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.

Atividade antiofídica de Camellia sinensis contra a ação tóxica de venenos botrópicos em preparação neuromuscular de ave / Antiophidic activity of Camellia sinensis against the toxic action of bothropic venoms in bird neuromuscular preparations

Introdução: venenos de serpentes do gênero Bothrops são capazes de induzir bloqueio neuromuscular em preparações isoladas de mamífero e ave. O tratamento específico é a soroterapia, porém, nem sempre se mostra eficaz contra a toxicidade induzida pelo veneno. Por outro lado, compostos vegetais têm sido estudados com finalidade antiofídica, como é o caso da Camellia sinensis (chá verde). Objetivo: o objetivo foi de avaliar a atividade do extrato alcoólico de Camellia sinensis contra a ação de diferentes venenos botrópicos em preparações neuromusculares de ave. Métodos: foram utilizadas preparações neuromusculares de biventer cervicis de pintainho (BC) que foram incubadas com: venenos de diferentes serpentes do gênero Bothrops (B. jararaca, B. jararacussu, B. alternatus e B. neuwiedi, 50-100 µg/mL), extrato alcoólico de Camellia sinensis (Cs) 100 µg/ml ou pré-tratadas com Cs (30min) seguida da adição dos diferentes venenos botrópicos. Resultados: ao final do experimento, foi observado bloqueio da resposta contrátil em 69,7 ± 4,3% (B. jararaca), 83,1 ± 10% (B. jararacussu), 88,1 ± 4,5% (B. alternatus) e 92,4 ± 5,6% (B. neuwiedi). Quando as preparações foram pré incubadas com o extrato de Cs, as proteções foram de: 62%, 89,2%, 75,1% e 78,2%, respectivamente. Conclusão: conclui-se que o extrato de Camellia sinensis se mostrou eficaz contra a neurotoxicidade induzida por venenos botrópicos

Introduction: venoms from snakes of the Bothrops genus are capable of inducing neuromuscular blockade in preparations isolated from mammals and birds. The specific treatment is serum therapy, but it is not always effective against the toxicity induced by the venom. On the other hand, some plant compounds have been studied for antiophidic purposes, as is the case with Camellia sinensis (green tea). Objective: evaluate the activity of the alcoholic extract from Camellia sinensis against the action of various bothropic venoms in bird neuromuscular preparations. Methods: Chick biventer cervicis (BC) neuromuscular preparations were used which were incubated with venoms from various snakes of the Bothrops genus (B. jararaca, B. jararacussu, B. alternatus and B. neuwiedi, 50-100 µg/ml), Camellia sinensis (Cs) alcoholic extract, 100 µg/ml, or previously treated with Cs (30 min), followed by the addition of the various bothropic venoms. Results: blockade of the contractile response was observed in 69.7 ± 4.3% (B. jararaca), 83.1 ± 10% (B. jararacussu), 88.1 ± 4.5% (B. alternatus) and 92.4 ± 5.6% (B. neuwiedi). When the preparations were previously incubated with Cs extract, results were 62%, 89.2%, 75.1% and 78.2%, respectively. Conclusion: Camellia sinensis extract was found to be effective against neurotoxicity induced by bothropic venoms.

Presynaptic Activity of an Isolated Fraction from Rhinella schneideri Poison.

Purpose: Rhinella schneideri is a toad found in many regions of the South America. The poison of the glands has cardiotoxic effect in animals and neuromuscular effects in mice and avian preparation. The purpose of this work was to identify the toxin responsible for the neuromuscular effect in avian and mice neuromuscular preparation. Methods: The methanolic extract from R. schneideri poison was fractioned by reversed phase HPLC. The purity and molecular mass were determined by LC/MS mass spectrometry. Chick biventer cervicis and mouse phrenic-nerve diaphragm were used as neuromuscular preparations to identify the toxin. Results: The purification resulted in 32 fractions, which 4 of them were active in neuromuscular preparation. The toxin of fraction 20 were chosen for better reproducibility of the whole extract activity and its molecular mass was 730.6 Da. The toxin produced facilitation of the muscle contraction followed by a complete neuromuscular blockade in chick biventer cervicis preparation in 90 min without interfering with the exogenous response to ACh and KCl. The quantal content was increased from 128 ± 13 (control) to 216 ± 44 (after 5 min and sustained until 60 min) in the presence of the toxin. Conclusion: In conclusion, our results demonstrated that the neuromuscular action of the poison of Rhinella schneideri is a multitoxin effect. More, the present work first isolated a 730.6 Da toxin that better represent the whole poison neuromuscular effect, to which is attributed a presynaptic action in avian and mouse neuromuscular preparation.

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.

Presynaptic activity of an isolated fraction from Rhinella schneideri poison

Purpose: Rhinella schneideri is a toad found in many regions of the South America. The poison of the glands has cardiotoxic effect in animals and neuromuscular effects in mice and avian preparation. The purpose of this work was to identify the toxin responsible for the neuromuscular effect in avian and mice neuromuscular preparation. Methods: The methanolic extract from R. schneideri poison was fractioned by reversed phase HPLC. The purity and molecular mass were determined by LC/MS mass spectrometry. Chick biventer cervicis and mouse phrenic-nerve diaphragm were used as neuromuscular preparations to identify the toxin. Results: The purification resulted in 32 fractions, which 4 of them were active in neuromuscular preparation. The toxin of fraction 20 were chosen for better reproducibility of the whole extract activity and its molecular mass was 730.6 Da. The toxin produced facilitation of the muscle contraction followed by a complete neuromuscular blockade in chick biventer cervicis preparation in 90 min without interfering with the exogenous response to ACh and KCl. The quantal content was increased from 128 ± 13 (control) to 216 ± 44 (after 5 min and sustained until 60 min) in the presence of the toxin. Conclusion: In conclusion, our results demonstrated that the neuromuscular action of the poison of Rhinella schneideri is a multitoxin effect. More, the present work first isolated a 730.6 Da toxin that better represent the whole poison neuromuscular effect, to which is attributed a presynaptic action in avian and mouse neuromuscular preparation.

Presynaptic activity of an isolated fraction from Rhinella schneideri poison

Purpose: Rhinella schneideri is a toad found in many regions of the South America. The poison of the glands has cardiotoxic effect in animals and neuromuscular effects in mice and avian preparation. The purpose of this work was to identify the toxin responsible for the neuromuscular effect in avian and mice neuromuscular preparation. Methods: The methanolic extract from R. schneideri poison was fractioned by reversed phase HPLC. The purity and molecular mass were determined by LC/MS mass spectrometry. Chick biventer cervicis and mouse phrenic-nerve diaphragm were used as neuromuscular preparations to identify the toxin. Results: The purification resulted in 32 fractions, which 4 of them were active in neuromuscular preparation. The toxin of fraction 20 were chosen for better reproducibility of the whole extract activity and its molecular mass was 730.6 Da. The toxin produced facilitation of the muscle contraction followed by a complete neuromuscular blockade in chick biventer cervicis preparation in 90 min without interfering with the exogenous response to ACh and KCl. The quantal content was increased from 128 ± 13 (control) to 216 ± 44 (after 5 min and sustained until 60 min) in the presence of the toxin. Conclusion: In conclusion, our results demonstrated that the neuromuscular action of the poison of Rhinella schneideri is a multitoxin effect. More, the present work first isolated a 730.6 Da toxin that better represent the whole poison neuromuscular effect, to which is attributed a presynaptic action in avian and mouse neuromuscular preparation.

Pharmacological Properties of Vochysia Haenkeana (Vochysiaceae) Extract to Neutralize the Neuromuscular Blockade Induced by Bothropstoxin-I (Lys49 Phospholipase A2) Myotoxin.

Purpose: Bothrops snakes are responsible for more than 70 % of snakebites every year in Brazil and their venoms cause severe local and systemic damages. The pharmacological properties of medicinal plants have been widely investigated in order to discover new alternative treatments for different classes of diseases including neglected tropical diseases as envenomation by snakebites. In this work, we have investigated the ability of Vochysia haenkeana stem barks extract (VhE) to neutralize the neuromuscular effects caused by Bothropstoxin-I (BthTX-I), the major phospholipase A2 (PLA2) myotoxin from B. jararacussu venom. Methods: The biological compounds of VhE were analysed under thin layer chromatography (TLC) and its neutralizing ability against BthTX-I was assessed through twitch-tension recordings and histological analysis in mouse phrenic nerve-diaphragm (PND) preparations. The antimicrobial activity of VhE was assessed against S. aureus, E. coli and P. aeruginosa strains. The aggregation activity of VhE was analysed under protein precipitation assay. Results: VhE showed the presence of phenolic compound visualized by blue trace under TLC. VhE abolished the neuromuscular blockade caused by BthTX-I applying the pre-toxin incubation treatment and partially neutralized the BthTX-I action under post-toxin incubation treatment; VhE contributed slightly to decrease the myotoxicity induced by BthTX-I. The neutralizing mechanism of VhE may be related to protein aggregation. VhE showed no antimicrobial activity. Conclusion: V. haenkeana extract which has no antimicrobial activity exhibited neutralizing ability against the neuromuscular blockade caused by BthTX-I and also contributed to decrease its myotoxicity. Protein aggregation involving phenolic compounds may be related in these protective effects.

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.

Translation and validation of Neck Bournemouth Questionnaire to Brazilian Portuguese.

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.

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.

The Facilitatory Effect of Casearia sylvestris Sw. (guaçatonga) Fractions on the Contractile Activity of Mammalian and Avian Neuromuscular Apparatus.

Many natural products influence neurotransmission and are used clinically. In particular, facilitatory agents can enhance neurotransmission and are potentially useful for treating neuromuscular diseases in which muscular weakness is the major symptom. In this work, we investigated the facilitatory effect of apolar to polar fractions of Casearia sylvestris Sw. (guaçatonga) on contractility in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) neuromuscular preparations exposed to indirect (via the nerve; 3 V stimuli) and direct (30 V stimuli) muscle stimulation in the absence and presence of pharmacological antagonists. Methanolic and ethyl acetate fractions, but not hexane or dichloromethane fractions, exerted a facilitatory effect on PND (indirect stimulation). The methanolic fraction was chosen for further assays to assess the involvement of: 1) presynaptic sites (axons or nerve terminals), 2) postsynaptic sites (cholinergic receptors, sarcolemma or T-tubules), and 3) the synaptic cleft (acetylcholinesterase enzyme). In preparations treated with d-tubocurarine, the methanolic fraction did not cause facilitation in response to direct stimuli; this fraction was also unable to reverse dantrolene-induced blockade (indirect stimulation). In curarized preparations, the methanolic fraction either restored neuromuscular transmission (mimicking the effect of neostigmine) or failed to cause any recovery of neurotransmission. In the presence of 3,4-diaminopyridine (3,4-DAP), the methanolic fraction decreased twitch amplitude, whereas at a high frequency of stimulation (40 Hz) there was an increase in tetanic tension. In BC preparations, the methanolic fraction did not affect contractures to exogenous acetylcholine or potassium chloride. Incubation with atropine showed there was certain modulation by prejunctional nicotinic receptors, whereas treatment with nifedipine showed that the neurofacilitation required the entry of extracellular calcium. Tetrodotoxin did not prevent the facilitatory effect of 3,4-DAP or neostigmine, but antagonized the response to the methanolic fraction. These findings indicate that neuronal sodium channels have an important role in the facilitatory response to the methanolic fraction, with extracellular calcium entry via calcium channels modulating this neurofacilitation. Possible modulation of prejunctional cholinoceptors was not excluded, particularly in view of certain antagonism by the methanolic fraction at muscarinic receptors. Since facilitation by the methanolic fraction involved enhanced acetylcholine release, use of this fraction could be potentially beneficial in neuromuscular diseases and in the reversal of residual paralysis in the post-operative period or after local anaesthesia.

The facilitatory effect of Casearia sylvestris Sw. (guaçatonga) fractions on the contractile activity of mammalian and avian neuromuscular apparatus.

Many natural products influence neurotransmission and are used clinically. In particular, facilitatory agents can enhance neurotransmission and are potentially useful for treating neuromuscular diseases in which muscular weakness is the major symptom. In this work, we investigated the facilitatory effect of apolar to polar fractions of Casearia sylvestris Sw. (guaçatonga) on contractility in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) neuromuscular preparations exposed to indirect (via the nerve; 3 V stimuli) and direct (30 V stimuli) muscle stimulation in the absence and presence of pharmacological antagonists. Methanolic and ethyl acetate fractions, but not hexane or dichloromethane fractions, exerted a facilitatory effect on PND (indirect stimulation). The methanolic fraction was chosen for further assays to assess the involvement of: 1) presynaptic sites (axons or nerve terminals), 2) postsynaptic sites (cholinergic receptors, sarcolemma or T-tubules), and 3) the synaptic cleft (acetylcholinesterase enzyme). In preparations treated with d-tubocurarine, the methanolic fraction did not cause facilitation in response to direct stimuli; this fraction was also unable to reverse dantrolene-induced blockade (indirect stimulation). In curarized preparations, the methanolic fraction either restored neuromuscular transmission (mimicking the effect of neostigmine) or failed to cause any recovery of neurotransmission. In the presence of 3,4-diaminopyridine (3,4-DAP), the methanolic fraction decreased twitch amplitude, whereas at a high frequency of stimulation (40 Hz) there was an increase in tetanic tension. In BC preparations, the methanolic fraction did not affect contractures to exogenous acetylcholine or potassium chloride. Incubation with atropine showed there was certain modulation by prejunctional nicotinic receptors, whereas treatment with nifedipine showed that the neurofacilitation required the entry of extracellular calcium. Tetrodotoxin did not prevent the facilitatory effect of 3,4-DAP or neostigmine, but antagonized the response to the methanolic fraction. These findings indicate that neuronal sodium channels have an important role in the facilitatory response to the methanolic fraction, with extracellular calcium entry via calcium channels modulating this neurofacilitation. Possible modulation of prejunctional cholinoceptors was not excluded, particularly in view of certain antagonism by the methanolic fraction at muscarinic receptors. Since facilitation by the methanolic fraction involved enhanced acetylcholine release, use of this fraction could be potentially beneficial in neuromuscular diseases and in the reversal of residual paralysis in the post-operative period or after local anaesthesia.

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

BACKGROUND: 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. METHODS: 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. RESULTS: Venom 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). CONCLUSION: These results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.

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

VdTX-1, a reversible nicotinic receptor antagonist isolated from venom of the spider Vitalius dubius (Theraphosidae).

Theraphosid spider venoms can block neurotransmission in vertebrate nerve-muscle preparations in vitro, but few of the components involved have been characterized. In this work, we describe the neuromuscular activity of venom from the Brazilian theraphosid Vitalius dubius and report the purification and pharmacological characterization of VdTX-1, a 728 Da toxin that blocks nicotinic receptors. Neuromuscular activity was assayed in chick biventer cervicis preparations and muscle responses to exogenous ACh and KCl were determined before and after incubation with venom or toxin. Changes in membrane resting potential were studied in mouse diaphragm muscle. The toxin was purified by a combination of filtration through Amicon® filters, cation exchange HPLC and RP-HPLC; toxin purity and mass were confirmed by mass spectrometry. Venom caused progressive neuromuscular blockade and muscle contracture; the blockade but not the contracture was reversible by washing. Venom attenuated contractures to exogenous ACh and KCl. Filtration yielded low (LM, <5 kDa) and high (HM, >5 kDa) fractions, with the latter reproducing the contracture seen in venom but with a slight and progressive twitch blockade. The LM fraction caused reversible blockade and attenuated contractures to ACh, but had no effect on contractures to KCl. VdTX-1 (728 Da) purified from the LM fraction was photosensitive and reduced the E(max) to ACh in biventer cervicis muscle without affecting the EC50; VdTX-1 also abolished carbachol-induced depolarizations. V. dubius venom contains at least two components that affect vertebrate neurotransmission. One component, VdTX-1, blocks nicotinic receptors non-competitively to produce reversible blockade without muscle contracture.

Beneficial effect of crotamine in the treatment of myasthenic rats.

INTRODUCTION: Crotamine is a basic, low-molecular-weight peptide that, at low concentrations, improves neurotransmission in isolated neuromuscular preparations by modulating sodium channels. In this study, we compared the effects of crotamine and neostigmine on neuromuscular transmission in myasthenic rats. METHODS: We used a conventional electromyographic technique in in-situ neuromuscular preparations and a 4-week treadmill program. RESULTS: During the in-situ electromyographic recording, neostigmine (17 µg/kg) caused short-term facilitation, whereas crotamine induced progressive and sustained twitch-tension enhancement during 140 min of recording (50 ± 5%, P < 0.05). On the treadmill evaluation, rats showed significant improvement in exercise tolerance, characterized by a decrease in the number of fatigue episodes after 2 weeks of a single-dose treatment with crotamine. CONCLUSIONS: These results indicate that crotamine is more efficient than neostigmine for enhancing muscular performance in myasthenic rats, possibly by improving the safety factor of neuromuscular transmission.

Mipartoxin-I, a novel three-finger toxin, is the major neurotoxic component in the venom of the redtail coral snake Micrurus mipartitus (Elapidae).

The major venom component of Micrurus mipartitus, a coral snake distributed from Nicaragua to northern South America, was characterized biochemically and functionally. This protein, named mipartoxin-I, is a novel member of the three-finger toxin superfamily, presenting the characteristic cysteine signature and amino acid sequence length of the short-chain, type-I, α-neurotoxins. Nevertheless, it varies considerably from related toxins, with a sequence identity not higher than 70% in a multiple alignment of 67 proteins within this family. Its observed molecular mass (7030.0) matches the value predicted by its amino acid sequence, indicating lack of post-translational modifications. Mipartoxin-I showed a potent lethal effect in mice (intraperitoneal median lethal dose: 0.06 µg/g body weight), and caused a clear neuromuscular blockade on both avian and mouse nerve-muscle preparations, presenting a post-synaptic action through the cholinergic nicotinic receptor. Since mipartoxin-I is the most abundant (28%) protein in M. mipartitus venom, it should play a major role in its toxicity, and therefore represents an important target for developing a therapeutic antivenom, which is very scarce or even unavailable in the regions where this snake inhabits. The structural information here provided might help in the preparation of a synthetic or recombinant immunogen to overcome the limited venom availability.

Neuromuscular activity of Bothrops alcatraz snake venom in chick biventer cervicis preparations.

Venom (10-100 µg/ml) from Bothrops alcatraz, a pitviper from the Alcatrazes Archipelago off the coast of southeastern Brazil, caused progressive, irreversible neuromuscular blockade in chick isolated biventer cervicis preparations. The venom also inhibited contractures to exogenous ACh (110 µM) and KCl (20 mM), caused myofiber damage and increased creatine kinase release. Commercial bothropic antivenom raised against mainland Bothrops species neutralized the neuromuscular activity, depending on the venom concentration.