Effects of estradiol on modulation of apoptotic signals in cerebral cortex and hippocampus of rats during chronic cerebral hypoperfusion

Iako se ishemijske bolesti mozga, koje se karakterišu pogoršanjem motornih i kognitivnih funkcija, uključujući smanjenje sposobnosti učenja i memorije, intenzivno proučavaju poslednjih 20 godina malo je poznato o molekulskim mehanizmima njihovog nastanka, (...)Although ischemic brain diseases, characterized by decline of motor and cognitive functions, including reducing the ability of learning and memory, have been intensively studied over the past 20 years, the molecular mechanisms of their generation, as well (...

Time-dependent expression of Bcl-2 and Bax proteins in cortical brain area of adult wistar rats after permanent bilateral occlusions

Model of permanent bilateral occlusion of common carotid arteries (2VO) is generally used to investigate mechanisms of chronic cerebral hypoperfusion that occurs in aging and other neurodegenerative processes. The aim of this study was to determine timedependent modulation of mitochondrial apoptotic signaling in cortical brain area following chronic cerebral hypoperfusion. Using Western blot technique we monitored the changes in the expression of proteins of Bcl-2 family (Bcl-2, Bax) 3, 7 and 90 days following the insult. According to our results the greatest impact of chronic cerebral hipoperfusion occurred on 7th day.Physical chemistry 2012 : 11th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 201

Time-Related Sex Diffrences in Cerebral Hypoperfusion-Induced Brain Injury

Although the model of cerebral hypoperfusion in rats has been a matter of many investigations over the years, the exact intracellular and biochemical mechanisms that lead to neuron loss and memory decline have not been clearly identified. In the current study, we examined whether cerebral hypoperfusion causes changes in hippocampal protein expression of apoptotic markers in the synaptosomal fraction and neurodegeneration in a time-dependent and sex-specific manner. Adult male and female Wistar rats were divided into two main groups, controls that underwent sham operation, and animals subjected to permanent bilateral occlusion of common carotid arteries. Both male and female rats were killed 3, 7 or 90 days following the insult. The obtained results indicate that the peak of processes that lead to apoptosis occured on postoperative day 7 and that they were more prominent in males, indicating that neuroprotective effects of certain substances (planned for future experiments), should be tested at this time point

Radiation-mediated modulations of extracelluar nucleotide hydrolysis in adult female rat brain

The present study was performed to investigate whether acute whole-body exposure of female adult rats to low dose (0.5 Gy) of ionizing irradiation (IR) is sufficient to alter ectonucleotidase enzyme activities in the brain. All measurements were done at time points 1, 24 and 72h after irradiation. Neuronal synaptic plasma membranes (SPMs) were isolated from whole brains and enzyme activities were determined by monitoring ATP, ADP and AMP hydrolysis in vitro. Our results indicate that whole-body IR is able to modulate investigated brain enzyme activities in a time-dependent manner.Physical chemistry 2012 : 11th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 201

Cell proliferation assay - Method optimisation for in vivo labeling of DNA in the rat forestomach

The study of cell proliferation is a useful tool in the fields of toxicology, pathophysiology and pharmacology. Cell proliferation and its degree can be evaluated using 5-bromo-2'deoxyuridine which is incorporated into the newly synthesized DNA. The aim of this study was the optimization of subcutaneous application of 5-bromo-2'-deoxyuridine implantation for continuous and persistent marking of proliferating cells in the rat forestomach. 3-tert-Butyl-4-hydroxyanisole was used as the agent that ensures cell proliferation. In order to determine the optimal dose for proliferating cells labeling, 5-bromo-2'-deoxyuridine doses of 50 mg, 100 mg, 200 mg or 350 mg were implemented 2 days prior to sacrifice by flat-faced cylindrical matrices. Immunohistochemical analysis using 5-bromo-2'-deoxyuridine in situ detection kit was performed for the detection of 5-bromo-2'-deoxyuridine labeled cells. The results showed that for adult rats, the optimum 5-bromo-2'-deoxyuridine dose is 200 mg per animal for subcutaneous application. The here described manner of 5-bromo-2'-deoxyuridine in vivo labeling provides a simple, efficient, and reliable method for cell labeling, and at the same minimizes stress to animals

Erratum to: 17 beta-Estradiol-Induced Synaptic Rearrangements Are Accompanied by Altered Ectonucleotidase Activities in Male Rat Hippocampal Synaptosomes (2016)

17 beta-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling cascades which induce formation of new dendritic spines in the hippocampus of males as in females, but the interaction with other metabolic processes, such as extracellular adenine nucleotides metabolism, are currently unknown. Extracellular adenine nucleotides play significant roles, controlling excitatory glutamatergic synapses and development of neural circuits and synaptic plasticity. Their precise regulation in the synaptic cleft is tightly controlled by ecto-nucleoside triphosphate diphosphohydrolase (NTPDase)/ecto-5-nucleotidase (eN) enzyme chain. Therefore, we sought to clarify whether a single systemic injection of E2 in male rats is accompanied by changes in the expression of the pre- and postsynaptic proteins and downstream kinases linked to E2-induced synaptic rearrangement as well as alterations in NTPDase/eN pathway in the hippocampal synaptosomes. Obtained data showed activation of mammalian target of rapamycin and upregulation of key synaptic proteins necessary for spine formation, 24 h after systemic E2 administration. In E2-mediated conditions, we found downregulation of NTPDase1 and NTPDase2 and attenuation of adenine nucleotide hydrolysis by NTPDase/eN enzyme chain, without changes in NTPDase3 properties and augmentation of synaptic tissue-nonspecific alkaline phosphatase (TNAP) activity. Despite reduced NTPDase activities, increased TNAP activity probably prevents toxic accumulation of ATP in the extracellular milieu and also hydrolyzes accumulated ADP due to unchanged NTPDase3 activity. Thus, our initial evaluation supports idea of specific roles of different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance.Erratum: [http://vinar.vin.bg.ac.rs/handle/123456789/1460

Effects of estradiol on modulation of apoptotic signals in cerebral cortex and hippocampus of rats during chronic cerebral hypoperfusion

Iako se ishemijske bolesti mozga, koje se karakterišu pogoršanjem motornih i kognitivnih funkcija, uključujući smanjenje sposobnosti učenja i memorije, intenzivno proučavaju poslednjih 20 godina malo je poznato o molekulskim mehanizmima njihovog nastanka, (...)Although ischemic brain diseases, characterized by decline of motor and cognitive functions, including reducing the ability of learning and memory, have been intensively studied over the past 20 years, the molecular mechanisms of their generation, as well (...

Effects of estradiol on modulation of apoptotic signals in cerebral cortex and hippocampus of rats during chronic cerebral hypoperfusion

Iako se ishemijske bolesti mozga, koje se karakterišu pogoršanjem motornih i kognitivnih funkcija, uključujući smanjenje sposobnosti učenja i memorije, intenzivno proučavaju poslednjih 20 godina malo je poznato o molekulskim mehanizmima njihovog nastanka, (...)Although ischemic brain diseases, characterized by decline of motor and cognitive functions, including reducing the ability of learning and memory, have been intensively studied over the past 20 years, the molecular mechanisms of their generation, as well (...

Effects of estradiol on modulation of apoptotic signals in cerebral cortex and hippocampus of rats during chronic cerebral hypoperfusion

Iako se ishemijske bolesti mozga, koje se karakterišu pogoršanjem motornih i kognitivnih funkcija, uključujući smanjenje sposobnosti učenja i memorije, intenzivno proučavaju poslednjih 20 godina malo je poznato o molekulskim mehanizmima njihovog nastanka, kao i odgovarajućim terapijama. Osnovni cilj ove doktorske disertacije je bio da se ispita efekat hroničnog tretmana polnim hormonom, estradiolom, na apoptotske puteve u kortikalnim i hipokampalnim ćelijama polno zrelih mužjaka pacova Wistar soja u stanju nametnute hronične moždane hipoperfuzije. Tome je predhodilo proučavanje efekta moždane hipoperfuzije na složenu kaskadu apoptotskih signalnih puteva u kori prednjeg mozga i hipokampusu pacova u različitim vremenskim tačkama. Za potrebe prvog dela eksperimenta jedna grupa pacova je lažno operisana, dok je druga grupa podvrgnuta operaciji trajnog podvezivanja zajedničkih karotidnih arterija. Jedinke obe grupe su ostavljene na preživljavanju 3, 7 ili 90 dana. Za potrebe drugog dela eksperimenta pacovi su podeljeni u dve grupe: životinje kojima su trajno podvezane zajedničke karotidne arterije, tretirane 7 dana lanenim uljem i jedinke podvrgnute operaciji trajnog podvezivanja zajedničkih karotidnih arterija i tretirane 7 dana estradiolom (33.3 g/kg/dan, rastvoren u lanenom ulju). Praćeni su sledeći parametri: obim neuralne smrti, fragmentacija DNK koja je karakteristična za apoptozu, kao i promene u ekspresiji proteina i iRNK markera procesa ćelijske smrti odnosno preživljavanja ćelija. Rezultati ove doktorske disertacije pokazuju da se u zadatim eksperimentalnim uslovima, u kori prednjeg mozga u ranim vremenskim tačkama javljaju neurodegenerativne promene koje prate ekspresiju apoptotskih molekula u neprečišćenoj membranskoj frakciji, dok se u kasnije aktiviraju adaptivni i kompenzatorni mehanizmi. U hipokampusu je uočen specifičan vremenski-zavisan trend ekspresije analiziranih pro- i anti-apoptotskih molekula koji dovode do pojave odložene ćelijske smrti. Na osnovu proučavanja efekata hroničnog tretmana malim dozama estradiola u stanju nametnute moždane hipoperfuzije može se zaključiti da ovaj potentni polni hormon ima kapacitet da oblikuje odgovor ispitivanih moždanih struktura...Although ischemic brain diseases, characterized by decline of motor and cognitive functions, including reducing the ability of learning and memory, have been intensively studied over the past 20 years, the molecular mechanisms of their generation, as well as appropriate treatments are still matter of controversy. The main goal of this PhD thesis was to investigate the effects of repeated estradiol treatment on apoptotic pathways in cortical and hippocampal cells of adult male Wistar rats in a state of imposed chronic cerebral hypoperfusion. This was preceded by the study of the effects of cerebral hypoperfusion on complex cascade of apoptotic signalling pathways in the cerebral cortex and hippocampus of rats at different time points. For the purposes of the first part of the experiment, one group of rats was sham operated, while the other group was subjected to permanent ligation of common carotid arteries. Animals were sacrificed 3, 7 or 90 days following the insult. For the second part of the experiment, the rats were divided into two groups: animals that underwent permanent common carotid artery occlusion, treated for 7 days with linseed oil and rats subjected to permanent ligation of the common carotid arteries, treated for 7 days with estradiol (33.3 μg/kg/day, dissolved in linseed oil). Parameters that were monitored are: volume of neuronal death, DNA fragmentation typical for apoptosis, as well as changes in mRNA and protein expressions of cell death or survival markers. The results obtained in this dissertation indicate that in cerebral cortex at two early investigated time points, the expression of apoptotic molecules in synaptosomal fraction is accompanied with neurodegenerative changes, while later adaptive and compensatory mechanisms are activated. In hippocampus, a specific time-dependent expression of analyzed pro- and anti-apoptotic molecules is associated with delayed apoptotic death. Furthermore, according to gained data, repeated estradiol treatment in state of imposed chronic cerebral hypoperfusion exerts a potent capacity to profile the response of investigated brain structures, through modulation of expression of mitochondrial apoptotic pathway markers. Also, results for synaptosomal, cytosolic, mitochondrial and nucleus fractions of both investigated brain structures, point that protective effects of estradiol are either less pronounced in synaptosomal fraction or ischemic changes in this cell fraction are significant, thus applied treatment is not sufficient to compensate the effects of the injury..

Time-related sex differences in cerebral hypoperfusion-induced brain injury

Although the model of cerebral hypoperfusion in rats has been a matter of many investigations over the years, the exact intracellular and biochemical mechanisms that lead to neuron loss and memory decline have not been clearly identified. In the current study, we examined whether cerebral hypoperfusion causes changes in hippocampal protein expression of apoptotic markers in the synaptosomal fraction and neurodegeneration in a time-dependent and sex-specific manner. Adult male and female Wistar rats were divided into two main groups, controls that underwent sham operation, and animals subjected to permanent bilateral occlusion of common carotid arteries. Both male and female rats were killed 3, 7 or 90 days following the insult. The obtained results indicate that the peak of processes that lead to apoptosis occured on postoperative day 7 and that they were more prominent in males, indicating that neuroprotective effects of certain substances (planned for future experiments), should be tested at this time point. [Projekat Ministarstva nauke Republike Srbije, br. 173044 i br. 41014