Effects of brief cerebral ischemia/reperfusion and dehydroepoandrosterone treatment on components of NMDA receptor-mediated signalling in rat hippocampus and prefrontal cortex

Stanje moždane ishemije/reperfuzije (I/R) karakteristično je za cerebrovaskularnaoboljenja kao što su moţdani udar i tranzitorni ishemijski atak(TIA). Promena signalizacije posredovana N-metil-D-aspartat (NMDA)receptorom ima značajnu ulogu u I/R-uzrokovanom oštećenju, dok jedehidroepiandrosteron (DHEA), pozitvni alosterični modulator NMDAreceptora, najčešće okarakterisan kao neuroprotektivan u tretmanu ishemičnihstanja.U ovoj disertaciji ispitivana je proteinska ekspresija komponentikompleksa NMDA receptora i njegove nishodne signalizacije u hipokampusu iprečeonoj kori muţjaka pacova Wistar soja 24 sata nakon podvezivanja obezajedničke karotidne arterije u trajanju od 15 min i/ili efekat tretmana DHEAprimenjenog 4 sata od hirurške procedure. Praćenje senzo-motorne funkcije iprisustvo infarktnih lezija na presecima celog mozga korišćeno je za validacijuI/R modela za proučavanje blaţih ishemičnih stanja poput TIA. Takođe,izvršeno je ispitivanje efekata I/R i tretmana na histo/morfološkom nivou.Odsustvo senzo-motornih deficita i infarktnih lezija kao i očuvanastruktura tkiva i morfologija neurona u hipokampusu i prečeonoj kori nakonI/R ukazuju na potencijalnu primenu modela u ispitivanju umerenihishemičnih stanja. Region-specifične promene signalnog puta NMDAreceptor/neuralna azot oksid sintaza/azot oksid nakon kratkotrajne I/R govoreu prilog većoj osetljivosti hipokampusa u odnosu na prečeonu koru. EfekatDHEA uočen je isključivo nakon I/R, vraćajući u prečeonoj kori parametare nakontrolnu vrednost, dok u hipokampusu promene Bax/Bcl -2 odnosa ipotencijala mitohondrijske membrane ukazuju na pokretanje pro-apoptotskihdešavanja. Stoga, prethodno okarakterisani neuroprotektivni efekat DHEAtreba dodatno istraţiti.Cerebral ischemia/reperfusion (I/R) is characteristic of cerebro-vasculardiseases such as stroke and transient ischemic attack (TIA). Alterations of Nmethyl-D-aspartate (NMDA) receptor-mediated signalling pathway are wellrecognized features of I/R-governed damage, while dehydroepiandrosterone(DHEA), its positive allosteric modulator, has been previously described asneuroprotective and potentially promising for therapy of ischemic conditions.In this doctoral dissertation changes of components of the NMDAreceptor complex and downstream signalling in hippocampus and prefrontalcortex were examined in male Wistar rats 24 h following 15 min bilateralcommon carotid artery occlusion. In addition, effects of 4 h post-surgical DHEAtreatment were investigated. Sensory-motor function and presence of infarctlesions were used to validate suitability of I/R model for studying mildischemic conditions such as TIA. Additionally, effects of I/R and treatmentwere evaluated on histo/morphological level.Absence of sensory-motor deficits and infarct lesions, as well aspreserved tissue structure and overall neuronal morphology in hippocampusand prefrontal cortex following I/R and treatment were detected indicatingsuitability of used I/R model for investigation of mild ischemic states. Regionspecificpostischemic changes of NMDA receptor/neuronal nitric oxidesynthase/nitric oxide following short-term I/R indicate higher sensitivity ofhippocampus compared to prefrontal cortex. DHEA effect was detectedexclusively following I/R, returning the parameters in prefrontal cortex to thecontrol value, while hippocampal Bax/Bcl-2 ratio and mitochondrial membranepotential changes indicate the initiation of pro-apoptotic events. Therefore,previously reported neuroprotective effect of DHEA should be considered withcaution and further investigated

Effects of brief cerebral ischemia/reperfusion and dehydroepoandrosterone treatment on components of NMDA receptor-mediated signalling in rat hippocampus and prefrontal cortex

Stanje moždane ishemije/reperfuzije (I/R) karakteristično je za cerebrovaskularnaoboljenja kao što su moţdani udar i tranzitorni ishemijski atak(TIA). Promena signalizacije posredovana N-metil-D-aspartat (NMDA)receptorom ima značajnu ulogu u I/R-uzrokovanom oštećenju, dok jedehidroepiandrosteron (DHEA), pozitvni alosterični modulator NMDAreceptora, najčešće okarakterisan kao neuroprotektivan u tretmanu ishemičnihstanja.U ovoj disertaciji ispitivana je proteinska ekspresija komponentikompleksa NMDA receptora i njegove nishodne signalizacije u hipokampusu iprečeonoj kori muţjaka pacova Wistar soja 24 sata nakon podvezivanja obezajedničke karotidne arterije u trajanju od 15 min i/ili efekat tretmana DHEAprimenjenog 4 sata od hirurške procedure. Praćenje senzo-motorne funkcije iprisustvo infarktnih lezija na presecima celog mozga korišćeno je za validacijuI/R modela za proučavanje blaţih ishemičnih stanja poput TIA. Takođe,izvršeno je ispitivanje efekata I/R i tretmana na histo/morfološkom nivou.Odsustvo senzo-motornih deficita i infarktnih lezija kao i očuvanastruktura tkiva i morfologija neurona u hipokampusu i prečeonoj kori nakonI/R ukazuju na potencijalnu primenu modela u ispitivanju umerenihishemičnih stanja. Region-specifične promene signalnog puta NMDAreceptor/neuralna azot oksid sintaza/azot oksid nakon kratkotrajne I/R govoreu prilog većoj osetljivosti hipokampusa u odnosu na prečeonu koru. EfekatDHEA uočen je isključivo nakon I/R, vraćajući u prečeonoj kori parametare nakontrolnu vrednost, dok u hipokampusu promene Bax/Bcl -2 odnosa ipotencijala mitohondrijske membrane ukazuju na pokretanje pro-apoptotskihdešavanja. Stoga, prethodno okarakterisani neuroprotektivni efekat DHEAtreba dodatno istraţiti.Cerebral ischemia/reperfusion (I/R) is characteristic of cerebro-vasculardiseases such as stroke and transient ischemic attack (TIA). Alterations of Nmethyl-D-aspartate (NMDA) receptor-mediated signalling pathway are wellrecognized features of I/R-governed damage, while dehydroepiandrosterone(DHEA), its positive allosteric modulator, has been previously described asneuroprotective and potentially promising for therapy of ischemic conditions.In this doctoral dissertation changes of components of the NMDAreceptor complex and downstream signalling in hippocampus and prefrontalcortex were examined in male Wistar rats 24 h following 15 min bilateralcommon carotid artery occlusion. In addition, effects of 4 h post-surgical DHEAtreatment were investigated. Sensory-motor function and presence of infarctlesions were used to validate suitability of I/R model for studying mildischemic conditions such as TIA. Additionally, effects of I/R and treatmentwere evaluated on histo/morphological level.Absence of sensory-motor deficits and infarct lesions, as well aspreserved tissue structure and overall neuronal morphology in hippocampusand prefrontal cortex following I/R and treatment were detected indicatingsuitability of used I/R model for investigation of mild ischemic states. Regionspecificpostischemic changes of NMDA receptor/neuronal nitric oxidesynthase/nitric oxide following short-term I/R indicate higher sensitivity ofhippocampus compared to prefrontal cortex. DHEA effect was detectedexclusively following I/R, returning the parameters in prefrontal cortex to thecontrol value, while hippocampal Bax/Bcl-2 ratio and mitochondrial membranepotential changes indicate the initiation of pro-apoptotic events. Therefore,previously reported neuroprotective effect of DHEA should be considered withcaution and further investigated

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Govori se o korištenju programa MAPLE u trigonometriji

Effect of dehydroepiandrosterone on cerebral ischemia/reperfusion

Moždana ishemija/reperfuzija (I/R), redukovan i ponovno uspostavljen protok krvi kroz mozak, predstavlja karakteristiku cerebro-vaskularnih oboljenja kao što su moždani udar i tranzitorni ishemijski atak. Izučavanje I/R na životinjskim modelima značajno je za razumevanje same patogeneze i potencijalnih terapeutika među kojima je svoje mesto našao i neurosteroid dehidroepiandrosteron (DHEA). DHEA može ostvarivati svoje neurobiološko dejstvo direktno vezivanjem za različite receptore ili indirektno putem metabolita ili intermedijera metabolickog puta. Mnogobrojne studije su pokazale njegov neuroprotektivan učinak u različitim patologijama centralnog nervnog sistema među kojima je svoje mesto zauzela i moždana I/R. U ovom radu osvrnućemo se na direktno delovanje DHEA u tretmanu ishemičnih stanja pružajući uvid u različite efekte koji su do sada pokazani u životinjskim modelima I/R kao i potencijalni značaj u modernoj medicini.Cerebral ischemia/reperfusion (I/R), reduced and re-established blood flow through the brain, represents characteristic of cerebrovascular diseases such as stroke and transient ischemic attack. I/R animal models enable understanding the pathogenesis itself as well as potential therapeutics, among which the neurosteroid dehydroepiandrosterone (DHEA) has found its place. DHEA can exert its neurobiological action directly by binding to various receptors or indirectly through metabolites or metabolic pathway intermediates. Numerous studies have shown its neuroprotective effect in various pathologies of the central nervous system, among which the cerebral I/R has taken its place. In this paper, we will focus on the direct actions of DHEA in the treatment of ischemic conditions, providing insight into the various effects shown so far in animal I/R models as well as potential significance in modern medicine

Prolonged Alprazolam Treatment Alters Components of Glutamatergic Neurotransmission in the Hippocampus of Male Wistar Rats—The Neuroadaptive Changes following Long-Term Benzodiazepine (Mis)Use

Alprazolam (ALP), a benzodiazepine (BDZ) used to treat anxiety, panic, and sleep disorders, is one of the most prescribed psychotropic drugs worldwide. The side effects associated with long-term (mis)use of ALP have become a major challenge in pharmacotherapy, emphasizing the unmet need to further investigate their underlying molecular mechanisms. Prolonged BDZ exposure may induce adaptive changes in the function of several receptors, including the primary target, gammaaminobutyric acid receptor type A (GABAAR), but also other neurotransmitter receptors such as glutamatergic. The present study investigated the potential effects of prolonged ALP treatment on components of glutamatergic neurotransmission, with special emphasis on N-Methyl-D-aspartate receptor (NMDAR) in the hippocampus of adult male Wistar rats. The study revealed behavioral changes consistent with potential onset of tolerance and involvement of the glutamatergic system in its development. Specifically, an increase in NMDAR subunits (NR1, NR2A, NR2B), a decrease in vesicular glutamate transporter 1 (vGlut1), and differential modulation of excitatory amino acid transporters 1 and 2 (EAAT1/2, in vivo and in vitro) were observed, alongside a decrease in α1-containing GABAAR following the treatment. By describing the development of compensatory actions in the glutamatergic system, the present study provides valuable information on neuroadaptive mechanisms following prolonged ALP intake

Streptozotocin, an FDA approved drug, affects the oxidative stress parameters and purinergic signaling components in primary rat astrocyte cultures

The antibiotic streptozotocin (STZ) is an FDA approved for pancreatic neuroendocrine tumors. It has also been used for rat model of diabetes induction, where it causes a progressive increase in BBB permeability, and activates glial cells. In intracerebroventricular injected STZ-induced AD model, the abnormal mitochondrial morphology, decrease ATP biosynthesis, accumulation of reactive oxygen species (ROS), disrupted homeostasis of brain insulin signaling and defect in cerebral glucose metabolism were observed. Streptozotocin has been used to induce mitochondrial, endoplasmic and in general oxidative stress in neuronal cells and in astrocytoma C6 cell line in vitro. Our study aimed to analyze the STZ effects on primary rat astrocyte cultures. The testing of STZ concentration range (1, 5, 10 and 20 mM) in MTT assay, excluded the 20 mM STZ which evoked a significant decrease in mitochondrial activity in astrocytes. As ROS are the most pronounced parameters elevated in STZ disease modeling, we analyzed GSH, SH groups and MDA 24 h after the STZ application. The 10 mM STZ lowered GSH levels, while SH groups showed a STZ dose dependent decrease. On the other hand, MDA showed a slight, but not significant increase following STZ concentration increase. Moreover, we investigated changes in the purinergic signaling system. Our results show the drop of CD73 activity 24 h after the 10 mM STZ treatment, accompanied by CD73 immunofluorescence decrease on the astrocyte membranes. Similarly, nucleoside triphosphate diphosphohydrolase 2 (NT2) was downregulated on astrocyte membranes. These results encourage further analysis of the P1 and P2 purinergic receptorsPoster Session: Neuroimmunoendocrine Interaction

Impaired olfactory performance and anxiety-like behavior in a rat model of multiple sclerosis are associated with enhanced adenosine signaling in the olfactory bulb via A1R, A2BR, and A3R

The present study shows that animals with experimental autoimmune encephalomyelitis (EAE) exhibit olfactory dysfunction and impaired general cognitive abilities, as well as anxiety-like behavior. Olfactory dysfunction occurs on average at 2 dpi, well before the onset of the first motor signs of EAE (8–10 dpi). After the initial olfactory dysfunction, the EAE animals show a fluctuation in olfactory performance that resembles the relapsing–remitting course of human MS. The study also shows severe neuroinflammation in the olfactory bulb (OB), with numerous infiltrated CD4+ T cells and peripheral macrophages in the superficial OB layers, marked microgliosis, and massive induction of TNF-α, IL-1β, and IL-6. Reduced tyrosine hydroxylase activity in the glomerular layer, pronounced granule cell atrophy, and reduced numbers of type B neuroblasts in the rostral migratory stream also indicate altered plasticity of the neuronal network in the OB. Considering the exceptionally high purinome expression in the OB, the possible involvement of purinergic signaling was also investigated. The study shows that macrophages infiltrating the OB overexpress A3R, while highly reactive microglia overexpress the adenosine-producing enzyme eN/ CD73 as well as A2BR, A3R, and P2X4R. Given the simultaneous induction of complement component C3, the results suggest that the microglial cells develop a functional phenotype of phagocytizing microglia. The study also demonstrates transcriptional and translational upregulation of A1R in mitral and tufted cells, which likely influence resting network activity in OB and likely contribute to olfactory dysfunction in EAE. Overall, our study shows that olfactory dysfunction and altered social and cognitive behavior in EAE are associated with increased adenosine signaling via A1R, A2BR, and A3R

Synthesis, Characterization, and Investigation of Anti-Inflammatory and Cytotoxic Activities of Novel Thiourea Derivatives of Naproxen

The objective of this study was to synthesize seven novel thiourea derivatives of naproxen (8–14), examine the anti-inflammatory activity of the newly synthesized compounds, investigate the cytotoxic potential of both sets of synthesized compounds (1–7 and 8–14), and select the most promising anti-inflammatory and antitumor drug candidates. The results of the in vivo anti-inflammatory study clearly showed that compounds 8 and 9 were capable of decreasing paw edema, as evident from a high percentage of inhibition (44.83% and 49.29%, respectively). In addition, the results of in vitro enzyme inhibition assays demonstrated that neither of the newly synthesized compounds reached 50% inhibition of 5-LOX at concentrations lower than 100 μM. In terms of antitumor potential, derivatives 3 and 8 exhibited strong cytotoxic effects on the HeLa cell line, suggesting the involvement of the extrinsic pathway of apoptosis. According to the overall results obtained for both sets of synthesized molecules, derivatives 4 and 8 can be underlined as molecules with the strongest anti-inflammatory activity, while derivatives 3 and 8 are the most promising cytotoxic agents

Intermittent Theta Burst Stimulation Ameliorates Cognitive Deficit and Attenuates Neuroinflammation via PI3K/Akt/mTOR Signaling Pathway in Alzheimer’s-Like Disease Model

Neurodegeneration implies progressive neuronal loss and neuroinflammation further contributing to pathology progression. It is a feature of many neurological disorders, most common being Alzheimer’s disease (AD). Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive stimulation which modulates excitability of stimulated brain areas through magnetic pulses. Numerous studies indicated beneficial effect of rTMS in several neurological diseases, including AD, however, exact mechanism are yet to be elucidated. We aimed to evaluate the effect of intermittent theta burst stimulation (iTBS), an rTMS paradigm, on behavioral, neurochemical and molecular level in trimethyltin (TMT)-induced Alzheimer’s-like disease model. TMT acts as a neurotoxic agent targeting hippocampus causing cognitive impairment and neuroinflammation, replicating behavioral and molecular aspects of AD. Male Wistar rats were divided into four experimental groups–controls, rats subjected to a single dose of TMT (8 mg/kg), TMT rats subjected to iTBS two times per day for 15 days and TMT sham group. After 3 weeks, we examined exploratory behavior and memory, histopathological and changes on molecular level. TMT-treated rats exhibited severe and cognitive deficit. iTBS-treated animals showed improved cognition. iTBS reduced TMT-induced inflammation and increased anti-inflammatory molecules. We examined PI3K/Akt/mTOR signaling pathway which is involved in regulation of apoptosis, cell growth and learning and memory. We found significant downregulation of phosphorylated forms of Akt and mTOR in TMT-intoxicated animals, which were reverted following iTBS stimulation. Application of iTBS produces beneficial effects on cognition in of rats with TMT-induced hippocampal neurodegeneration and that effect could be mediated via PI3K/Akt/mTOR signaling pathway, which could candidate this protocol as a potential therapeutic approach in neurodegenerative diseases such as AD

Ecto-5'-nucleotidase marks amoeboid microglial cells in the rat model of neurodegeneration

Adenosine 5'-triphosphate (ATP) and adenosine are versatile signaling molecules involved in many pathophysiological processes in the nervous system. They can be released from all types of brain cells in the extracellular space and activates purinergic receptors. Signaling via extracellular ATP is regulated by cell-surface located ectonucleotidases. Extracellular AMP resulting from the hydrolysis of ATP and ADP can in turn be hydrolyzed into adenosine by ecto-5'-nucleotidase (eN). We examined the involvement of purinergic signaling components in the rat model of trimethyltin (TMT)-induced hippocampal neurodegeneration (8mg/kg, single ip), which results in behavioral and neurological dysfunction similar as in Alzheimer's disease models. Enzyme histochemistry and immunohistochemistry (ir) showed that products of AMPase activity and eN-ir were accumulated in the neuronal strata, infiltrating within neuronal cell layers, depicting individual round-shaped elements that covered neuronal layers with pronounced cell death mostly at the late stage of TMT-induced neurodegeneration. Co-localization with Iba1+ specifically marked eN at amoeboid microglial cells. Neither of the tested pro-inflammatory cytokines (IL-1β, TNF-α, IL10) and C3 nor polarization marker iNOS was found in association with those Iba1/eN+ -cells. Iba1-ir cells co-localized with Arg1-ir and phagocytic marker CD68- ir. Marked induction of P2Y12R-, P2Y6R-, and P2X4-mRNA at the early stage of TMT-induced neurodegeneration might reflect the migration, and chemotaxis of microglia, while induction of P2X7R at amoeboid cells probably modulates their phagocytic role. These findings may contribute to a better understanding of the involvement of purinergic signaling components in the progression of neurodegenerative disorders that could be target molecules for development of novel therapies.Poster Session: Neuroimmunoendocrine Interaction