Ultrastructural aspects of mouse nerve-muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX-I and Bbil-TX: Unknown myotoxic effects.

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve-muscle preparations exposed to Bothrops venoms and their phospholipase A2 toxins (PLA2 ) can exhibit a neurotoxic pattern as increase in frequency of miniature end-plate potentials (MEPPs) as well as in amplitude of end-plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA2 toxins (BthTX-I and Bbil-TX) in mouse isolated nerve-phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX-I and Bbil-TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic-like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu.

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes.

Phrenic nerve diabetic neuropathy in rats: unmyelinated fibers morphometry.

We have demonstrated that phrenic nerves' large myelinated fibers in streptozotocin (STZ)-induced diabetic rats show axonal atrophy, which is reversed by insulin treatment. However, studies on structural abnormalities of the small myelinated and the unmyelinated fibers in the STZ-model of neuropathy are limited. Also, structural changes in the endoneural vasculature are not clearly described in this model and require detailed study. We have undertaken morphometric studies of the phrenic nerve in insulin-treated and untreated STZ-diabetic rats and non-diabetic control animals over a 12-week period. The presence of neuropathy was assessed by means of transmission electron microscopy, and morphometry of the unmyelinated fibers was performed. The most striking finding was the morphological evidence of small myelinated fiber neuropathy due to the STZ injection, which was not protected or reversed by conventional insulin treatment. This neuropathy was clearly associated with severe damage of the endoneural vessels present on both STZ groups, besides the insulin treatment. The STZ-diabetes model is widely used to investigate experimental diabetic neuropathies, but few studies have performed a detailed assessment of either unmyelinated fibers or capillary morphology in this animal model. The present study adds useful information for further investigations on the ultrastructural basis of nerve function in diabetes.

Ultrastructural morphology and morphometry of phrenic nerve in rats.

Despite numerous literature reports on the morphometry of the myelinated fibers of phrenic nerves in rats, a systematic study of the longitudinal and lateral symmetry of the unmyelinated fibers morphometry is not available. In this study, we have undertaken ultrastructural and morphometric studies of the phrenic nerve in adult rats, assessing two different levels (proximal and distal) from both right and left sides. Phrenic nerves of adult male Wistar rats were prepared for epoxy resin embedding and transmission electron microscopy. Morphometric analysis was performed with the aid of computer software, which took into consideration the unmyelinated fiber number, density, area, and diameter, as well as ratio between myelinated and unmyelinated fibers, and the percentage of the fascicular area occupied by the myelinated and unmyelinated fibers. Comparison of data from proximal and distal segments on the same side and from the same levels between sides was performed. Differences were considered significant when P < 0.05. The most important finding is that morphometric parameters of the phrenic nerve unmyelinated fibers in adult rats are both longitudinally and laterally symmetric. This study adds important morphometric information about the unmyelinated fibers of the phrenic nerves in adult rats for proximal and distal levels on both sides of the animal.

Toxin 2 (PhTx2), a neurotoxic fraction from Phoneutria nigriventer spider venom, causes acute morphological changes in mouse skeletal muscle.

Phoneutria nigriventer (Labidognatha, Ctenidae) is a spider found in the warm regions of South America. Bites by this species cause intense local pain, autonomic dysfunction and paralysis. PhTx2, a neurotoxic fraction of the venom of this species, interferes with the physiology of sodium channel function. The present study describes the morphological changes in mouse phrenic nerve and diaphragm muscle after 15, 30, 45 and 60 min of incubation with 1 microg of PhTx2/ml. Light and transmission electron microscopy showed that PhTx2 caused progressive myonecrosis which involved swelling of the sarcoplasmic reticulum, mitochondrial damage, disorganization of the sarcomeres, zones of hypercontracted myofibrils and rupture of the plasma membrane. The intramuscular fascicles of the phrenic nerve showed vacuolated myelinated axons and Schwann cells. The neuromuscular junctions had vesicle-depleted nerve terminals with swollen mitochondria. The axolema was frequently invaginated and sequestered portions of the axoplasm, or was sometimes interrupted at the site of the synaptic gutter. The post-synaptic junctional folds were shallow and disperse. These morphological alterations in the muscle and nerve fibres were similar to those caused by osmotic disturbances and agree with the ability of PhTx2 to increase the permeability of sodium channels. An increase in sodium influx would probably be accompanied by an influx of water and an elevation in the concentration of cytosolic calcium as a result of calcium release by the sarcoplasmic reticulum and/or mitochondria and the entry of extracellular calcium. The morphological effects caused by PhTx2 were comparable to those seen with Phoneutria nigriventer whole venom which is known to activate and to delay the inactivation of sodium channels. We conclude that PhTx2 is probably the main toxic fraction responsible for such morphological alterations.

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 micrograms/ml) depolarization in the isolated mouse phrenic nerve-diaphragm preparations incubated at 37 degrees C. d-Tubocurarine (10 micrograms/ml) and (alpha beta ungarotoxin (3-5 micrograms/ml) were able to partially protect against the depolarization induced by the venom (10 micrograms/ml), suggesting the involvement of subsynaptic cholinergic receptors. This venom (10 micrograms/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" terminals with omega-shaped axolemmal indentations and a decreased number of synaptic vesicles 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 degrees C) 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.

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. (AU)

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.