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Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures

BACKGROUND: L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage i...

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Autores principales: Primini, Eduardo Octaviano, Liberato, José Luiz, Fontana, Andreia Cristina Karklin, dos Santos, Wagner Ferreira
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Centro de Estudos de Venenos e Animais Peçonhentos 2019
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533932/
https://www.ncbi.nlm.nih.gov/pubmed/31131008
http://dx.doi.org/10.1590/1678-9199-JVATITD-1488-18
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author Primini, Eduardo Octaviano
Liberato, José Luiz
Fontana, Andreia Cristina Karklin
dos Santos, Wagner Ferreira
author_facet Primini, Eduardo Octaviano
Liberato, José Luiz
Fontana, Andreia Cristina Karklin
dos Santos, Wagner Ferreira
author_sort Primini, Eduardo Octaviano
collection PubMed
description BACKGROUND: L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage implicated in many neurological disorders. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; therefore, there is a need to develop novel treatments. In this regard, previous studies have shown that neuroactive compounds obtained from the venom of the spider Parawixia bistriata have neuroprotective effects in vitro and in vivo. In this sense, this work aimed to evaluate potential neuroprotective effects of fraction RT10, obtained from this spider venom, on primary cultures of neuron and glial cells subjected to glutamate excitotoxicity insults. METHODS: Primary cultures of neurons and glia were obtained from the cerebral tissue of 1-day-old postnatal Wistar rats. After 7 days in vitro (DIV), the cultures were incubated with fraction RT10 (0.002; 0.02; 0.2 and 2 µg/µL) or riluzole (100 µM) for 3-hours before application of 5 mM L-Glu. After 12 hours, the resazurin sodium salt (RSS) test was applied to measure metabolic activity and proliferation of living cells, whereas immunocytochemistry for MAP2 was performed to measure neuronal survival. In addition, the cells were immunolabeled with NeuN and GFAP in baseline conditions. RESULTS: In the RSS tests, we observed that pre-incubation with RT10 before the excitotoxic insults from L-Glu resulted in neuroprotection, shown by a 10% reduction in the cell death level. RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%. Moreover, qualitative analysis of neuronal morphology (by MAP2 staining, expressed as fluorescence intensity (FI), an indirect measure of neuronal survival) indicate that RT10 reduced the toxic effects of L-Glu, as shown by a 38 % increase in MAP2 fluorescence when compared to L-Glu insult. On the other hand, the riluzole treatment resulted in 17% increase of MAP2 fluorescence; therefore, the neuroprotection from RT10 was more efficacious. CONCLUSION: RT10 fraction exhibits neuroprotective effects against L-Glu excitotoxicity in neuron-glia cultured in vitro.
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spelling pubmed-65339322019-05-24 Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures Primini, Eduardo Octaviano Liberato, José Luiz Fontana, Andreia Cristina Karklin dos Santos, Wagner Ferreira J Venom Anim Toxins Incl Trop Dis Research BACKGROUND: L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage implicated in many neurological disorders. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; therefore, there is a need to develop novel treatments. In this regard, previous studies have shown that neuroactive compounds obtained from the venom of the spider Parawixia bistriata have neuroprotective effects in vitro and in vivo. In this sense, this work aimed to evaluate potential neuroprotective effects of fraction RT10, obtained from this spider venom, on primary cultures of neuron and glial cells subjected to glutamate excitotoxicity insults. METHODS: Primary cultures of neurons and glia were obtained from the cerebral tissue of 1-day-old postnatal Wistar rats. After 7 days in vitro (DIV), the cultures were incubated with fraction RT10 (0.002; 0.02; 0.2 and 2 µg/µL) or riluzole (100 µM) for 3-hours before application of 5 mM L-Glu. After 12 hours, the resazurin sodium salt (RSS) test was applied to measure metabolic activity and proliferation of living cells, whereas immunocytochemistry for MAP2 was performed to measure neuronal survival. In addition, the cells were immunolabeled with NeuN and GFAP in baseline conditions. RESULTS: In the RSS tests, we observed that pre-incubation with RT10 before the excitotoxic insults from L-Glu resulted in neuroprotection, shown by a 10% reduction in the cell death level. RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%. Moreover, qualitative analysis of neuronal morphology (by MAP2 staining, expressed as fluorescence intensity (FI), an indirect measure of neuronal survival) indicate that RT10 reduced the toxic effects of L-Glu, as shown by a 38 % increase in MAP2 fluorescence when compared to L-Glu insult. On the other hand, the riluzole treatment resulted in 17% increase of MAP2 fluorescence; therefore, the neuroprotection from RT10 was more efficacious. CONCLUSION: RT10 fraction exhibits neuroprotective effects against L-Glu excitotoxicity in neuron-glia cultured in vitro. Centro de Estudos de Venenos e Animais Peçonhentos 2019-05-16 /pmc/articles/PMC6533932/ /pubmed/31131008 http://dx.doi.org/10.1590/1678-9199-JVATITD-1488-18 Text en This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Primini, Eduardo Octaviano
Liberato, José Luiz
Fontana, Andreia Cristina Karklin
dos Santos, Wagner Ferreira
Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures
title Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures
title_full Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures
title_fullStr Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures
title_full_unstemmed Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures
title_short Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures
title_sort neuroprotective properties of rt10, a fraction isolated from parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533932/
https://www.ncbi.nlm.nih.gov/pubmed/31131008
http://dx.doi.org/10.1590/1678-9199-JVATITD-1488-18
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