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.

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.

Influência de anestésicos locais sobre o bloqueio neuromuscular produzido pelo rocurônio: ação da lidocaína e da mistura enantiomérica em excesso de 50 por cento de bupivacaína na junção neuromuscular / Influencia de anestésicos locales sobre el bloqueo neuromuscular producido por el rocuronio: acción de la lidocaína y de la mezcla enantiomérica en exceso de 50 por ciento de bupivacaína en la junción neuromuscular / Influence of local anesthetics on the neuromuscular blockade produced by rocuronium: effects of lidocaine and 50 percent enantiomeric excess bupivacaine on the neuromuscular junction

Justificativa e objetivos: Os efeitos dos anestésicos locais (AL) na transmissão neuromuscular e sua influência no bloqueio neuromuscular produzido por bloqueadores neuromusculares competitivos são ainda alvo de pouca investigação. O objetivo do estudo foi avaliar in vitro os efeitos da lidocaína e da mistura enantiomérica em excesso de 50 por cento de bupivacaína (S75-R25) no bloqueio neuromuscular...

Background and objetives: The effects of local anesthetics (LA) on neuromuscular transmission and their influence on the neuromuscular blockade produced by competitive neuromuscular blockers have not been fully investigated. The objective of this study was to evaluate, in vitro, the effects of lidocaine and 50 percent enantiomeric excess bupivacaine (S75-R25) on the neuromuscular blockade produced by rocuronium...

Justificativa y objetivos: Los efectos de los anestésicos locales (AL), en la transmisión neuromuscular y su influencia en el bloqueo neuromuscular producido por bloqueadores neuromusculares competitivos, todavía no se ha investigado lo suficiente. El objetivo del estudio, fue evaluar in vitro los efectos de la lidocaína y de la mezcla enantiomérica en exceso de 50 por ciento de bupivacaína (S75-R25) en el bloqueo neuromuscular...

A study of the myotoxicity of bothropstoxin-i using manganese in mouse phrenic nerve-diaohragm and extensor digitorum longus preparations

Bothropstoxin-I (BthTX-I) from Bothrops jararacussu snake venom has a predominantly postsynaptic action that is responsible for this toxin´s myotoxicity. However, BthTX-I also has a presynaptic action that is counteracted by Mn2+, a reversible neuromuscular blocker that acts predominantly presynaptically. In this work, we used two nerve-muscle preparations (mouse phrenic nerve-diaphragm - PND and extensor digitorum longus - EDL) to investigate the ability of Mn2+ to protect against the myotoxicity of BthTX-I. The preparations were incubated with Tyrode solution (control), BthTX-I, or Mn2+ alone. BthTX-I (1.4 µM) produced irreversible blockade in both preparations, whereas the blockade by Mn2+ (0.9 mM) was total and reversible in PND but just partially reversible in EDL. Pretreating the preparations with Mn2+ resulted in 100% and 80% protection against BthTX-I-induced blockade, respectively. However, when Mn2+ (0.9 or 1.8 mM) and BthTX-I (1.4 µM) were co-incubated for 30 min before testing, the blockade was faster and sustained. Washing the preparations resulted in complete, sustained recovery in those exposed to 1.8 mM Mn2+ but not to 0.9 mM Mn2+. Morphological analysis showed that the extent of fiber damage by BthTXI (1.4 µM) was 82% (PND) and 68.5% (EDL), and that Mn2+ (0.9 mM) afforded 40% protection in both preparations and reduced the increase in muscle fiber cross-sectional area by 20% and 15%, respectively, compared to BthTX-I alone. Mn2+ (0.9 mM) significantly attenuated the release of creatine kinase by BthTXI. The low creatine kinase activity resulted from a protective action of Mn2+ on the sarcolemma and from direct inactivation of the released enzyme. These results show that Mn2+ prevents membrane disruption by BthTX-I and can protect against the myotoxicity and neurotoxicity caused by this toxin.

Effects of manganese ions in the neuromuscular junction

The neurotoxic effects of manganese occured mainly from its inhalation are well described in the literature. In this study we have been demonstrated the potent and reversible effect of manganese ions (Mn²+) on neuromuscular transmission using conventional myographic. The Mn²+ mechanism wich causes reversible blockade at neuromuscular junction was investigated in isolated mouse phrenic nerve-diaphragm (PND) preparations. As much Mn²+ 0.9 mM as Mn²+ 1.8 mM produced rapid neuromuscular blockade (50% in < 4 min), but only the lower concentration reversed spontaneously. The use of d-tubocurarine (5.8 µM), 3,4-diaminopyridine 3,4-DAP, 0.09 mM) and dantrolene (10 µM) excluded the involvment of nicotinic receptors, K+ channels and ryanodine receptors, respectively, as the potential target for this ion. Manganese acts an early competitive antagonist and after as an agonist of Ca²+, indicating that this ion may probably act via Ca²+ cannels...

Electrophysiological and ultrastructural analysis of the neuromuscular blockade and miotoxicity induced by the Micrurus nigrocinctus snake venom

Micrurus nigrocinctus is the most abundant coral snake in Central America. The venom of this specie induced a concentration-dependent (10-20 mug/ml) depolarization in the isolated mouse phrenic nerve-diaphragm preparations incubated ate 37 degree. D-Tubocurrarine (10 mug/ml) and alpha betaungarotoxin (3-5 mug/ml) were able to partially protect against the depolarization induced by the venom (10 mug/ml), suggesting the involvement of subsynaptic cholinergic receptors. This venom (10 mug/ml) also increased the frequency and amplitude of miniature end-plate potentials (mepps) during the first 10-20 min of incubation. Subsequently, the mepps progressively decreased and disappeared after 60 min. These responses were accompanied by ultrastructural changes involving the nerve terminals, the subsynaptic junctional folds and the muscle mitochondria. The synaptic gutter was shallow and, very often, "shrunken" terminal with omega-shaped axolemmal identations and a decreased number of synaptic vecicles were present. A common finding was the presence of numerous finger-like, membrane-bounded bodies interposed between the terminal and the Schwann cells or postsynaptic sarcolemma. The preincubation of the venom with specific antivenom or the incubation of the preparations at room temperature (24-26 degree) reduced the number and intensity of the ultrastructural alterations. The last finding suggests the involvement of a enzymatic process, probably a phospholipase A2, present in the venom. There was a good correlation between the electrophysiological and ultrastructural effects induced by the venom which allow us to conclude that M. nigrocinctus venom has a presynaptic action in the initial stages of intoxication followed by sub- and postsynaptic effects, the last being the most important cause of neuromuscular blockade. A direct action of the venom on muscle fibers may also contributes to the irreversible blockade.