Action of heparin and acetylcholine modulators on the neurotoxicity of the toad Rhinella schneideri (Anura: Bufonidae) in Brazil / Acción de la heparina y moduladores de acetilcolina en la neurotoxicidad del sapo Rhinella schneideri (Anura: Bufonidae) en Brasil

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 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.

Methanolic extract of Rhinella schneideri (Anura: Bufonidae) poison causes ultrastructural changes in nerve terminals of phrenic nerve-diaphragm preparations in mice / Extracto metanólico de Rhinella schneideri (Anura: Bufonidae) causa cambios ultraestructurales en las terminaciones nerviosas de preparaciones de nervio frénico de ratón

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.

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.

Translation and validation of Neck Bournemouth Questionnaire to Brazilian Portuguese / Tradução e validação do Neck Bournemouth Questionnaire para o português do Brasil

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.

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.

Determinação da metemoglobina em voluntários fumantes e não fumantes / Determination of methemoglobin in smokers and non smokers volunteers

Objetivo ­ Determinar a concentração de metemoglobina (MetHb) em voluntários fumantes e não fumantes, e comparar com as possíveis alterações bioquímicas. Métodos ­ Os sujeitos do estudo foram voluntários do município de Sorocaba/SP (n=30), sendo metade para o grupo de fumantes (n=15) e outra metade para o grupo de não fumantes (n=15). Foi realizada a extração de saponina utilizando a droga vegetal Quilaia sp., para a realização de hemólise nas amostras de sangue e, então, dosar a metemoglobina dos voluntários. Amostras de sangue foram coletadas diretamente em tubos comerciais à vácuo e a metemoglobina foi determinada através da técnica que dispensa o uso do cianeto, evitando risco tóxico. Resultados ­ Os resultados estão apresentados em Média ± Desvio Padrão da porcentagem de MetHb: Grupo Fumantes foi de 3,4 ± 0,82% e o Grupo Não Fumantes foi de 8,3 ± 4,9% (valor normal: 1,9 a 3,8%), sendo diferentes significativamente. Os valores para a dosagem de glicemia e colesterol total não apresentaram diferença significativa, quando comparado o grupo de Fumantes com o Não Fumantes. Conclusões ­ Conclui-se que os valores de MetHb se apresentaram alterados em indivíduos fumantes, porém serão necessários maiores estudos acerca da comparação da alteração de MetHb com glicemia e/ou colesterol total.

Objective ­ To determine the concentration of methemoglobin (MetHb) in smoker and non-smoker volunteers comparing with possible biochemical alteration. Methods ­ The study were performed with volunteers of city Sorocaba/SP (n=30), separated in two groups: smokers group (n=15) and non-smokers group (n=15). The extraction of saponin was performed using the plant Quilaia sp., for hemolysis in the blood samples and, so, measure mehtemoglobin levels in the volunteers. Blood samples were collected directly in commercial tubes of vacuum and the methemoglobin was determined through of method described that dispenses the use of cyanide, avoiding the toxic risk. Results ­ The results are express in mean ± standard deviation of percentage MetHb: Non-smokers Group 3.4 ± 0.82%; Smokers Group 8.3 ± 4.9% (normal values 1.9%-3.8%), being different significantly. The glycemic and total cholesterol results, do not present significant difference when compared the smokers group with non-smokers group. Conclusions ­ The MetHb results presented are altered in smoker volunteers, however larger studies are needed on the comparison of the change of MetHb with glycaemia and/or total cholesterol.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella 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. The 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.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella 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. The 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.

Correlação entre os diferentes perfis sorológicos para a infecção pelo vírus da hepatite B e alterações das enzimas hepáticas: análise de fatores preditivos para adoecimento / Correlation between different serological profiles for hepatitis B infection and liver enzymatic alterations: analysis of predictive factors for illness

Objetivo: correlacionar os diferentes perfis sorológicos para a infecção pelo vírus da hepatite B e alterações de enzimas hepáticas. Métodos: foram colhidos os resultados de banco de dados laboratoriais, tendo como base a realização de sorologia para hepatite B (pesquisa de antígenos circulantes e seus anticorpos) e bioquímicos (dosagens das aminotransferases, quando presentes de γ-GT e fosfatase alcalina). Resultados: no presente estudo, 63,8% dos indivíduos infectados eram do gênero masculino; 8,4% dos indivíduos cujos dados foram analisados encontravam-se em fase aguda e 58,47% apresentavam-se com perfil compatível com fase de recuperação. As mulheres, apesar de no presente estudo serem menos propícias à infecção, apresentaram maior tendência ao que se entende por lesão hepática. Dentre os parâmetros bioquímicos avaliados no estudo, mostraram-se mais eficientes, com análise de tendências calculadas R2 próximas de 1 e podendo ser utilizados como critério de acompanhamento clínico, respectivamente a γ-GT (R2 0,94965); ALT (R2 0,8773); AST (R2 0,82645). A FAL apresentou resultados contraditórios, não permitindo ser utilizada como marcador bioquímico de evolução clínica (R2 inferior a 0,8). Conclusão: diante da maior incidência da infecção pelo HBV em indivíduos do gênero masculino, considera-se que esse dado não está necessariamente relacionado com o gênero, mas sim com um comportamento de risco mais característico das populações masculinas. A γ-GT e as aminotransferases apontaram como os melhores parâmetros para evolução clínica desde a fase aguda à recuperação. A fosfatase alcalina correspondeu às fases de infecção pelo HBV de maneira contraditória, não permitindo ser utilizada para acompanhamento clínico da infecção pelo HBV

Objective: to correlate the different serological profiles for infection of hepatitis B and liver enzymatic alterations. Methods: the results were collected directly from laboratory database, based on the serology for hepatitis B (study of circulating antigens and their antibodies) and biochemical (aminotransferase dosages, when present of γ-GT (GGT) and alkaline phosphatase). Results: in this study, 63,8% of the infected individuals were male, 8,4% of subjects whose data were analyzed were at the acute phase and 58,47% was shown with a profile compatible with the recovery phase. For women, though in the present study are less favorable to infection, were more likely to what is meant by hepatic injury. Among the biochemical parameters evaluated in the study, were more efficient, trend analysis calculated R2 close to 1 and can be used as criteria for clinical monitoring, respectively γ-GT (R2 0,94965), ALT (R2 0,8773); AST (R2 0,82645). The FAL showed contradictory results, not allowing it to be used as a biochemical marker of clinical evolution (R2 less than 0,8). Conclusion: although the higher incidence of HBV infection in male individuals, it is considered that this data is not necessarily related to genre, but to a risk behavior more characteristic of male populations. The γ-GT and aminotransferases pointed as the best parameters for clinical evolution from the acute phase to recovery. Alkaline phosphatase corresponded to the phase of HBV infection in a contradictory way, not allowing it to be used for clinical monitoring of HBV infection

Heparin and commercial bothropic antivenom against the paralyzing effect of Bothrops jararacussu snake venom

The crude venom of Bothrops jararacussu (Bjssu) is known to induce muscular paralysis in vitro. Many studies have shown that various substances, including heparin, neutralize the damage caused by snake venom. In the present study, the ability of heparin (Hep) and commercial bothropic antivenom (CBA) to neutralize neuromuscular effects of Bjssu venom, at different time-points, was analyzed. Mouse phrenic nerve-diaphragm preparation was used through a conventional myographic technique, following five different protocols: Group 1 was incubated with Bjssu (40 µg/mL) without any other treatment; Groups 2 and 3 were pretreated with heparin (1 µL/mL) and CBA (120 µL/mL), respectively, for 15 minutes before venom addition; Group 4 after 50 percent neuromuscular blockade induced by Bjssu crude venom received 1 µL/mL of heparin while Group 5 received a mixture of Hep:CBA:Bjssu. Control preparations (Tyrode) were treated with Hep and CBA (mean ± SEM; n = 3-6). After 120 minutes of venom incubation, Group 1 preparations presented twitch-tension of 12 ± 2 percent. However, in Groups 2 and 3, the neutralizations were 92 ± 1.9 percent and 81 ± 6 percent, respectively. The heparin addition, after 50 percent neuromuscular blockade by Bjssu, produced 40 ± 6 percent muscular response after 120 minutes of incubation. Hep:CBA:Bjssu mixture displayed a protective effect of 84 ± 10 percent against venom action. In conclusion, heparin and commercial bothropic antivenom efficiently neutralized the neurotoxic effects caused by B. jararacussu crude venom, even at different incubation time-points.