Abstract
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.
Highlights
L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions
RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%
Primini et al Journal of Venomous Animals and Toxins including Tropical Diseases (2019) 25:e148818 survival) indicate that RT10 reduced the toxic effects of L-Glu, as shown by a 38 % increase in Microtubule associated protein 2 (MAP2) fluorescence when compared to L-Glu insult
Summary
L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; 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. L-Glu transporters are responsible for glutamate clearance in the brain; they can be targeted for the development of therapies for CNS disorders, in which glutamate excitotoxicity plays an important role, such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), autism, stroke, Huntington’s disease and epilepsy [2,3,4,5]. We investigated a potential neuroprotective effect of RT10, a pool of molecules from P. bistriata venom, in primary cultures of neuron and glial cells exposed to excitotoxic insults by the application of a high concentration of L-Glu
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