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.

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.

Early histopathological changes in bone marrow of Paracoccidioides brasiliensis-infected mice

The involvement of bone marrow in the pathology of experimental P. brasiliensis infection in BALB/c mice was investigated. The histopathological features of bone marrow induced by the fungus were correlated with hematological changes in peripheral blood from 1 to 28 days post-infection. Intense lymphopenia and moderate neutrophilia were detected. The early changes in bone marrow included (i) maturation arrest characterized by an increase immature blood cell precursors, mainly of granulocytic origin, (ii) intense vascular congestion when compared with the vessels of normal marrow, and (iii) an increased number of megakaryocytes. The normal histological pattern of bone marrow was restored by 28 days post-infection. No histologically recognizable lesion, such as granuloma formation or an abnormal cellular infiltrate, which could indicate the presence of the P. brasiliensis in bone marrow, was observed. In addition, special stains were unable to detect the fungus. The mechanisms responsible for the alterations described here are still unclear but are probably related to more general phenomena affecting the host rather than the direct damage of the precursors cells by P. brasiliensis.

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.

Influence of protein malnutrition on histochemically-delected compounds in the liver of rats

Weanling (21-day-old) Norvegicus Wistar male rats were submitted to protein malnutrition for 120 days, and after this period the livers were studied with morphohistochemical techniques. When compared to controls, protein-deprived rats showed a wide cytoplasmic vacuolization of hepatocytes due to fatty degeneration. They also had lower rates of glucose-6-phosphatase activity, resulting in higher glycogen stores. There also minor changes in elastic and reticular fibrils. Other histochemically-delected compounds, such as glycosaminoglycans, reducing radicals and iron stores have not apparently changed, despite impaired protein intake. Morphohistochemical changes exhibited a zonal lobular heterogeneity. The results suggest that protein malnutrition can alter rat liver structure and function by affecting carbohydrate, protein and lipid metabolism. This study also highlights the hepatic lobular metabolic heterogeneity and its modulation when submitted to adverse conditions.