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

 

Eduardo Octaviano Primini¹, José Luiz Liberato^1 2, Andreia Cristina Karklin Fontana³, Wagner Ferreira dos Santos^1 2

 

1 Neurobiology and Venoms Laboratory, Department of Biology, College of Philosophy, Sciences and Literature of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

2 Institute for Neurosciences and Behavior - INeC, Ribeirão Preto, SP, Brazil.

3 Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA, USA.

 

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.

 

Keywords: L-Glutamate; excitotoxicity; neuroprotection; Parawixia bistriata; RT10

 

Received: August 03, 2018.

Accepted: January 15, 2019.

 

Correspondence: wagnerf@usp.br

 

Competing interests

The authors declare that they have no competing interests.

 

Authors' contributions

EOP was responsible for carrying out the experiments, extraction and analysis of spider venom and writing of the manuscript. JLL was responsible for experiment design, data analysis and writing and correction of the manuscript. ACKF was responsible for manuscript correction, including English assistance and discussion of the results. WFS was the major contributor to the project, coordinated the whole team, assisted in the project design, contributed to writing the manuscript and analyzing the data, searched for funding, assisted in the project design and advised EOP. All authors read, corrected and approved the final manuscript.