Resumen Introducción: Rhinella schneideri está ampliamente distribuida en Suramérica y su veneno es caracterizado por inducir cardiotoxicidad y neurotoxicidad. Objetivo: En este trabajo, investigamos estrategias farmacológicas para atenuar la neurotoxicidad periférica inducida por el veneno de R. schneideri en preparaciones neuromusculares de aves. Métodos: Los experimentos fueron realizados usando preparaciones de biventer cervicis de pollos sometidas a estimulación de campo para el registro de las contracciones musculares o expuestas a la acetilcolina y al cloruro de potasio para la respuesta contractural. Resultados: El veneno (10 µg/ml) provocó un bloqueo neuromuscular completo en las preparaciones después de aproximadamente 70 min de incubación (tiempos para 50 y 90 % de bloqueo: 15 ± 3 min y 40 ± 2 min, respectivamente; P < 0.05, N = 5); las contracturas en respuesta a la acetilcolina y el KCl exógenos no fueron afectadas por el veneno, indicando que no hay una interacción especifica con receptores postsinápticos o miotoxicidad respectivamente. El bloqueo neuromuscular causado por el veneno (10 µg/ml) no fue prevenido por la heparina (5 y 150 UI/ml) bajo condiciones pre y post-tratamiento. La incubación a bajas temperaturas (23-25 ºC) abolió el bloqueo neuromuscular; después de aumentar la temperatura a 37 ºC, el bloqueo neuromuscular total fue levemente más lento que el visto en preparaciones directamente incubadas a 37 ºC (tiempos para 50 y 90 % de bloqueo: 23 ± 2 min y 60 ± 2.5 min, respectivamente; P < 0.05, N= 4). Neostigmina (3.3 µM) no revirtió el bloqueo neuromuscular, mientras que 3.4-diaminopiridina (91.6 µM) produjo una reversión parcial y sostenida de las respuestas neuromusculares (29 ± 7.8 % de la reversión máxima alcanzada en aproximadamente 40 min de incubación; P < 0.05, N = 4). Conclusiones: El veneno de R. schneideri indujo neurotoxicidad periférica potente in vitro, el cual puede ser revertido por 3.4-diaminopiridina.

Abstract Introduction: Rhinella schneideri is a toad widely distributed in South America and its poison is characterized by inducing cardiotoxicity and neurotoxicity. Objective: In this work, we investigated pharmacological strategies to attenuate the peripheral neurotoxicity induced by R. schneideri poison in avian neuromuscular preparation. Methods: The experiments were carried out using isolated chick biventer cervicis preparation subjected to field stimulation for muscle twitches recordings or exposed to acetylcholine and potassium chloride for contracture responses. Results: Poison (10 μg/ml) produced complete neuromuscular blockade in chick biventer cervicis preparation within approximately 70 min incubation (times for 50 and 90 % blockade: 15 ± 3 min and 40 ± 2 min, respectively; P < 0.05, N= 5); contracture responses to exogenous acetylcholine and KCl were unaffected by poison indicating no specificity with postsynaptic receptors or myotoxicity, respectively. Poison (10 μg/ml)-induced neuromuscular blockade was not prevented by heparin (5 and 150 IU/ml) under pre- or post-treatment conditions. Incubation at low temperature (23-25 °C) abolished the neuromuscular blockade; after raising the temperature to 37 °C, the complete neuromuscular blockade was slightly slower than that seen in preparations directly incubated at 37 °C (times for 50 and 90 % blockade: 23 ± 2 min and 60 ± 2.5 min, respectively; P < 0.05, N= 4). Neostigmine (3.3 μM) did not reverse the neuromuscular blockade in BC preparation whereas 3,4-diaminopyridine (91.6 μM) produced a partial and sustained reversal of the twitch responses (29 ± 7.8 % of maximal reversal reached in approximately 40 min incubation; P < 0.05, N= 4). Conclusions: R. schneideri poison induces potent peripheral neurotoxicity in vitro which can be partially reversible by 3,4-diaminopyridine.

Resumen Rhinella schneideri (o Bufo paracnemis), conocido popularmente en Brasil como sapo cururu, también se encuentra en otros países de América del Sur. Los efectos cardiovasculares de este veneno son ampliamente conocidos y recientemente se demostró que es capaz de afectar la unión neuromuscular en la preparación aislada de aves y ratones. En este trabajo, utilizamos microscopía electrónica de transmisión para investigar la ultraestructura de la terminación nerviosa motora y pliegues de unión postsináptica de preparaciones de nervio frénico-hemidiafragma incubadas durante 5 o 60 min con el extracto metanólico de R. schneideri (50 μg/mL). Además, se examinó el estado de los receptores de acetilcolina (AChR) mediante la ubicación de inmunofluorescencia de TRITC-α-bungarotoxina en la membrana de la placa terminal. Los resultados muestran que 5 min de incubación con el extracto de secreción de glándula disminuyeron significativamente (32 %) el número de vesículas sinápticas en el terminal del nervio motor, pero no disminuyeron la densidad electrónica en la parte superior de los pliegues de unión donde se concentran los receptores nicotínicos. Sin embargo, 60 min de incubación condujeron a una recarga significativa de los terminales nerviosos en las vesículas sinápticas, mientras que la inmunorreactividad del AChR no fue tan marcada como en el control y después de 5 min de incubación. Las fibras musculares estaban bien conservadas, pero los axones motores intramusculares no. Los hallazgos corroboraron los datos farmacológicos ya que la disminución en el número de vesículas sinápticas (5 min) seguida de recuperación (60 min) está de acuerdo con el aumento transitorio de la frecuencia de MEPPs, lo que significa una mayor liberación de neurotransmisores. Estos datos apoyan el modo de acción presináptico predominante de R. schneideri, pero no excluyen la posibilidad de una acción postsináptica secundaria dependiendo del tiempo en que la preparación esté expuesta al veneno.

Abstract Rhinella schneideri (or Bufo paracnemis), popularly known in Brazil as cururu toad, is also found in other countries in South America. The cardiovascular effects of this poison are largely known and recently was shown that it is capable to affect the neuromuscular junction on avian and mice isolated preparation. In this work, we used transmission electron microscopy to investigate the ultrastructure of the motor nerve terminal and postsynaptic junctional folds of phrenic nerve-hemidiaphragm preparations incubated for either 5 or 60 min with the methanolic extract of R. schneideri (50 µg/mL). In addition, the status of the acetylcholine receptors (AChR) was examined by TRITC-α-bungarotoxin immunofluorescence location at the endplate membrane. The results show that 5 min of incubation with the gland secretion extract significantly decreased (32 %) the number of synaptic vesicles into the motor nerve terminal, but did not decrease the electron density on the top of the junctional folds where nicotinic receptors are concentrated; however, 60 min of incubation led to significant nerve terminal reloading in synaptic vesicles whereas the AChR immunoreactivity was not as marked as in control and after 5 min incubation. Muscle fibers were well-preserved but intramuscular motor axons were not. The findings corroborated pharmacological data since the decrease in the number of synaptic vesicles (5 min) followed by recovery (60 min) is in accordance with the transient increase of MEPPs frequency meaning increased neurotransmitter release. These data support the predominant presynaptic mode of action of the R. schneideri, but do not exclude the possibility of a secondary postsynaptic action depending on the time the preparation is exposed to poison. Rev. Biol. Trop. 66(3): 1290-1297. Epub 2018 September 01.

Resumo Objetivo: Traduzir o Neck Bournemouth Questionnaire para o português do Brasil, adaptá-lo culturalmente e verificar a sua validade e confiabilidade. Métodos: O desenvolvimento da versão brasileira do Neck Bournemouth Questionnaire (NBQ-Brasil) foi baseado nas diretrizes propostas por Guillemin. O processo aplicado consistiu em tradução, retrotradução, revisão por um comitê e pré-teste. Participaram deste estudo 61 voluntários que apresentavam dor cervical; 35 deles participaram durante a fase de pré-teste para verificar a compreensão do instrumento e os 26 restantes durante a análise psicométrica. A avaliação psicométrica incluiu a análise da confiabilidade interavaliadores e intra-avaliador e da validade do construto (correlação entre o NBQ-Brasil, o SF-36, a escala numérica de dor e o Neck Disability Index). Resultados: Alguns termos e algumas expressões foram alterados para se obter equivalência cultural com o NBQ-Brasil durante a fase de tradução. O NBQ mostrou uma CCI intra-avaliador de 0,96 e CCI interavaliadores de 0,87. A análise da validade do construto mostrou correlações moderadas com o SF-36 e correlação forte com a escala numérica de dor e o Neck Disability Index. Conclusão: O Neck Bournemouth Questionnaire foi traduzido e adaptado culturalmente para o idioma português e demonstrou ser válido e confiável para avaliar a dor cervical dos pacientes.

Abstract Objective: To translate the Neck Bournemouth Questionnaire to Brazilian Portuguese, cross-culturally adapt, and to verify its validity and its reliability. Methods: The development of the Brazilian version of Neck Bournemouth Questionnaire (Brazil-NBQ) was based on the guideline proposed by Guillemin. The applied process consisted of translation, back-translation, committee review and pre-test. Sixty-one volunteers presenting neck pain participated in this study. Thirty-five of them participated during pre-testing phase to verify the instrument comprehension, and the remaining 26 took part during psychometric analysis. Psychometric evaluation included interrater and intrarater reliability and construct validity (correlation among Brazil-NBQ, SF-36, Numerical rating score and Neck Disability Index). Results: Some terms and expressions were changed to obtain cultural equivalence for Brazil-NBQ during the translation phase. The NBQ showed an intrarater ICC of 0.96 and interrater ICC of 0.87. Construct validity analysis showed moderate correlations with SF-36 and strong correlation with Numerical rating score and Neck Disability Index. Conclusion: Neck Bournemouth Questionnaire was translated and culturally adapted to Portuguese language, and it demonstrated to be valid and reliable to evaluate patients’ neck pain.

BackgroundRhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity.MethodsThe neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.ResultsVenom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p< 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p< 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively).ConclusionThese results indicate that the methanolic extract of R. schneiderivenom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.