An Investigation of the Nature of the Neural and Local Mechanisms That Regulate Vascular Functioning

1. In the pithed rat, stimulation of the autonomic outflow at different levels between C1 and L3 produced characteristic increases in blood pressure and heart rate. Stimulation between C6 and T1 and between T2 and T4 produced large increases in heart rate with only small increases in blood pressure. Stimulation at T8-9 produced the largest increase in blood pressure and a large increase in heart rate. 2. Spinal nerve stimulation at T8-9 characteristically produced pressor responses during stimulation and a secondary, pressor response that persisted after stimulation ceased. These responses were frequency- dependent so that the response at 20 Hz (supramaximal voltage, 0.4 ms pulse-width, 100 pulses) was larger than that at 2 Hz (supramaximal voltage, 0.4 ms pulse-width, 100 pulses). 3. The pressor response to stimulation at T8-9 to 2 Hz and 20 Hz probably resulted from stimulation of nerve fibres to blood vessels and also to the adrenal glands, which contributed both to the component of the pressor response that occurred during stimulation and to the secondary component that persisted after stimulation ceased. This was indicated by experiments in which adrenalectomy reduced the first component of the pressor response that occurred during stimulation at T8-9 and abolished the second component that persisted after stimulation. 4. The effect of propranolol (2 mg kg-1) on both components of the pressor response to spinal nerve stimulation at T8-9 was complex and dependent on the basal blood pressure of the pithed rat. In most pithed rats, propranolol enhanced both components of the pressor response to nerve stimulation. This was most marked at 2 Hz and most obvious in the secondary poststimulation component, which probably resulted from adrenaline released from the adrenals. 5. In some experiments where the blood pressure was low (<18-22 mm Hg) propranolol did not enhance the pressor response to spinal nerve stimulation. This may have been because in these few rats propranolol impaired cardiac output and as a result the ability of the heart to pump blood through the constricted blood vessels diminished, so that despite nerve stimulation-induced peripheral vasoconstriction, blood pressure did not increase. 6. alpha,beta-methylene ATP (alpha,beta-mATP,2x0.05 and 5x0.1 mg kg-1) produced pressor responses that showed tachyphylaxis. There was no evidence that alpha,beta-mATP desensitized the pressor response to spinal nerve stimulation at T8-9.7. Captopril inhibited pressor responses to nerve stimulation and to exogenous NA (9 mug kg-1). This result suggests that angiotensin contributes to the pressor response to spinal nerve stimulation. An alternative explanation of this effect is that bradykinin breakdown might be inhibited by captopril so that more nitric oxide (NO), released by bradykinin, might be available to act as a physiological antagonist of NA. 8. There was no evidence of a prazosin-resistant pressor response to spinal nerve stimulation at T8-9 in the pithed rat. 9. L-NAME (45 mg kg-1) increased the basal blood pressure in the anaesthetized rat and also, but to a lesser extent, in the pithed rat in the presence of propranolol (2 mg kg-1). There was little difference in the extent to which L-NAME increased the basal blood pressure in the anaesthetized and the pithed rats in the absence of propranolol. In pithed rats but not in anaesthetised rats, the increment in the basal blood pressure caused by L-NAME was proportional to the level of the basal blood pressure. It appears that endogenous NO was continuously produced in both anaesthetized and pithed rats and had a role in keeping the basal blood pressure low. The greater increase in the basal blood pressure produced by 1 L-NAME (45 mg kg-1) in anaesthetized rats was probably due to the withdrawal of the inhibitory effect of NO on the sympathetic tone, which was thereafter able to operate unopposed and raise the blood pressure. Such a mechanism would not occur in the pithed rat. (Abstract shortened by ProQuest.)

A comparison of in vitro methods for assessing the potency of therapeutic antisera against the venom of the coral snake Micrurus nigrocinctus

Therapeutic antisera against Micrurus nigrocinctus venom were tested for protection against lethality, as well as for inhibition of the nicotinic acetylcholine receptor (AchR)-binding and neutralization of phospholipase A2 (PLA2) activities of the homologous venom. Protection against venom lethality did not correlate with inhibition of AchR-binding activity, whereas there was a significant correlation between antisera potency and inhibition of PLA2 activity (r = 0.82, n = 10, P < 0.02). Inhibition of PLA2 activity could be useful in assessing the protective efficacy of M. nigrocinctus antisera during antivenom production. Micrurus nigrocinctus nigrocinctus venom proteins were fractionated by cation-exchange chromatography on Mono S FPLC and fractions assayed for lethality, AchR-binding and PLA2 activities. Antisera were titrated by enzyme-linked immunoassay (ELISA) against a crude M. n. nigrocinctus venom, two FPLC lethal fractions containing AchR-binding activity, and two toxins purified from M. n. nigrocinctus venom. No correlation was found between protective efficacy and the ELISA titer against any of these antigens. Compared to other elapid venoms that contain few toxins as major components, M. n. nigrocinctus venom appears to be more complex and its lethal effect is likely to be due to the combined effect of several neurotoxins.Universidad de Costa Rica/[74l-93-301]/UCR/Costa RicaInternational Foundation for Science/[F/0883-4]/IFS/SueciaSwedish Agency for Research Cooperation with Developing Countries//SAREC/SueciaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Antivenoms for the treatment of snakebite envenomings: The road ahead

The parenteral administration of antivenoms is the cornerstone of snakebite envenoming therapy. Efforts are made to ensure that antivenoms of adequate efficacy and safety are available world-wide. We address the main issues to be considered for the development and manufacture of improved antivenoms. Those include: (a) A knowledge-based composition design of venom mixtures used for immunization, based on biochemical, immunological, toxicological, taxonomic, clinical and epidemiological data; (b) a careful selection and adequate management of animals used for immunization; (c) well-designed immunization protocols; (d) sound innovations in plasma fractionation protocols to improve recovery, tolerability and stability of antivenoms; (e) the use of recombinant toxins as immunogens to generate antivenoms and the synthesis of engineered antibodies to substitute for animal-derived antivenoms; (f) scientific studies of the contribution of existing manufacturing steps to the inactivation or removal of viruses and other zoonotic pathogens; (g) the introduction of novel quality control tests; (h) the development of in vitro assays in substitution of in vivo tests to assess antivenom potency; and (i) scientifically-sound pre-clinical and clinical assessments of antivenoms. These tasks demand cooperative efforts at all main stages of antivenom development and production, and need concerted international partnerships between key stakeholders.Universidad de Costa Rica//UCR/Costa RicaInternational Foundation for Science//IFS/SueciaCiencia y Tecnología para el Desarrollo//CYTED/EspañaConsejo Superior de Investigaciones Científicas//CRUSA-CSIC/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

The use of the chick biventer cervicis preparation to assess the protective activity of six international reference antivenoms on the neuromuscular effects of snake venoms in vitro

The protective activity of some antivenoms on the neuromuscular activity in vitro of six snake venoms was established in order to test the feasibility of using a simple isolated nerve-muscle preparation to compare different antivenoms. Six venoms designated as International Reference Venoms by the World Health Organization (WHO) were used: Echis carinatus (Iran), Echis carinatus (Mali), Naja naja kaouthia, Notechis scutatus, Trimeresurus flavoviridis, and Vipera russelli (Thailand). The chick biventer cervicis nerve-muscle preparation was used to detect neurotoxic and myotoxic effects. The ratio of the amount of antivenom needed to neutralize a dose of venom (w/w) was calculated in order to quantify the potencies of the antivenoms. The antivenom to venom ratios were 1000 for Echis carinatus (Mali) and for Notechis scutatus; 100 for Naja naja kaouthia and Vipera russelli; and 10 for Echis carinatus (Iran) and Trimeresurus falvoviridis. It is concluded that in vitro assays using the chick biventer cervicis preparation could be used to compare the relative potencies of different antivenoms at neutralizing myotoxic and neurotoxic toxins in snake venoms

Screening of snake venoms for neurotoxic and myotoxic effects using simple in vitro preparations from rodents and chicks

Eight snake venoms designated by the WHO as International Reference Venoms, and one additional venom were assessed for neurotoxic and myotoxic effects in vitro using the chick biventer cervicis and the rat and mouse phrenic nerve-diaphragm preparations. The objective was to determine whether any of the preparations could be used to detect evidence of neurotoxic or myotoxic activity prior to a more detailed examination. Bungarus multicinctus venom at concentrations above 1 microgram ml-1 selectively blocked neuromuscular transmission, with no direct effect on muscle fibres. Naja naja kaouthia and Notechis scutatus venoms selectively blocked neuromuscular transmission at low concentrations, but at higher concentrations both venoms caused direct effects on skeletal muscle resulting in contractures, loss of tension following direct stimulation and a loss in sensitivity to elevated [K+]0. Vipera russelli (Thailand) venom also blocked neuromuscular transmission but it was less potent than the venoms of B. multicinctus, N. n. kaouthia and N. scutatus. It also caused contractures in the chick biventer cervicis muscle. The venoms of Echis carinatus (Iran and Mali), Crotalus atrox, Bothrops atrox asper and Trimeresurus flavoviridis had limited neuromuscular blocking activity, and most of these venoms blocked [K+]0 and cholinoceptor stimulation in the chick muscle. Although both chick and rodent muscles allowed the assessment of neurotoxic and myotoxic activity, the chick biventer cervicis was simpler and more robust in use than either of the rodent phrenic nerve-diaphragm preparations. We propose that the chick biventer cervicis muscle could be used as a standard preparation for the screening of snake venoms for neurotoxic and myotoxic effects, and that it may be possible to use this preparation as a means to check that antivenoms can neutralize neurotoxic and direct myotoxic actions of venoms

Species and regional variations in the effectiveness of antivenom against the in Vitro neurotoxicity of death adder (Acanthophis) venoms

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 mug/ml), as well as the effectiveness of antivenom. (1-5 units/ml; 10 min prior to the addition of 10 mug/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 m-M) and carbachol (20 muM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Scram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied. (C) 2001 Academic Press

Isolation and pharmacological characterization of a phospholipase A(2) myotoxin from the venom of the Irian Jayan death adder (Acanthophis rugosus)

1. It has long been thought that death adder venoms are devoid of myotoxic activity based on studies done on Acanthophis antarcticus (Common death adder) venom. However, a recent clinical study reported rhabdomyolysis in patients following death adder envenomations, in Papua New Guinea, by a species thought to be different to A. antarcticus. Consequently, the present study examined A. rugosus (Irian Jayan death adder) venom for myotoxicity, and isolated the first myotoxin (acanmyotoxin-1) from a death adder venom. 2. A. rugosus (10–50 μg ml(−1)) and acanmyotoxin-1 (MW 13811; 0.1–1 μM) were screened for myotoxicity using the chick directly (0.1 Hz, 2 ms, supramaximal V) stimulated biventer cervicis nerve-muscle (CBCNM) preparation. A significant contracture of skeletal muscle and/or inhibition of direct twitches were considered signs of myotoxicity. This was confirmed by histological examination. 3. High phospholipase A(2) (PLA(2)) activity was detected in both A. rugosus venom (140.2±10.4 μmol min(−1) mg(−1); n=6) and acanmyotoxin-1 (153.4±11 μmol min(−1) mg(−1); n=6). Both A. rugosus venom (10–50 μg ml(−1)) and acanmyotoxin-1 (0.1–1 μM) caused dose-dependent inhibition of direct twitches and increase in baseline tension (n=4–6). In addition, dose-dependent morphological changes in skeletal muscle were observed. 4. Prior incubation (10 min) of CSL death adder antivenom (5 units ml(−1); n=4) or inactivation of PLA(2) activity with 4-bromophenacyl bromide (1.8 mM; n=4) prevented the myotoxicity caused by acanmyotoxin-1 (1 μM). 5. Acanmyotoxin-1 (0.1 μM; n=4) displayed no significant neurotoxicity when it was examined using the indirectly (0.1 Hz, 0.2 ms, supramaximal V) stimulated CBCNM preparation. 6. In conclusion, clinicians may need to be mindful of possible myotoxicity following death adder envenomation in Irian Jaya