with MF if compared with their SD controls (p=0,030 for males, p=0,017 for females). However, inflammation is increasing in males and females in case of LG treatment compared with their HFHSD controls (p=0,009 for males). Male LG group shows an increase compared to all other male groups, without statistical significance. There is a statistically higher Iba1 expression in males MF than female MF group (p=0,006). GFAP staining showed an increased astrocytes volume in MF females compared to SD female control (p=0,049). We also observed increase in MF female group compared with HFHSD female group (p=0,049). Bielschowski and Congo Red stainings were analyzed in motor cortex and smaller number of plaques was observed in control SD groups. All other male and female groups showed higher number of plaques. The only distinction we observed was bigger plaques formation in case of MF male group. In piriform cortex we observed no plaques in both control SD groups. Male HFHSD group as well as MF and LG groups showed overall higher density of plaques than female groups. MF male group again showed bigger plaques than other animal groups.
Bielschowski staining of motor cortex showed small number of plaques in control SD male and female group and higher number of plaques in all other male and female groups. Piriform cortex showed no visible plaque formation in male and female control SD groups and small number of plaques was seen in all other male and female groups.
Conclusion HFHSD and aging increase risks for neurodegeneration and inflammation in the brain. Inflammation and neurodegeneration are two major hallmarks of Alzheimers disease. Both Metformin and Liraglutide treatments had stronger protective effects of inflammation in the brain of female animals than in males. Using histological staining for plaque formation we observed the presence of neurodegeneration in all animal groups except SD male and female control groups. These results are inconclusive on extent of the brain damage and further analyses are needed to confirm the profile of neurodegeneration.
Acknowledgements: This study was supported by Cedars Sinai Medical Center's International Research and Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of Health, Science and Technology (RECOOP HST Association) and internal research grant from J. J. Strossmayer University of Osijek, Croatia.
The study was performed at the Department of Pharmacodynamics and Biopharmacy, University of Szeged, Hungary during 2015.
The importance of autophagy in the antiapoptotic effect of resveratrol Ulakcsai Zs, Bagaméry F, Szökő É, Tábi T
Department of Pharmacodynamics, Faculty of Pharmacy, Semmelweis University, Hungary Introduction: Previously we have demonstrated that resveratrol dose dependently prevented caspase 3 activation secondary to serum deprivation in non-transformed cells.
Aim: In the present work we aimed to assess the role of the probable molecular targets of resvereatrol, i.e. estrogen or aromatic hydrocarbon receptors and autophagy in its cytoprotective effect.
Methods: Apoptosis was induced by serum deprivation in primary mouse embryonic fibroblasts. Caspase 3 activation was assayed by using its fluorogenic substrate. Reactive oxygen species production and depolarization of the mitochondrial membrane were measured by fluorescence methods. The involvement of the receptors and autophagy in the effect of resveratrol were also analyzed using special agonists and antagonists.
Results: We found that neither aromatic hydrocarbon receptors nor estrogen receptors play an important role in the antiapoptotic effect of resveratrol. An autophagy inhibitor, chloroquine abolished the preventive effect of resveratrol, which indicates the importance of autophagy. In the presence of serum deprivation resveratrol induced a highly significant depolarization in mitochondrial membrane potential. Reactive oxygen species production was elevated by serum deprivation and further increased after resveratrol treatment.
Conclusion: We have demonstrated that resveratrol can protect primary fibroblasts against serum deprivation induced apoptosis by provoking mild intracellular stress and upregulating autophagy.
Ethical approval: All animal procedures were approved by the ethics committee of the Semmelweis University (22.1/1375/7/2010).
Acknowledgement:
Thank you for Cedars Sinai Medical Center’s International Research and Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of Health, Science and Technology (RECOOP HST Association) for their support of our organization as
Application of THY1 – YFP neural stem cells in regenerative neuroscience Alić I1,2, Kosi N2, Kapuralin K2, Gajović S2, Pochet R2, Mitrečić D2*
1Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine,
University of Zagreb, Zagreb, Croatia
2Laboratory for Stem Cells, Croatian Institute for Brain Research, School of Medicine,
University of Zagreb, Zagreb, Croatia
Corresponding author: [email protected] Key words: neural stem cells, transgenic mice, stroke
Introduction: This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in all parts of neurons. In this study, we have determined and compared expression pattern of: 1) Thy1 - YFP positive cells during in vitro differentiation of neural stem cells (NSC), 2) during embryonic development of Thy1 – YFP mouse embryo and 3) after transplantation of Thy1 – YFP cells into the stroke-affected mouse brain.
Materials and Methods: Neural stem cells were isolated from the forebrain of 14.5 old mouse embryos and cultured as neurospheres. Immunocytochemistry and RT-PCR were performed after cells differentiation. Embryonic slices were prepared for immunohistochemistry. In third part of this study, PKH26 labeled NSC were transplanted into the stroke-affected mouse brain obtained by MCAO method.
Results: THY1 - YFP expression was described during differentiation of NSC and observed in neural progenitors as well as in mature neurons. Neural progenitors were nestin positive while mature cell were not. THY1 – YFP positive cells were positive for mature neuronal markers (MAP2, β3-tubulin and NeuN) during the whole differentiation period, while astrocytes which were GFAP positive were not expressing YFP. During embryonic development THY1 – YFP positive cells were observed from E12.5 and the expression steadily increased to the end of pregnancy. Positive cells were present in both, central and peripheral nervous system. After transplantation, cells survived and incorporated into the stroke-affected brain.
Conclusion: Our results showed that mature neurons as well as neuronal progenitor cells do express THY1 – YFP construct in in vitro conditions, during embryonic development and after transplantation into the stroke-affected mouse brain. This suggests that in addition to analyses of neuronal differentiation in B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this strain can be successfully used in studies where tracing of cells with neuronal fate is needed. Source: The work has been supported by projects: YoungBrain (EU-ESF), the Croatian National Foundation (02.05/40), Foundation Adris awarded to D.M. and by FP7 Glowbrain project (REGPOT–2012–CT2012–316120) awarded to S.G.
Ethical approval: All experiments on animals described in this work received approval of the Internal Review Board of the Ethical Committee of the School of Medicine, University of Zagreb. Approval number (640-01/12-17/33) and date (22.05.2013.)
ARHGAP25 Rac-GAP has an important role in autoantibody-induced model of rheumatoid arthritis
T. F. Svanya, P. Lévai
Semmelweis University, Faculty of Medicine, Department of Physiology, Budapest Corresponding author: Tim F. Svanya [email protected]
Keywords: neutrophils, arthritis, ARHGAP25, inflammation, GAP
Introduction: Small G-proteins of the Rac family play a central role in regulation of numerous cellular functions. ARHGAP25, a GTP-ase activating protein (GAP), is known to be responsible for Rac inactivation. According to our previous results, it is mainly expressed in leukocytes and plays a major role in the function of neutrophilic granulocytes, such as transmigration through endothelium, production of reactive oxygen species (ROS) and phagocytosis. In addition, it plays a regulatory role in actin cytoskeleton reorganization. Methods: Arhgap25-/- (KO) mouse strain was purchased from the Knockout Mouse Project
(KOMP). Arthritis was induced in male KO and wild type mice by intraperitoneal injection of 150µl K/BxN mouse strain-derived serum. For control non-arthritogenic BxN serum was used. For the following 10 days ankle thickness was measured and a clinical score, indicating the severity of inflammation by subjective evaluation of edema and hyperemia, was determined. The loss of function was investigated by placing the mice on a wire-grid measuring the latency to fall upon turning over.
Results: Absence of ARHGAP25 caused a significant decrease in clinical scores, as well as in ankle thickness compared to wild type mice. Similar results were observed in the functional test; ARHGAP25-/- animals spent longer time on the grid. In mice treated with control serum
neither inflammation, nor loss of function could be observed, regardless of the genotype. Discussion: Our previous work, investigating the effect of ARHGAP25 on neutrophil responses in cell-based models, indicated enhanced effector functions in the absence of ARHGAP25. However, the present study shows a less severe arthritis induced by autoantibodies due to yet not elucidated mechanisms.
Conclusion: Our current results indicate, that beyond the elementary phagocyte functions, ARHGAP25 is a crucial player of inflammation in a human disease-related complex model. Acknowledgement
Supervisors: Dr. Roland Csépányi-Kömi, Prof. Dr. Erzsébet Ligeti Research support: Hungarian Research Fund OTKA 108382
Institutional Animal Care and Use Committee Approval: 22.1/323/3/2011. Issuing date: 8th of March 2011 by Pest Megyei Kormányhivatal Élelmiszerlánc-Biztonsági és Állategészségügyi Igazgatósága
The use of formalin-fixed paraffin-embedded samples in early diagnosis of diseases Reviewed and accepted but could not present
B. Döncző1, M. Szigeti1,2, Gy. Ostoros2,3, A. Gacs3, J. Tóvári3, A. Guttman1,2
1Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen,
Hungary
2MTA-PE Translational Glycomics Group, Pannon University, Veszprám, Hungary 3National Institute of Oncology, Department of Experimental Pharmacology, Budapest,
Hungary
Corresponding author: Andras Guttman [email protected] Key words: FFPE, glycosylation, capillary electrophoresis
Introduction: Formalin - fixed paraffin - embedded (FFPE) samples are generally used in histology as well as to archive clinical and pathological samples. FFPE tissue collections, with their accompanying clinical outcome information, are invaluable resources for translational studies of cancer and other diseases. However, this huge sample collection was mainly used so far for nucleic acid analysis. The objective of this work is to show that formalin fixed samples hold a great promise in glycomics studies. Information from these samples could be useful in early diagnosis of some diseases such as cancer.
Methods: The carbohydrate moiety of intact and formalin treated standard glycoproteins as well as human serum was removed by endoglycosidase digestion. The released glycans were labeled with a charged fluorophore and analyzed by capillary electrophoresis - laser induced fluorescent detection (CE-LIF). Glycans from tumor biopsy samples and their FFPE counterparts were extracted with radioimmunoprecipitation assay (RIPA) buffer followed by the same glycan removal, labeling and analysis process was used as described above.
Results: and Discussion: Preliminary experiments with standard glycoproteins showed identical glycosylation patterns before and after formalin treatment. Human serum was also treated and analyzed to compare the control to formalin treated and formalin fixed paraffin embedded formats. The results show practically no differences among the peak patterns. In case of tumor tissues, the effect of formalin fixation and paraffin embedding was investigated on the global N-glycosylation profile of mouse samples, also with very little changes observed. Conclusion: Our results suggested that the sugar moiety of glycoproteins remained intact after formalin treatment, thus, can be used to discover disease associated glycosylation changes at the molecular, cellular and tissue levels. The ability to effectively profile N-glycans form FFPE samples offers new opportunities to understand disease associated glycan profile changes, even retrospectively from large hospital archives.
Acknowledgement: The study was supported by Cedars Sinai Medical Center’s International Research and Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of Health, Science and Technology (RECOOP HST Association) and the
participating Cedars – Sinai Medical Center - RECOOP Research Centers (CRRC).
Ethical Committee Approval: All animal-model protocols were carried out in accordance with the Guidelines for Animal Experiments and were approved by the Institutional Ethics Committee at the National Institute of Oncology, Budapest, Hungary (permission number: 22.1/722/3/2010)
The role of gamma aminobutyric acid (GABA) in the regulation of endocervical epithelium secretory activity
Skelin M, Gajović S, Ćurlin M
Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Croatia Corresponding author: Marija Ćurlin ([email protected])
Key Words: endocervical, mucus, gaba, cell culture
Introduction: Endocervical secretory epithelium produces cervical mucus, a glycoprotein gel that accepts, filters, prepares, stores and releases sperm for transport to the oviduct for fertilization, as well as provides a barrier to the infection. The mechanisms of mucus secretion in the cervix are poorly investigated. We have proposed a new model of regulation of mucus secretion that involves action potential independent activity of GABA molecules.
Methods: To test this hypothesis, we analyzed mouse and human endocervical tissue for the presence of GABA receptors by immunohistochemistry.
Reuslts: In accordance with our model, the results showed that GABAR-A is present in the endocervical tissue of both human and mouse. These results present a scientific novelty and show that our model is realistically set up.
Discussion: The main goal of our current efforts is to establish a 3D epithelial cell culture in order to investigate the effect of estrogen on GABA signaling pathway action. We will test the expression of estrogen receptors in cells that show positive signal with the markers of GABA release. By applying inhibitors or activators of various receptors and by immunohistochemical and electron microscopy analysis we will investigate the effect of estrogen on mucin exocytosis.
Conclusion: Preliminary results support our hypothesis and further research will enable us to explore the molecular mechanism of endocervical epithelium secretory activity.
Ethical approval: All experiments received approval of the Internal Review Board of the Ethical Committee of the School of Medicine, University of Zagreb. All experiments were carried out in accordance with the EU Directive 2010/63/EU and 2004/23/EC.
Source: The work has been supported by FP7 Glowbrain project (REGPOT–2012–CT2012– 316120), and Croatian Science Foundation Fellowship to M.S.
Analyses of impact circadian dysfunctions on the occurrence of disautotonia in medical student population
Zvir M., Beliak A., Zayachkivska O.
Physiology Department, Lviv National Medical University, Lviv, Ukraine Corresponding author: Maryana Zvir [email protected]
Key words: chronic stress, disautotonia, circadian dysfunctions, medical students
Introduction. Chronic stress and modern lifestyle cause disautotonia that leads to health problems. WHO develops the International Classification of Diseases, version 11 that includes stress-spectrum diseases. Our studies showed that stress symptoms are more frequent among medical students (MS) and it makes important search of new non-invasive diagnostic stress markers.
Methods: 50 MS were interviewed using questionnaire that included stress perception, physical activity, sleep quality and time spent on IT devices. Anthropometric data, heart rate variability (HRV) and the saliva microcrystallization (SM) by dehydration of mix saliva were analyzed.
Results: examinees tend to have body mass index with mean 21.7; overweight was in 4%. Males had slightly lower muscle mass of 39.2% (normal value (N) above 40%) and higher total fat content - 21.4% (N - up to 20.0%). 68% of subjects spent >7 hrs/day sitting and 22% were physically active. The time of using gadgets >6 hrs/day was 63%. The good sleep quality was in 54% vs poor in 46% participants. 67% of MS confirmed high stress level and in 30% was reflected preference sympathetic activity by HRV
.
Type I SM was found in 16%; II and III - 70%; IV - 14% of students and it correlated with decrease in adaptive abilities.Discussion: Modern MS are a special population group, characterized by enormous workload, circadian dysfunctions, which increase stress level in their life. Such high prevalence of stress, found in our study, is alarming, because even in mild form it leads to negative effects on health and academic performance. Such studies may clarify the notion of modern lifestyle and it non- pharmacological correction.
Conclusion.
C
ircadian dysfunction, high level of stress and increased sitting time are the early triggers for disautotonia in MS. Further studies are required to assess saliva secretotome by proteinomic applying for evaluation early biomarkers of disautotonia.Plasma membrane Ca2+-pump selective regulators – calix[4]arene C-90 and imidazo[1,2-a]azepine – change parameters of uterus smooth muscles contraction Mazur1 Iu.Iu., Veklich1 T.O., Shkrabak1 O.A., Gerashchenko2 I.V., Kosterin1 S.O. 1 Department of Muscle Biochemistry, Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv, Ukraine
2 Institute of Pharmacology and Toxicology of the National Academy of Medical Sciences, Kyiv, Ukraine
Corresponding authorIuliia Mazur [email protected]
Key words: myometrium, calix[4]arene, contractility, imidazo[1,2-a]azepine, oxytocin Introduction. Disfunction of plasma membrane Mg2+,ATP-dependent Ca2+-pump (PMCA)
activity is often observed in diseases of smooth muscle (SM), i.e., uterus SM: preterm labor, atony, dysmenorrhea, hypo- and hypertonus, prolonged labour. Specific and target influence on PMCA activity may help to improve those pathological conditions. For this purpose we use calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenyl-sulfonylimino)-methylamino- 25,26,27,28-tetrapropoxycalix[4]arene) and imidazo[1,2-a]azepine derivate (IFT-35).
Methods. Calix[4]arene C-90 was characterized with infrared spectroscopy and nuclear magnetic resonance methods. PMCA enzymatic and Ca2+-transport activity was determined on
swine myometrium plasma membrane vesicle by measurements of Pi and by fluorescent probe