The role of purinergic signaling system trimethyltin- induced neurodegeneration and neuroinflammation of hippocampus of female rats

Neurodegeneracija je proces koji se odlikuje progresivnim gubitkom nervnog tkiva, posebno nervnih ćelija, nervnih nastavaka i mijelina, koji se uočava kod velikog broja neuroloških bolesti, a za koji do danas nije razvijena adekvatna terapija, niti su u brojnim bolestima otkriveni uzroci. S obzirom na to da neurodegeneracija podrazumeva oštećenje tkiva, ona je nužno praćena neuroinflamatornom aktivacijom mikroglije i astrocita, što može doprineti progresiji inicijalne patologije. Predmet istraživanja ove doktorske disertacije bio je ispitivanje promena komponenti purinske signalizacije u hipokampusu ženki pacova u modelu neurodegeneracije i neuroinflamacije izazvane trimetil kalajem (TMK). TMK je neurotoksin koji dovodi do selektivne smrti neurona limbičkog sistema, naročito u hipokampusu, što rezultuje histopatološkim i bihejvioralnim promenama koje verno reprodukuju neke od glavnih karakteristika mnogih neurodegenerativnih bolesti. Prvi set rezultata dobijenih u okviru ove disertacije pokazuje da tokom prve tri nedelje nakon delovanja TMT javlja progresivno umiranje nervnih ćelija, što je praćeno snažnom i specifičnom aktivacijom astrocita i mikroglije. Aktivirani astrociti u hipokamusu ispoljavaju morfološku i funkcionalnu dihotomiju, tako što astrociti CA1 regiona hipokampusa pokazuju odlike hipertrofiranih ćelija, dok astrociti hilarnog regiona pokazuju odlike atrofije. Aktivirane mikroglijske ćelije pojačanao eksprimiraju početni enzim purinske kaskade, NTPDaza1/CD39, dok mikroglijske ćelije ameobidnog oblika, na mestima aktivne neurodegeneracije, pojačano eksprimiraju završni enzim purinske kaskade, eN/CD73. Reaktivni astrociti pojačano eksprimiraju A1R, A2AR i P2Y1R receptore kao i C3, iNOS, NF-kB, koji ukazuju na njihov proinflamacijski karakter. U mikroglijskim ćelijama, povećana ekspresija purinskih receptora P2Y12R, P2Y6R i P2X4R na genskom nivou, ukazuje na migratorni fenotip aktivirane mikroglije tokom rane faze neurodegeneracije, dok je povećana proteinska ekspresija P2X7R verovato povezana sa fagocitozom ameboidne mikroglije. Pojačana ekspresija navedenih komponenti purinske transmisije na mikrogliji (NTPDaze1/CD39, eN/CD73 kao i P2X7R) i astrocitima (A1R, A2AR, P2Y1R) u neurodegeneraciji i neuroinflamaciji izazvanoj TMK, kao i smanjena ekspresija A1R, P2Y12R, ukazuju na ulogu purinske signalizacije u neurodegeneraciji i neuroinflamaciji.Neurodegeneration reffers to a progressive loss of neuronal tissue, specifically nerve cells, nerve endings and myelin sheet, and it is common for many neurological disorders. There is no known effective drug or therapeutic approach for virtually all neurodegenerative diseases and cause for many of them has not been discovered yet. Neurodegenerative disorders are often accompanied by neuroinflammatory activation of microglia and astrocytes which can further contribute to pathology progression. The main goal of this dissertation was to examine changes in the main components of purinergic signaling in trimethyltin-induced (TMT) neurodegeneration and neurofinflammation of hippocampus of female rats. TMT is a potent neurotoxin which causes selective neuronal death of lymbic system, especially hippocampus which results in histopathological and behavioral changes resulting in some of the main common characteristics of neurodegenerative disorders. First set of the results of this doctoral dissertation shows that the first three weeks after TMT intoxication are accompanied by extensive neuronal death in CA sectors of hippocampus, followed by reactive astro- and microgliosis. Reactive astrocytes in hippocampus showed morphological and functional dichotomy. Astrocytes of CA1 region were characterized by hypetrhophic morphology while astrocytes od hilar region had atrophy-like morphology. Activated microglial cells showed increased expression of NTPDase1/CD39, while ameboid microglia in the site of active neurodegeneration showed eN/CD73. Reactive astrocytes showed increase expression of A1R, A2AR i P2Y1R and C3, iNOS, NF-kB, which points to their proinflammatory phenotype. Increased gene expression of P2Y12R, P2Y6R i P2X4R points toward migratory phenotype of microglia during early phase of neurodegeneration, while late expression of P2X7R is probably related to phagocitic properties of amoeboid microglial cells. Increased expression of microglial (NTPDase1/CD39, eN/CD73 and P2X7R) and astrocytic (A1R, A2AR, P2Y1R) purinergic compontents in TMT-induced neurodegeneration and neuroinflammation as well as loss of homeostatic A1R, P2Y12R receptors in neurons definitely points to an active role of purinergic signaling in neurodegenerative process

An increase of hydro-aggregate's installed power and efficiency factor before the revitalization phase

The revitalization of hydro-aggregates should be performed before the life-time expiry of most their parts. But, as in our practice aggregates have been under exploitation much longer then theoretical life-time advices (40, even 50 years), there is a need to estimate the equipment's condition through more sophisticated monitoring, providing the maximum level of safety and availability with minimum investment expenses, before the revitalization finally takes place. That estimation is based on appropriating calculations and measurements needed to take the competent decision. Results of that analysis can be also useful for estimation of the aggregate's power and parameters after the revitalization. In this paper the results of appropriate calculations and measurements for the Hydropower station HE Zvornik are given

An increase of hydro-aggregate's installed power and efficiency factor before the revitalization phase

The revitalization of hydro-aggregates should be performed before the life-time expiry of most their parts. But, as in our practice aggregates have been under exploitation much longer then theoretical life-time advices (40, even 50 years), there is a need to estimate the equipment's condition through more sophisticated monitoring, providing the maximum level of safety and availability with minimum investment expenses, before the revitalization finally takes place. That estimation is based on appropriating calculations and measurements needed to take the competent decision. Results of that analysis can be also useful for estimation of the aggregate's power and parameters after the revitalization. In this paper the results of appropriate calculations and measurements for the Hydropower station HE Zvornik are given

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

Microglial- and Astrocyte-Specific Expression of Purinergic Signaling Components and Inflammatory Mediators in the Rat Hippocampus During Trimethyltin-Induced Neurodegeneration

The present study examined the involvement of purinergic signaling components in the rat model of hippocampal degeneration induced by trimethyltin (TMT) intoxication (8 mg/kg, single intraperitoneal injection), which results in behavioral and neurological dysfunction similar to neurodegenerative disorders. We investigated spatial and temporal patterns of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5′ nucleotidase (eN/CD73) activity, their cell-specific localization, and analyzed gene expression pattern and/or cellular localization of purinoreceptors and proinflammatory mediators associated with reactive glial cells. Our study demonstrated that all Iba1+ cells at the injured area, irrespective of their morphology, upregulated NTPDase1/CD39, while induction of eN/CD73 has been observed at amoeboid Iba1+ cells localized within the hippocampal neuronal layers with pronounced cell death. Marked induction of P2Y12R, P2Y6R, and P2X4-messenger RNA at the early stage of TMT-induced neurodegeneration might reflect the functional properties, migration, and chemotaxis of microglia, while induction of P2X7R at amoeboid cells probably modulates their phagocytic role. Reactive astrocytes expressed adenosine A1, A2A, and P2Y1 receptors, revealed induction of complement component C3, inducible nitric oxide synthase, nuclear factor-kB, and proinflammatory cytokines at the late stage of TMT-induced neurodegeneration. An increased set of purinergic system components on activated microglia (NTPDase1/CD39, eN/CD73, and P2X7) and astrocytes (A1R, A2AR, and P2Y1), and loss of homeostatic glial and neuronal purinergic pathways (P2Y12 and A1R) may shift purinergic signaling balance toward excitotoxicity and inflammation, thus favoring progression of pathological events. 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 the development of novel therapies

Ketamine’s Amelioration of Fear Extinction in Adolescent Male Mice Is Associated with the Activation of the Hippocampal Akt-mTOR-GluA1 Pathway

Fear-related disorders, including post-traumatic stress disorder (PTSD), and anxiety disorders are pervasive psychiatric conditions marked by persistent fear, stemming from its dysregulated acquisition and extinction. The primary treatment for these disorders, exposure therapy (ET), relies heavily on fear extinction (FE) principles. Adolescence, a vulnerable period for developing psychiatric disorders, is characterized by neurobiological changes in the fear circuitry, leading to impaired FE and increased susceptibility to relapse following ET. Ketamine, known for relieving anxiety and reducing PTSD symptoms, influences fear-related learning processes and synaptic plasticity across the fear circuitry. Our study aimed to investigate the effects of ketamine (10 mg/kg) on FE in adolescent male C57 BL/6 mice at the behavioral and molecular levels. We analyzed the protein and gene expression of synaptic plasticity markers in the hippocampus (HPC) and prefrontal cortex (PFC) and sought to identify neural correlates associated with ketamine’s effects on adolescent extinction learning. Ketamine ameliorated FE in the adolescent males, likely affecting the consolidation and/or recall of extinction memory. Ketamine also increased the Akt and mTOR activity and the GluA1 and GluN2A levels in the HPC and upregulated BDNF exon IV mRNA expression in the HPC and PFC of the fear-extinguished mice. Furthermore, ketamine increased the c-Fos expression in specific brain regions, including the ventral HPC (vHPC) and the left infralimbic ventromedial PFC (IL vmPFC). Providing a comprehensive exploration of ketamine’s mechanisms in adolescent FE, our study suggests that ketamine’s effects on FE in adolescent males are associated with the activation of hippocampal Akt-mTOR-GluA1 signaling, with the vHPC and the left IL vmPFC as the proposed neural correlates

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

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

Long-term alprazolam treatment may cause tolerance development by modulating components of glutamatergic neurotransmission in the hippocampus of male Wistar rats

The benzodiazepine alprazolam (ALP) is commonly prescribed to treat anxiety, panic, and sleep disorders. However, ALP is often abused for prolonged periods of time, leading to severe side effects such as tolerance, dependence, and withdrawal syndrome. Previous literature data suggest that neuroadaptive changes at synaptic receptors, such as gammaaminobutyric acid receptor type A (GABAAR) and glutamatergic receptors, may be responsible for the occurrence and development of the aforementioned side effects. Therefore, the present study investigated the potential effects of prolonged ALP treatment (2 mg/kg, ip.) on the α1-subunit containing GABAAR and components of glutamatergic neurotransmission in the hippocampus of adult male Wistar rats. The study revealed behavioral changes consistent with a possible onset of tolerance and associated changes in the GABAergic and glutamatergic systems. The primary target of ALP, the α1-subunit containing GABAAR, was decreased indicating its potential downregulation by prolonged agonist (ALP) action. Considering studied glutamatergic components, an increase in NMDAR subunits, a decrease in vGlut1, and differential modulation of excitatory amino acid transporters 1 and 2 (EAAT1/2, in vivo and in vitro) were observed. These changes may all together indicate a compensatory mechanism due to the sustained suppression of glutamatergic neurons by enhanced inhibitory impulses from GABAergic neurons. The data presented provide valuable and, to our knowledge, the first information on components of glutamatergic neurotransmission after prolonged ALP treatment and their potential impact on the development of side effects. However, further research is needed to examine the observed changes in detail.Poster Session: Bran Stimulation & Signalling, Phase Separation and Open Dat