Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2011Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood. Methodology/Principal Findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A2 (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration. Conclusions/Significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.This study was supported by NeTropica (grant 2-N-2008), by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A7-604). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Nature of the postsynaptic action of crotoxin at guinea-pig diaphragm end-plates

Crotoxin is known to desensitize the nicotinic receptor of Torpedo marmorata and Electrophorus electricus electroplaques. The aim of the present study was to elucidate whether the postsynaptic effect of crotoxin at a mammalian muscle end-plate is also caused by receptor desensitization or results from a curaremimetic action. For this purpose, we investigated the action of 4-aminopyridine (4-AP) on crotoxin-induced blockade of miniature end-plate potentials (m.e.p.p.s) and of the depolarization of end-plates produced by carbachol. The experiments were carried out in guinea-pig diaphragms bathed in Tyrode solution at 37 degrees C and gassed with 95% O-2, 5% CO2. The potentials were measured with conventional techniques using glass microelectrodes. Even at low concentrations, crotoxin blocked the m.e.p.p.s and this blockade was antagonized by 4-AP. Neostigmine was without effect. 4-AP did not restore the m.e.p.p.s blocked by either d-tubocurarine (dTc) or beta-bungarotoxin (beta-BTX). 4-AP also antagonized the crotoxin-induced blockade of the end-plate depolarization produced by carbachol. These results show that the postsynaptic effect of crotoxin at the guinea-pig muscle end-plate also results from nicotinic receptor desensitization.91334

MODE OF ACTION OF THE CORAL SNAKE MICRURUS-SPIXII VENOM AT THE NEUROMUSCULAR-JUNCTION

The venom of Micrurus spixii, a coral snake dispersed throughout the Amazon Valley induces neuromuscular blockade in the rat phrenic nerve-diaphragm preparation and in the chick biventer cervicis nerve-muscle preparation. In both tissues, the venom does not depress the twitch response elicited by direct muscle stimulation. The depolarization produced by carbachol in the rat diaphragm and the contractures induced by acetylcholine (ACh) in the denervated rat hemidiaphragm and in the chick biventer cervicis muscle are inhibited by low venom concentrations. The frequency and, in several experiments, the amplitude of the miniature end-plate potentials (m.e.p.p.s) are increased by the venom. The m.e.p.p. amplitude always decreases before their disappearance. 3,4-Diaminopyridine (3,4-DAP) antagonizes the effect of the venom on neuromuscular transmission in the rat phrenic nerve-diaphragm and on the depolarization produced by carbachol in this preparation. It also antagonizes the venom-induced blockade of the m.e.p.p.s and increases the end-plate potential (e.p.p.) after the blockade produced by the venom in the rat phrenic nerve-diaphragm. The 3,4-DAP antagonistic effect on the postsynaptic action of the venom suggests that desensitization of the end-plate receptors is induced by the venom and is the main cause of the neuromuscular blockade that it produces.41193

EFFECTS INDUCED BY BOTHROPSTOXIN, A COMPONENT FROM BOTHROPS-JARARACUSSU SNAKE-VENOM, ON MOUSE AND CHICK MUSCLE PREPARATIONS

Bothropstoxin, a 13,700 mol.wt myotoxic phospholipase homologue isolated from the venom of Bothrops jararacussu and devoid of PLA2, proteolytic or hemolytic activities, inhibited muscle twitch tension, evoked either directly or indirectly through stimulation of the motor nerve in the mouse phrenic-diaphragm preparations. The compound action potential of the muscle was also abolished with a similar time course. In addition, the toxin (0.7 mM) evoked membrane depolarization which was inhibited in the presence of 10 mM Ca2+. In chick biventer cervicis muscle, the toxin (2 mM) induced a contracture that reached its maximum amplitude in 44.8 +/- 15.6 min (n = 6) and was not blocked by either d-tubocurarine or tetrodotoxin. The time to maximum amplitude was reduced to 5.5 +/- 1.0 min (n = 4) in nominally Ca2+-free Krebs solution and was completely abolished in Ca2+-free Krebs solution containing 1 mM EGTA.30101203121

Actions of Androctonus australis and Leiurus quinquestriatus venoms in the rat isolated atria and anesthetized rats; effect of magnesium and lidocaine

In rat isolated atria, Androctonus australis (Aa), Leiurus quinquestriatus quinquestriatus (Lqq), and L.q. hebraceus (Lqh) venoms produced intense contracture, alterations in the force and frequency of the spontaneous atrial contractions, and delayed afterdepolarizations (DAD). It was shown by means of tetrodotoxin-induced blockade of neurotransmitter release that the contracture and DAD were produced by the action of the venoms on the atrial cell membrane (direct action) while alterations in the force and frequency of the spontaneous atrial contractions were caused by acetylcholine and norepinephrine released by the venoms (indirect action). The irregularities in the spontaneous contractions and DAD were suppressed by magnesium and lidocaine which, however, caused only a small reduction in the intensity of the atrial contracture. The venom-induced DAD was also abolished by ryanodine and intensified by an increase in [Ca2+](0). In anesthetized rats, Aa, Lqq, and Lqh venoms induced hypertension, arrhythmias, and T wave inversion. The arrhythmias included bradycardia, ventricular and supraventricular extrasystoles, unsustained and sustained ventricular tachycardia with torsade de pointes episodes. Magnesium and lidocaine abolished them, and magnesium also counteracted the hypertension. These results suggest that magnesium and lidocaine, particularly magnesium, may be useful in the treatment of the arrhythmias, hypertension, and other disorders associated with Buthinae scorpion envenomation.11111

Novel effects of guanidine on the neuromuscular junction

1. The effects of guanidine on the isolated mouse phrenic nerve diaphragm(MPND) and chick biventer cervicis (CBC) neuromuscular preparations were determined by myographic and electrophysiological methods. 2. Guanidine at concentrations of 5-10 mM induced an initial facilitation followed by neuromuscular blockade in both preparations. In the isolated MPND such blockade was associated with the abolition of miniature end-plate potentials (MEPPs), but in the CBC the acetylcholine-induced contracture remained unimpaired. After guanidine removal, a heretofore undescribed pronounced facilitation of neuromuscular transmission associated with an increase in MEPP frequency was observed. Simultaneously, the muscular contractions exhibited delayed relaxation and aftercontractions. 3. The K+ channel opener, cromakalim (100-200 mu M) inhibited both the well-described initial and the novel postremoval facilitatory effects of guanidine in a concentration-dependent manner. These findings are consistent with the proposal that guanidine blocks K+ channels in motor nerve endings. 4. The guanidine-induced NMB was reverted by increasing the Ca2+ concentration (1.8-5 mM) in the nutritive solution. 5. Tetrodotoxin (TTX, 1.56 mu M) did not influence the increase in MEPPS frequency induced by guanidine (10 mM) but did reduce the rise in MEPPS frequency observed after guanidine removal. 6. The present findings indicate that the effects of guanidine on the neuromuscular junction are more complex than currently described because they include a neuromuscular blockade and a postremoval facilitation previously unreported in the literature. (C) 1997 Elsevier Science Inc.28459960