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

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.(AU)

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 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)...(AU)

Neurotransmitter evaluation in the hippocampus of rats after intracerebral injection of TsTX scorpion toxin

TsTX is an -type sodium channel toxin that stimulates the discharge of neurotransmitters from neurons. In the present study we investigated which neurotransmitters are released in the hippocampus after TsTX injection and if they are responsible for electrographic or histopathological effects. Microdialysis revealed that the toxin increased glutamate extracellular levels in the hippocampus; however, levels of gamma-aminobutyric acid (GABA), glycine, 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were not significantly altered. Neurodegeneration in pyramidal cells of hippocampus and electroencephalographic alterations caused by the toxin were blocked by pretreatment with riluzole, a glutamate release inhibitor. The present results suggest a specific activity of TsTX in the hippocampus which affects only glutamate release.

Efeitos da associação do riluzol ao dantrolene em ratos submetidos ao trauma medular agudo

Objetivou-se avaliar os efeitos da associação do riluzol ao dantrolene em ratos submetidos ao trauma medular agudo experimental. Foram utilizados 29 ratos machos adultos da variedade Wistar com peso médio de 350g divididos em cinco grupos: CN (Controle negativo), CP (Controle positivo), R (riluzol 4 mg/kg), D (dantrolene 10 mg/kg) e R+D (riluzol 4 mg/kg + dantrolene 10 mg/kg). À exceção do grupo CN, que foi submetido apenas à laminectomia de T12, todos os outros grupos foram submetidos à laminectomia de T12 e posteriormente ao trauma medular agudo compressivo, utilizando-se aparelho estereotáxico, com peso de 40,5g, durante cinco minutos. Quinze minutos após a laminectomia os grupos CN, CP e D receberam placebo,i.p, e R e R+D riluzol,i.p. Após 1 hora a laminectomia, os grupos CN, CP e R receberam placebo,i.p, e os grupos D e R+D dantrolene,i.p. Os animais foram submetidos à avaliação clínica-neurológica, diariamente durante os sete dias subseqüentes ao trauma, para observação da função motora em campo aberto e classificação conforme escala proposta por Basso, Beatie e Bresnahan. Após oito dias os animais foram eutanasiados e amostras de sangue, para análises hematológicas e bioquímicas, e da medula espinhal, para avaliação histológica, imunoistoquímica (anti-NeuN) e TUNEL, foram coletadas. Os animais submetidos ao trauma apresentaram paraparesia não ambulatória e apenas o grupo R+D demonstrou recuperação motora após os sete dias de avaliação pelo teste BBB (P<0,05). Não houve diferença estatística entre os valores dos perfils hematológico e bioquímico, com exceção dos valores de uréia que se apresentaram mais elevados nos grupos traumatizados. A contagem de células marcadas com anti-NeuN demonstrou maior número de neurônios íntegros nos animais que receberam os tratamentos, principalmente nos animais que receberam a associação de riluzol e dantrolene (P<0,05). Na técnica TUNEL observou-se menor número de células apoptóticas nos animais dos grupos R, D e R+D quando comparados ao grupo CP, sem que, contudo, fosse observada diferença entre a associação e a monoterapia. De acordo com esses resultados conclui-se que a associação do riluzol ao dantrolene sódico nas doses de 4mg/kg e 10 mg/kg, respectivamente, promoveu efeito neuroprotetor em ratos submetidos ao trauma medular agudo, associado à melhora da função motora e diminuição da apoptose neural

The objective of this study was to evaluate the association effect of riluzole and dantrolene in the spinal cord injured rats. Twenty nine male Wistar rats, 350 g, were randomly distributed into five groups: CN (Sham), CP (Placebo), R (Riluzole, 4mg/kg), D (Dantrolene,10mg/kg) and R+D (Riluzole 4mg/kg + Dantrolene 10mg/kg). All animals underwent laminectomy of T12 and, except CN, to an acute spinal cord compressive trauma, using a stereotaxic device with a 40,5g rod for five minutes. Fifteen minutes after the laminectomy, CN, CP and D received placebo, i.p, and R, R+D, riluzole, i.p. One our after the laminectomy, CN, CP and R received placebo,i.p, and D and R+D, dantrolene,i.p. Neurologic examination, performed daily for seven days, of the animals consisted of Basso, Beatie, Bresnahan (BBB) test for the evaluation of motor function. After eight days the animals were euthanized and blood samples for hematological and biochemical analysis, and spinal cord, for histological, immunohistochemical (anti-NeuN) and TUNEL evaluation, were collected. Traumatized animals showed no ambulatory paraparesis and only the R+D group showed motor recovery after seven days of evaluation by BBB test (P<0,05). There was no statistical difference between the values of hematologic and biochemical perfils, with the exception of urea values that were higher in traumatized animals. The counting of cells stained with anti-NeuN showed a higher number of intact neurons in treated animals, mainly on those that received R+D (P<0,05). In the TUNEL technique a lower number of apoptotic cells were observed in R, D and R+D groups compared with CP. Meanwhile no diferrence in the number of apoptotic cells were observed between R+D and R, D. Acording to this results we concluded that the association of riluzole and dantrolene, 4mg/kg and 10mg/kg respectively, promoted synergistic neuroprotective effects in spinal cord injured rats, associated with improvement of motor function and decreased of the neural apoptosis

Avaliação da capacidade motora em campoaberto de ratos submetidos a trauma medularagudo e tratados com a associação de riluzol edantrolene / Evaluation of the motor capacity of rats, in an open field, submitted to acute spinalcord trauma and treated with the association of riluzole and dantrolene

O riluzol e o dantrolene são fármacos que apresentam potencial efeito neuroprotetor. Não há relatosda associação destes fármacos para tratamento de trauma medular, porém acredita-se que esta possaoferecer efeito neuroprotetor sinérgico. Assim, objetivou-se avaliar a função motora em campo abertode ratos submetidos ao trauma medular agudo e tratados com riluzol (4mg/kg),dantrolene (10 mg/kg) e placebo (volume equivalente). Foram utilizados 25 ratos Wistar (±420 g), divididos em 5 grupos:GI (controle negativo), GII (placebo), GIII (riluzol), GIV (dantrolene) e GV (riluzol+dantrolene).Realizou-se laminectomia de T12. Os grupos GII, GIII, GIV e GV também foram submetidos a traumamedular compressivo (40,5g) por 5 minutos. Quinze minutos após o trauma GI, GII e GIV receberamplacebo, e GIII e GV riluzol. Após 1 hora ao trauma, os grupos GI, GII e GIII receberam placebo, e osgrupos GIV e GV dantrolene. Realizou-se observação da função motora 1 dia antes e nos 7 dias subsequentesao procedimento cirúrgico.Não houve diferença significativa entre GI e GV. Conclui-se quea associação do riluzol ao dantrolene é capaz de promover recuperação motora em ratos submetidosao trauma medular agudo, sugerindo efeito neuroprotetor.(AU)

Riluzole and Dantrolene are drugs which have potential neuroprotective effect. There are no reportsof the association of these drugs for the spinal cord trauma treatment, but it is believed that canprovide synergistic neuroprotective effect. Therefore, the aimed of this study was to evaluate ratsmotor function, in an open field, submitted to acute spinal cord trauma and treated with riluzole(4mg/kg), dantrolene (10mg/kg) and placebo (equivalent volume). 25 Wistar rats (±420 g) were dividedinto five groups: GI (negative control), GII (placebo), GIII (riluzole), GIV (dantrolene) and GV(riluzole+dantrolene). Laminectomy was performed from T12. GII, GIII, GIV and GV groups werealso submitted to compressive spinal cord trauma (40,5g) for 5 minutes. Fifteen minutes after the traumaGI,GII and GIV received placebo, and GIII and GV riluzole. One hour after the trauma, GI, GII andGIII received placebo and GIV and GV dantrolene. Observation of the motor function one day before and seven days after the surgical procedure was performed. There was no significant difference between GI andGV after seven days observation. So, the combination of riluzole and dantrolene is able to promote motor recoveryin rats subjected to acute spinal cord trauma, suggesting a neuroprotective effect.(AU)

Avaliação da capacidade motora em campoaberto de ratos submetidos a trauma medularagudo e tratados com a associação de riluzol edantrolene / Evaluation of the motor capacity of rats, in an open field, submitted to acute spinalcord trauma and treated with the association of riluzole and dantrolene

O riluzol e o dantrolene são fármacos que apresentam potencial efeito neuroprotetor. Não há relatosda associação destes fármacos para tratamento de trauma medular, porém acredita-se que esta possaoferecer efeito neuroprotetor sinérgico. Assim, objetivou-se avaliar a função motora em campo abertode ratos submetidos ao trauma medular agudo e tratados com riluzol (4mg/kg),dantrolene (10 mg/kg) e placebo (volume equivalente). Foram utilizados 25 ratos Wistar (±420 g), divididos em 5 grupos:GI (controle negativo), GII (placebo), GIII (riluzol), GIV (dantrolene) e GV (riluzol+dantrolene).Realizou-se laminectomia de T12. Os grupos GII, GIII, GIV e GV também foram submetidos a traumamedular compressivo (40,5g) por 5 minutos. Quinze minutos após o trauma GI, GII e GIV receberamplacebo, e GIII e GV riluzol. Após 1 hora ao trauma, os grupos GI, GII e GIII receberam placebo, e osgrupos GIV e GV dantrolene. Realizou-se observação da função motora 1 dia antes e nos 7 dias subsequentesao procedimento cirúrgico.Não houve diferença significativa entre GI e GV. Conclui-se quea associação do riluzol ao dantrolene é capaz de promover recuperação motora em ratos submetidosao trauma medular agudo, sugerindo efeito neuroprotetor.

Riluzole and Dantrolene are drugs which have potential neuroprotective effect. There are no reportsof the association of these drugs for the spinal cord trauma treatment, but it is believed that canprovide synergistic neuroprotective effect. Therefore, the aimed of this study was to evaluate ratsmotor function, in an open field, submitted to acute spinal cord trauma and treated with riluzole(4mg/kg), dantrolene (10mg/kg) and placebo (equivalent volume). 25 Wistar rats (±420 g) were dividedinto five groups: GI (negative control), GII (placebo), GIII (riluzole), GIV (dantrolene) and GV(riluzole+dantrolene). Laminectomy was performed from T12. GII, GIII, GIV and GV groups werealso submitted to compressive spinal cord trauma (40,5g) for 5 minutes. Fifteen minutes after the traumaGI,GII and GIV received placebo, and GIII and GV riluzole. One hour after the trauma, GI, GII andGIII received placebo and GIV and GV dantrolene. Observation of the motor function one day before and seven days after the surgical procedure was performed. There was no significant difference between GI andGV after seven days observation. So, the combination of riluzole and dantrolene is able to promote motor recoveryin rats subjected to acute spinal cord trauma, suggesting a neuroprotective effect.