Direct vs. mediated effects of scorpion venom: an experimental study of the effects of a second challenge with scorpion venom
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To assess the respective roles of venom and of catecholamines following scorpion envenomation and to verify whether a second challenge with scorpion venom induces the same consequences than a first one.
Design and setting
Controlled animal study in a university research laboratory.
Anesthetized and ventilated dogs.
Fifteen dogs received intravenously a sublethal dose of scorpion venom (0.05 mg/kg). In the reenvenomated group (n=5) a second venom challenge with one-half sublethal venom dose was performed 30 min after the first one. The control group (n=10) received saline. Five additional animals served as sham.
Measurements and results
Plasma toxin and catecholamine levels and a set of usual hemodynamic measurements were repeatedly measured in the first hour following envenomation. In the reenvenomated group another set of measurements was performed 5 min after the second challenge. Changes in toxin, catecholamines, and the main hemodynamic parameters were compared between the study groups. Initial peak toxin levels were similar in the two groups. They induced a striking increase in circulating catecholamines, a fall in heart rate, and an increase in mean arterial and pulmonary artery occluded pressures and in systemic vascular resistance. In the reenvenomated group the second challenge with scorpion venom achieved a toxin blood level similar to the first peak. However, it was not associated with a significant effect either on catecholamines release or on hemodynamics. Subsequent trends in hemodynamic changes were similar to those observed in the control group.
These data emphasize the limited role of direct effects of scorpion venom on the cardiovascular system and the key role of catecholamines.
KeywordsScorpion envenomation Hemodynamics Dog
Scorpion envenomation has a high attendant morbidity and mortality due mostly to acute heart failure presenting either as pulmonary edema or cardiogenic shock [1, 2]. Shortly following scorpion envenomation a catecholamine outpouring seems to account for its main cardiovascular features (increase in vascular resistance, hypertension, cardiac dysfunction, and pulmonary edema) [1, 2, 3, 4]. In addition to the role of catecholamines, a direct role of scorpion toxins on the cardiovascular system has been repeatedly reported, although this issue has recently been seriously questioned [5, 6, 7, 8, 9, 10]. However, this represents one of the most challenging issues in scorpion envenomation since it has major therapeutic implications. Indeed, the demonstration of a direct role of scorpion toxins on the cardiovascular system would provide substantial grounds to the use of scorpion antivenom to neutralize circulating levels of venom at any moment in the course of scorpion envenomation. Moreover, despite its short elimination half-life scorpion venom has been detected in blood several hours after envenomation in some circumstances . On the other hand, there is compelling evidence suggesting that scorpion venom does no more than the ignition of a myriad of circulating mediators with potent cardiovascular action, which in turn elicit the main cardiorespiratory consequences of severe scorpion envenomation. In addition to catecholamines, many other mediators have been shown to be involved, including neuropeptide Y, endothelin, and cytokines [3, 8, 12, 13].
To further delineate the respective role of direct and mediated effects of scorpion venom we conducted an experimental study to determine whether a second challenge with scorpion venom administered 30 min after the first one induces similar cardiovascular effects. We hypothesized that since the physiological response to prolonged or repeated sympathetic stimulation is reduced because of exhaustion of the catecholamines stores, a second challenge with scorpion venom would not be associated with a substantial release of catecholamines, and that any further alteration in cardiovascular status would therefore be due to the direct effects of scorpion toxins.
Materials and methods
All conditions of animal anesthesia, catheterization, and killing conformed with the international guidelines and were approved by the local institutional review board on clinical and animal research.
Twenty mongrel dogs weighing 13.8±3 kg were perfused with saline throughout the study period. Anesthesia was performed with pentobarbital (10 mg/kg for the induction, renewed as required thereafter). Animals were intubated and mechanically ventilated (tidal volume=10–15 ml/kg, frequency=20 cycles/min, FIO2=50% and adjusted as mandated by blood gazes measurements). Right heart catheterization by a Swan-Ganz 5F-catheter was then carried out by denuding the femoral vein, and the pulmonary artery catheter was positioned through the check of vessels pressures. An arterial catheter was introduced in the femoral artery.
The pressures in the systemic artery, pulmonary artery, occluded pulmonary artery, and the right atrium were recorded. Cardiac output measurements were performed by the thermodilution technique. The usually derived parameters were calculated: cardiac output adjusted to animal weight, stroke volume (ml/kg), and systemic and pulmonary vascular resistance. Blood samples were also withdrawn for the dose of catecholamines and of scorpion toxin Aah-1.
Three groups of animals were studied in this experiment. The first group (n=5) served as sham preparation. The second group (n=10; control) had one challenge with scorpion venom allowing a characterization of the main hemodynamic and neurohormonal consequences of scorpion envenomation. In the third group (n=5; reenvenomation) two injections with scorpion venom were made, the first after baseline values has been obtained and a second challenge after 30 min. In envenomated animals a first dose of 0.05 mg/kg body weight of the purified venom toxic fraction (G50 fraction of the scorpion Androctonus australis hector) was injected by the forearm vein after verification of the preparation stability (systemic artery pressure and heart rate variation of less than 10% for at least 10 min). The above hemodynamic parameters were measured at baseline (prior to the venom injection) and at 5, 30, and 60 min following venom injection. In the reenvenomation group (n=5) a second injection of a one-half dose venom (0.025 mg/kg) was injected after 30 min. This period was chosen because catecholamine stores were thought to be exhausted following the first venom challenge. In addition to the above points of time, another hemodynamic set of measurements was made after 35 min in the reenvenomation group (5 min after the second venom challenge). Blood samples (for the dose of catecholamines and scorpion toxin Aah-1) were withdrawn at each of these points of time and at 15 min.
All blood samples were collected and centrifuged at 4°C. Plasma was stored at −20°C until analysis of plasma catecholamines were quantified by radioenzymatic assay (CAT-A-KITTM assay system; TRK895, Amersham; detection limit 20 pg/ml). The scorpion toxin Aah-1 levels were measured by a recombinant scFv/streptaridum-binding peptides fusion, protein method (detection limit: 0.3 ng/ml) . Collected data in the control group were previously published in a study evaluating the effects of scorpion antivenom administered at various doses and at different times of experimental evenomation .
Data are presented as medians with interquartile range. Intragroup comparisons were made with Friedman’s test while between groups comparisons for each point of time used the Kruskall-Wallis tests. Comparison between the hemodynamic effects of the two venom challenges in the reenvenomated group was made with the Wilcoxon test. Differences at the level of p<0.05 were considered statistically significant.
Scorpion toxin levels in blood
Hemodynamic and neurohormonal changes following scorpion venom administration
change in hemodynamic parameters after the first and the second venom challenge. No statistical difference was observed in comparisons between control and reenvenomation groups (HR heart rate, MAP mean arterial pressure, SV stroke volume, PAOP pressure of occluded pulmonary artery)
SVR (dyne s−1 m−5)
Effects of the second challenge with scorpion venom
Comparison in the reenvenomated group of absolute variations following initial and second venom challenges (HR heart rate, MAP mean arterial pressure, SV stroke volume, PAOP pressure of occluded pulmonary artery)
Variation 5 min after initial envenomation
Variation 5 min after reenvenomation
Despite similar toxin levels achieved in blood by a first and a second challenge with scorpion venom we did not observe similar pathophysiological effects. Indeed, the catecholamine storm and the major hemodynamic changes usually associated with experimental scorpion envenomation (increases in vascular resistance in left ventricular filling pressure and in systemic pressure and a decrease in heart rate) were not observed following the second venom challenge. Obviously our findings cannot firmly rule out a direct toxic effect on cardiovascular and neurohormonal effects of envenomation; however, they do emphasize the major role of catecholamine blood levels.
Two possible explanations may explain our findings. The first is that scorpion toxin does not account by itself for the observed cardiovascular impairment following envenomation, acting rather through the release of mediators. The second is that following scorpion envenomation there is a period during which no further impairment in hemodynamic status is induced (refractory period). We hypothesize that this refractory period concerns either the stimulation of the sympathetic system (saturation of venom receptors) or that of adrenergic receptors (a phenomenon of sympathetic receptors’ saturation or downregulation). The lack of catecholamine increase following the second venom challenge lessens the putative role of the sympathetic receptors’ downregulation and emphasizes the major role of the lack of catecholamine release underlying the lack of a protracted hemodynamic impairment. The lack of a secondary increase in circulating catecholamines observed in our study may be due to saturation of scorpion venom receptors achieving a persistent maximum effect. It may also be related to the fact that the physiological response to prolonged or repeated sympathetic stimulation is reduced because of an exhaustion of the catecholamines stores . Hence all assumptions converge toward the key role of catecholamines in the occurrence of hemodynamic consequences of severe scorpion envenomation. Although speculative, opinions differ regarding the mechanisms underlying catecholamine release following scorpion envenomation. Peripheral sympathetic stimulation, central spinal and sympathetic preganglionic stimulation, hypothalamic stimulation, and adrenal medullary secretory effect, alone or in combination, have been advocated .
In experimental models scorpion venom has been shown to achieve significant levels of concentration in many tissues of which the heart, lungs, and kidneys, suggesting a direct action of scorpion venom on these tissues . Scorpion venom is indeed a potent activator of the sodium, potassium, and calcium channels at the cellular level and therefore has the potential for direct action on many tissues. Nevertheless, the fact that the sympathetic system is usually stimulated following scorpion envenomation represents a confounding factor in any attempt to separate direct venom effects on the cardiovascular system from those mediated by catecholamine release. Few experimental studies have addressed the issue of respective effects of direct and mediated cardiovascular consequences of scorpion envenomation. Almeida et al.  and Couto et al.  studied the direct effects of scorpion venom on the isolated pig heart and atria. They recorded a dose-dependent inotropic effect of scorpion venom on isolated pig heart. At the atrium level the addition of toxin purified from Tityus serrulatus scorpion venom induced a transient increase in contractile force that was prevented by a β-blocker (metoprolol) reflecting the adrenergic origin of this inotropic effect, emphasizing again the difficulty even in the experimental setting to separate the direct effects of venom from those mediated by the catecholaminergic release. Our study provides additional evidence in favor of the mediator theory although its experimental design does not allow ruling out direct toxic effects since catecholamines-induced hemodynamic disturbances are prominent.
The issue addressed by the current study is both pathophysiologically and clinically relevant. Apart from its contribution to the understanding of the pathophysiology of scorpion envenomation, our study helps to answer one of the recurrent questions surrounding the treatment of severe scorpion envenomation: should we systematically antagonize scorpion toxin that persists in the blood of envenomated patients [11, 16, 17, 18, 19, 20, 21, 22, 23, 24]? The answer to this question leads to two questions. Firstly, what is the relevance of measured toxin levels in comparison to their toxic levels in scorpion envenomation. Secondly, can pathophysiological features of scorpion envenomation be reproduced without any limit of time each time scorpion toxin is administered? Our study provides a piece of answer to the second issue. It suggests that during the first 30 min following scorpion envenomation circulating scorpion toxin (even at levels similar to that associated with severe envenomation’s features) is no longer able to induce harm. Determining the duration of this refractory period was beyond the scope of the current study, but this parameter is probably important to delineate. Whether serum antivenom should be administered beyond the 30-min limit remains an open question. Nevertheless, our findings suggest that antagonizing catecholamine effects on receptors would be more promising than neutralizing the venom.
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