In Situ Effects of Doxycycline on Neuromuscular Junction in Mice.

BACKGROUND: The antibacterial mechanism of doxycycline is known, but its effects on the nerve-muscle system are still not unclear. OBJECTIVE: The aim of the study was to combine molecular targets of the neuromuscular machinery using the in situ neuronal blocker effect of doxycycline, a semisynthetic second-generation tetracycline derivative, on mice neuromuscular preparations. METHODS: The effects of doxycycline were assessed on presynaptic, synaptic cleft, and postsynaptic neurotransmission, along with the muscle fiber, using the traditional myographic technique. Precisely, the effects of doxycycline were categorized into "all" or "nothing" effects depending on the concentration of doxycycline used; "all" was obtained with 4 µM doxycycline, and "nothing" was obtained with 1-3 µM doxycycline. The rationale of this study was to apply known pharmacological tools against the blocker effect of 4 µM doxycycline, such as F55-6 (Casearia sylvestris), CaCl2 (or Ca2+), atropine, neostigmine, polyethylene glycol (PEG 400), and d-Tubocurarine. The evaluation of cholinesterase enzyme activity and the diaphragm muscle histology were performed, and protocols on the neuromuscular preparation submitted to indirect or direct stimuli were complementary. RESULTS: Doxycycline does not affect cholinesterase activity nor causes damage to skeletal muscle diaphragm; it acts on ryanodine receptor, sarcolemmal membrane, and neuronal sodium channel with a postjunctional consequence due to the decreased availability of muscle nicotinic acetylcholine receptors. CONCLUSION: In conclusion, in addition to the neuronal blocker effect of doxycycline, we showed that doxycycline acts on multiple targets. It is antagonized by F55-6, a neuronal Na+-channel agonist, and Ca2+, but not by neostigmine.

The Influence of Silver Nanoparticles Against Toxic Effects of Philodryas olfersii Venom.

PURPOSE: A silver nanoparticle obtained by reducing salts with solid dispersion of curcumin (130 nm, 0.081 mg mL-1) was used to counteract against the toxic - edematogenic, myotoxic, and neurotoxic - effects of Philodryas olfersii venom. METHODS: The edematogenic effect was evaluated by plasma extravasation in rat dorsal skin after injection of 50 µg per site of venom alone or preincubated with 1, 10, and 100 µL of AgNPs; the myotoxicity was evaluated by measuring the creatine kinase released into the organ-bath before the treatment and at the end of each experiment; and neurotoxicity was evaluated in chick biventer cervicis using the conventional myographic technique, face to the exogenous acetylcholine (ACh) and potassium chloride (KCl) added into the bath before the treatment and after each experiment. Preliminarily, a concentration-response curve of AgNPs was carried out to select the concentration to be used for neutralizing assays, which consists of neutralizing the venom-induced neuromuscular paralysis and edema by preincubating AgNPs with venom for 30 min. RESULTS: The P. olfersii venom-induced edema (n=6) and a complete neuromuscular blockade (n=4) that includes the total and unrecovered block of ACh and KCl contractures. AgNPs produced a concentration-dependent decrease the venom-induced edema (n=6) from 223.3% to 134.4% and to 100.5% after 10 and 100 µL AgNPs-preincubation, respectively. The preincubation of venom with AgNPs (1 µL; n=6) was able to maintain 46.5 ± 10.9% of neuromuscular response under indirect stimuli, 39.2 ± 9.7% of extrinsic nicotinic receptors functioning in absence of electrical stimulus and 28.3 ± 8.1% of responsiveness to potassium on the sarcolemmal membrane. The CK release was not affected by any experimental protocol which was like control. CONCLUSION: AgNPs interact with constituents of P. olfersii venom responsible for the edema-forming activity and neuromuscular blockade, but not on the sarcolemma membrane-acting constituents. The protective effect of the studied AgNPs on avian preparation points out to molecular targets as intrinsic and extrinsic nicotinic receptors.

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.

New pharmacological insights of Galactia glaucescens.

Galactia glaucescens leaves are popularly used against snakebites in Brazil. The hydroethanolic extract from aerial parts of G. glaucescens (HEGg) was assayed against the neurotoxicity and myotoxicity induced by Bothrops jararacussu venom. A traditional myographic technique was applied for neurotoxicity and the resulting muscles were treated routinely by light microscopy analysis for myotoxicity. Additionally, the antimicrobial potential of HEGg was evaluated against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains, as well as Rutin was isolated for the first time in this specie using chromatographic and spectroscopic methods and its antiophidian property was assessed. HEGg totally prevents the neurotoxicity and myotoxicity effects caused by B. jararacussu, but did not show any antimicrobial effect. Concluding, HEGg and Rutin were able to counteract the toxic effects of the venom and confirmed the antiophidian potential, but not antimicrobial, of G. glaucescens as an alternative for neutralization of B. jararacussu venom.

Neutralising ability of Terminalia fagifolia extract (Combretaceae) against the in vitro neuromuscular effects of Bothrops jararacussu venom.

The ability of Terminalia fagifolia hydroalcoholic extract (Tf-HE) to neutralise the paralysis and myotoxicity induced by Bothrops jararacussu venom was assayed using mouse phrenic nerve-diaphragm (PND) preparation and two varieties of chick biventer cervicis (BC) preparations. Tf-HE 100 µg/mL and 500 µg/mL were tested against 40 and 200 µg of venom/mL in PND and BC preparations, respectively, using pre- and post-venom incubation treatments. The effects of Tf-HE against the myotoxicity caused by venom were evaluated via histological analysis (PND) and creatine kinase (CK) release (BC). Tf-HE was able to reverse the venom paralysis in both preparation types. The contractures to exogenous ACh in BC preparations showed that Tf-HE may act on extrinsic, preserving those intrinsic postsynaptic receptors. There was a positive correlation between CK and morphological changes. The high non-hemolytic saponin content can explain the Tf-HE efficacy against the toxic effects of B. jararacussu venom in vertebrate neuromuscular preparations.

A Highly Polar Phytocomplex Involving Rutin is Responsible for the Neuromuscular Facilitation Caused by Casearia sylvestris (guaçatonga).

BACKGROUND: Of the various biological activities ascribed to extracts from Casearia sylvestris (guaçatonga), its facilitatory activity, i.e., ability to increase skeletal muscle contractile amplitude, has promising therapeutic applications. In this work, we investigated the components responsible for the previously described neurofacilitation caused by C. sylvestris leaves. METHODS: The methanolic fraction of C. sylvestris leaves was initially fractionated by column chromatography and partitioned in a MeOH:H2O gradient. The resulting fractions were analyzed by analytical HPLC and yielded fraction 5:5 (F55) that was subjected to solid phase extraction and preparative HPLC. Of the seven resulting subfractions, only F55-6 caused muscle facilitation. Subfractions F55-6 and F55-7 (similar in composition to F55-6 by TLC analysis, but inactive) were analyzed by 1H-NMR to identify their constituents. RESULTS: This analysis identified a rutin-glycoside phytocomplex that caused neurofacilitation, a property that commercial rutin alone did not exhibit. CONCLUSION: F55-6 apparently caused neurofacilitation by the same mechanism (presynaptic action) as the methanolic fraction since its activity was also inhibited in tetrodotoxin-pretreated preparations.

Evaluation of Betulin Mutagenicity by Salmonella/Microsome Test.

Purpose: Betulin is a pentacyclic triterpene found in the outer barks of innumerous plants. This secondary metabolite is easily isolated from plants with the major interest in converting it to betulinic acid, which pharmacological properties were much more exploited than betulin. But, investments in the own betulin have been grown since no chemical step is necessary. In this study we focused the precursor betulin in order to evaluate its mutagenicity by Salmonella/microsome assay (Ames test). Methods: The Ames test was carried out using a commercial betulin exposed to Salmonella typhimurium strains TA98, TA100, TA102, and TA97a, in experiments with (+S9) and without (-S9) metabolic activation. Results: Betulin was unable to increase the number of revertants (+S9 and -S9 metabolic activation) showing the absence of any mutagenic effect by Ames test. Conclusion: This study allowed attribute safety to betulin being important for exploiting its pharmacological uses.

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.

Safety assessment of the royal sun mushroom, Agaricus brasiliensis (higher Basidiomycetes) intake during rat pregnancy.

In this study, we investigated the reproductive capacity of pregnant rats exposed to daily orally administered powder-dehydrated reconstituted of Agaricus brasiliensis (=Agaricus blazei sensu Murrill), the fetal organogenesis, and the development of the pups. Pregnant rats were exposed for the entire gestational period to water (control) and A. brasiliensis at 300 or 600 mg/kg/day. Fertility and body weight of dams were monitored. Pups were monitored for body weight, offspring vitality, morphology, and physical and neurobehavioral development. An increase in sternebrae agenesis was observed at the 600 mg/kg/day dose of A. brasiliensis, while incomplete ossification of sternebrae was seen even at a 300 mg/kg/day dose. In conclusion, this study is the first to demonstrate the impact of maternal exposure to A. brasiliensis on the fetal organogenesis and development of offspring in a rat model. The 600 mg/kg/day dose showed some negative effects, and low toxicity was observed at the 300 mg/kg/day dose.

Vellozia flavicans Mart. ex Schult. hydroalcoholic extract inhibits the neuromuscular blockade induced by Bothrops jararacussu venom.

BACKGROUND: Snakebite is a significant public health issue in tropical countries. In Brazil, some of the most common snake envenomations are from Bothrops. Bothrops bites trigger local and systemic effects including edema, pain, erythema, cyanosis, infections, and necrosis. Vellozia flavicans is a plant from the Brazilian "cerrado" (savanna) that is popularly used as an anti-inflammatory medicine. Since inflammation develops quickly after Bothrops bites, which can lead to infection, the aim of the present study was to observe possible anti-snake venom and antimicrobial activities of V. flavicans (Vf). METHODS: The chromatographic profile of the main constituents from the Vf leaf hydroalcoholic extract was obtained by thin-layer chromatography (TLC). The anti-snake venom activity was measured by Vf's ability to neutralize the in vitro neuromuscular blockade caused by Bothrops jararacussu venom (Bjssu) in a mouse phrenic nerve-diaphragm model (PND). After a 20 min incubation, preparations of PND were added to Tyrode's solution (control); Vf (0.2, 0.5, 1, and 2 mg/mL); 40 µg/mL Bjssu; pre-incubation for 30 min with Bjssu and 1 mg/mL Vf; and a Bjssu pretreated preparation (for 10 min) followed by 1 mg/mL Vf. Myographic recording was performed, and the contractile responses were recorded. The antimicrobial activity (minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC]) was obtained for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, using gentamicin and vancomycin as positive controls. RESULTS: TLC analysis yielded several compounds from Vf, such as flavonoids (quercetin) and phenolic acids (chlorogenic acid). Bjssu completely blocked the contractile responses of PND preparations, while Vf preserved 97% (±10%) of the contractile responses when incubated with Bjssu. In the PND pretreated with Bjssu, Vf was able to inhibit the neuromuscular blockade progress. MIC and MBC of Vf ranged from 2.5 to 5.0 mg/mL for P. aeruginosa and S. aureus strains, while no antimicrobial activity was observed for E. coli and E. faecalis. CONCLUSIONS: The hydroalcoholic extract from Vf leaves was able to neutralize and decrease the in vitro neuromuscular blockade caused by Bjssu. However, it did not show significant antimicrobial activity against the tested bacteria.