Inhibition Of Indirect Twitches Research Articles

A Comparison of the Efficacy of Antivenoms and Varespladib against the In Vitro Pre-Synaptic Neurotoxicity of Thai and Javanese Russell's Viper (Daboia spp.) Venoms.

The heterogeneity in venom composition and potency in disparate Eastern Russell's viper (Daboia siamensis) populations has repercussions for the efficacy of antivenoms. This is particularly pronounced in geographical areas in which the venom of the local species has not been well studied and locally produced antivenoms are unavailable. In such cases, alternative therapies following envenoming, which are not limited by species specificity, may be employed to complement antivenoms. We studied the neuromuscular activity of D. siamensis venom from Thailand and Java (Indonesia) and the ability of Thai antivenoms and/or Varespladib to prevent or reverse these effects. Both Thai and Javanese D. siamensis venoms displayed potent pre-synaptic neurotoxicity but weak myotoxicity in the chick biventer cervicis nerve-muscle preparation. Whilst the neurotoxicity induced by both venoms was abolished by the prior administration of Thai D. siamensis monovalent antivenom or pre-incubation with Varespladib, Thai neuro-polyvalent antivenom only produced partial protection when added prior to venom. Pre-synaptic neurotoxicity was not reversed by the post-venom addition of either antivenom 30 or 60 min after either venom. Varespladib, when added 60 min after venom, prevented further inhibition of indirect twitches. However, the subsequent addition of additional concentrations of Varespladib did not result in further recovery from neurotoxicity. The combination of Thai monovalent antivenom and Varespladib, added 60 min after venom, resulted in additional recovery of twitches caused by either Thai or Javanese venoms compared with antivenom alone. In conclusion, we have shown that Varespladib can prevent and partially reverse the pre-synaptic neurotoxicity induced by either Thai or Javanese D. siamensis venoms. The efficacy of Thai D. siamensis monovalent antivenom in reversing pre-synaptic neurotoxicity was significantly enhanced by its co-administration with Varespladib. Further work is required to establish the efficacy of Varespladib as a primary or adjunct therapy in human envenoming.

Isolation and Characterization of Two Postsynaptic Neurotoxins From Indian Cobra (Naja Naja) Venom.

The Indian Cobra (Naja naja) is among the “Big Four” responsible for most of the snakebite envenoming cases in India. Although recent proteomic studies suggest the presence of postsynaptic neurotoxins in N. naja venom, little is known about the pharmacology of these toxins. We isolated and characterized α-Elapitoxin-Nn2a (α-EPTX-Nn2a; 7020 Da) and α-Elapitoxin-Nn3a (α-EPTX-Nn3a; 7807 Da), a short-chain and long-chain postsynaptic neurotoxin, respectively, which constitute 1 and 3% of N. naja venom. α-EPTX-Nn2a (100–300 nM) and α-EPTX-Nn3a (100–300 nM) both induced concentration-dependent inhibition of indirect twitches and abolished contractile responses of tissues to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior incubation of tissues with Indian polyvalent antivenom (1 ml/0.6 mg) prevented the in vitro neurotoxic effects of α-EPTX-Nn2a (100 nM) and α-EPTX-Nn3a (100 nM). The addition of Indian polyvalent antivenom (1 ml/0.6 mg), at the t90 time point, could not reverse the in vitro neurotoxicity of α-EPTX-Nn2a (100 nM). The in vitro neurotoxicity of α-EPTX-Nn3a (100 nM) was partially reversed by the addition of Indian polyvalent antivenom (1 ml/0.6 mg), as well as repeated washing of the tissue. α-EPTX-Nn2a displayed non-competitive antagonism of concentration-response curves to carbachol, with a pA2 of 8.01. In contrast, α-EPTX-Nn3a showed reversible antagonism of concentration-response curves to carbachol, with a pA2 of 8.17. De novo sequencing of α-EPTX-Nn2a and α-EPTX-Nn3a showed a short-chain and long-chain postsynaptic neurotoxin, respectively, with 62 and 71 amino acids. The important observation made in this study is that antivenom can reverse the neurotoxicity of the clinically important long-chain neurotoxin, but not the short-chain neurotoxin, from N. naja venom.

TheCardiovascularandNeurotoxicEffectsofthe VenomsofSixBonyandCartilaginousFishSpecies.

Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii and Himantura toshi, and the bony fish Platycephalus fucus, Girella tricuspidata, Mugil cephalus, and Dentex tumifrons. All venoms (10–100 µg/kg, i.v.), except G. tricuspidata and P. fuscus, induced a biphasic response on mean arterial pressure (MAP) in the anesthetised rat. P. fucus venom exhibited a hypotensive response, while venom from G. tricuspidata displayed a single depressor response. All venoms induced cardiovascular collapse at 200 µg/kg, i.v. The in vitro neurotoxic effects of venom were examined using the chick biventer cervicis nerve-muscle (CBCNM) preparation. N. kuhlii, H. toshi, and P. fucus venoms caused concentration-dependent inhibition of indirect twitches in the CBCNM preparation. These three venoms also inhibited responses to exogenous acetylcholine (ACh) and carbachol (CCh), but not potassium chloride (KCl), indicating a post-synaptic mode of action. Venom from G. tricuspidata, M. cephalus, and D. tumifrons had no significant effect on indirect twitches or agonist responses in the CBCNM. Our results demonstrate that envenoming by these species of fish may result in moderate cardiovascular and/or neurotoxic effects. Future studies aimed at identifying the molecules responsible for these effects could uncover potentially novel lead compounds for future pharmaceuticals, in addition to generating new knowledge about the evolutionary relationships between venomous animals.

Species differences in the neuromuscular activity of post-synaptic neurotoxins from two Australian black snakes (Pseudechis porphyriacus and Pseudechis colletti)

Bites by Australian black snakes (Pseudechis spp.) do not cause neurotoxicity in human envenoming. This is unusual as in vitro neurotoxicity has been reported for all Pseudechis spp. venoms. The present study aimed to identify, isolate and characterise neurotoxins from the venoms of Pseudechis porphyriacus and Pseudechis colletti to elucidate the reason for the lack of neurotoxicity in humans. α-Elapitoxin-Ppr1 and α-elapitoxin-Pc1 were isolated from P. porphyriacus and P. colletti, respectively, using reverse-phase high performance liquid chromatography. Each toxin consisted of 62 amino acids with molecular weights of 6746.5Da and 6759.6Da, respectively. α-Elapitoxin-Ppr1 and α-elapitoxin-Pc1 caused concentration-dependent (0.1–0.3μM) inhibition of indirect twitches in the chick biventer cervicis nerve-muscle preparation. Both toxins inhibited contractile responses to exogenous ACh and CCh, but not KCl, suggesting a post-synaptic mode of action at the nicotinic acetylcholine receptor (nAChR). CCh concentration–response curves obtained in the presence or absence of α-elapitoxin-Ppr1 or α-elapitoxin-Pc1 indicated pA2 values of 6.97±0.03 and 7.04±0.07, respectively. Neither α-elapitoxin-Ppr1 (0.1μM) nor α-elapitoxin-Pc1 (0.1μM) had a significant effect on the electrically-induced twitches of the rat isolated phrenic nerve-diaphragm preparation. When the venom with the toxin removed (10μg/ml) was added to both the rat and chick preparations, the inhibition was significantly less than that caused by the intact whole venoms (10μg/ml). The current study shows that α-elapitoxin-Ppr1 and α-elapitoxin-Pc1 act as pseudo-irreversible antagonists at the nAChR of the skeletal neuromuscular junction and that the avian preparation is more sensitive to the neurotoxic effects of these toxins than the mammalian preparation.

Neuromuscular activity of the venoms of the Colombian coral snakes Micrurus dissoleucus and Micrurus mipartitus: An evolutionary perspective

The venoms of coral snakes (genus Micrurus) are known to induce a broad spectrum of pharmacological activities. While some studies have investigated their potential human effects, little is known about their mechanism of action in terms of the ecological diversity and evolutionary relationships among the group. In the current study we investigated the neuromuscular blockade of the venom of two sister species Micrurus mipartitus and Micrurus dissoleucus, which exhibit divergent ecological characteristics in Colombia, by using the chick biventer cervicis nerve-muscle preparation. We also undertook a phylogenetic analysis of these species and their congeners, in order to provide an evolutionary framework for the American coral snakes. The venom of M. mipartitus caused a concentration-dependant inhibition (3–10 μg/ml) of nerve-mediated twitches and significantly inhibited contractile responses to exogenous ACh (1 mM), but not KCl (40 mM), indicating a postsynaptic mechanism of action. The inhibition of indirect twitches at the lower venom dose (3 μg/ml) showed to be triphasic and the effect was further attenuated when PLA2 was inhibited. M. dissoleucus venom (10–50 μg/ml) failed to produce a complete blockade of nerve-mediated twitches within a 3 h time period and significantly inhibited contractile responses to exogenous ACh (1 mM) and KCl (40 mM), indicating both postsynaptic and myotoxic mechanisms of action. Myotoxic activity was confirmed by morphological studies of the envenomed tissues. Our results demonstrate a hitherto unsuspected diversity of pharmacological actions in closely related species which exhibit divergent ecological characteristics; these results have important implications for both the clinical management of Coral snake envenomings and the design of Micrurus antivenom.