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Effects of antivenom serotherapy on hemodynamic pathophysiology in dogs injected with L. quinquestriatus scorpion venom
PII: S0041-0101(98)00011-7
Toxicon, Vol. 36, No. 7, pp. 963±971, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0041-0101/98 $19.00 + 0.00
EFFECTS OF ANTIVENOM SEROTHERAPY ON HEMODYNAMIC PATHOPHYSIOLOGY IN DOGS INJECTED WITH L. QUINQUESTRIATUS SCORPION VENOM ARIEL TARASIUK,* SOFIA KHVATSKIN and SHAUL SOFER Pediatric Intensive Care Unit, Division of Pediatrics, Soroka Medical Center and Department of Physiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84101 Beer-Sheva, Israel (Received 22 September 1997; accepted 5 January 1998)
A. Tarasiuk, S. Khvatskin and S. Sofer. Eects of antivenom serotherapy on hemodynamic pathophysiology in dogs injected with L. quinquestriatus scorpion venom. Toxicon 36, 963±971, 1998.ÐIn dogs, scorpion venom causes an immediate increase in cardiac output that declines below baseline values within one hour. We tested the hypotheses that antivenom given before venom injection may prevent changes in cardiac output, while antivenom given after the inotropic stage of envenomation cannot reverse cardiac output decline. Twenty-®ve anesthetized, mechanically ventilated dogs were given 0.1 mg/kg IV venom of the scorpion Leiurus quinquestriatus. The dogs were randomized into 4 groups: 5 dogs were given venom alone (control group) and 6 dogs were given 6 ml of antivenom one minute before venom injection while 8 and 6 dogs were given 6 ml of antivenom 20 and 60 min after venom injection, respectively. Parameters re¯ecting respiratory and circulatory functions were measured for 180 min after venom injection. Scorpion venom caused a gradual decrease in heart rate, an initial elevation of systemic and pulmonary blood pressure and cardiac output followed by a decline in these parameters. PO2, pH and HCOÿ 3 gradually decreased, while PCO2 gradually increased from baseline. Antivenom given before venom injection prevented all the eects induced by the venom. Antivenom given at 20 and 60 min after venom injection had no eect on cardiac output and HCOÿ 3 decline, but caused an increase in heart rate, PO2 and pH and a decrease in PCO2. We assume that antivenom clears free toxins from the circulation, and since cardiac output and HCOÿ 3 did not improve after this clearance, we conclude that following intravenous venom injection, heart and circulation are rapidly aected by the toxins or by other substances released by the venom which do not respond to antivenom. Improvements in respiration and heart rate with antivenom given after venom injection may be secondary to reversion of cholinergic eects of the venom. Improvement in respiration may be also explained by reversion of the toxic eects on Ca2+ activated K+ channels of bronchial smooth muscle. All these eects may be * Author to whom correspondence should be addressed. 963
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secondary to clearance of toxins by the antivenom. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
Scorpion envenomation is a common medical hazard in various parts of the world. Severe intoxication may include cardiac and respiratory dysfunction leading to multisystem organ failure and death. The pathogenesis of respiratory and heart failure are as yet not completely elucidated but may involve a direct eect of the toxins on heart and lung tissues and/or secondary eects of neurotransmitters and other substances released by the venom (Gueron and Yarom, 1970; Freire-Maia et al., 1973; Freire-Maia et al., 1974; Gueron et al., 1980; Sofer and Gueron, 1988; Gueron et al., 1990; Sofer et al., 1991; Abroug et al., 1995; Sofer, 1995). Most researchers and clinicians dealing with victims of scorpion sting advocate administration of speci®c serum immunoglobulines as the mainstay of therapy (FreireMaia and Campos, 1987, 1989; Ismail, 1994, 1995; Dehesa-Davila and Possani, 1994; Freire-Maia et al., 1994; Gateau et al., 1994). The eectiveness of antiserum in the treatment of envenomation has been questioned over the last two decades for several reasons (Gueron and Ovsyshcher, 1987; Gueron et al., 1992; El-Amin, 1992; Bawaskar and Bawaskar, 1994; Sofer et al., 1994): (1) Most patients presenting to a medical facility after having been stung exhibit signs and symptoms of systemic intoxication, and it is assumed that at this stage of envenomation symptoms are mostly related to neurotransmitters released by the venom rather than to the venom itself. (2) No single control study has been published which documents the eectiveness of antivenom above placebo in a prospective clinical trial. (3) Although some ``rescued'' animal studies showed ecacy of antiserum given after venom injection (Ismail et al., 1992; Ismail and AbdElsalam, 1996; Kri® et al., 1996; Revelo et al., 1996), most animal studies that document the eectiveness of antiserum, used antivenom concomitantly with venom or before injection of venom. (4) It has been shown that following venom injection, low molecular weight toxins of venom rapidly enter the blood stream and organs, while the heavy chain antivenom molecules are diused slowly into ``shallow'' and ``deep'' compartments (Ismail et al., 1983). Furthermore, antivenom contains animal proteins which may cause allergic reactions, anaphylaxis, and serum sickness (Cupo et al., 1991; Dudin et al., 1991; Bond, 1992). In spite of these reservations, retrospective epidemiological studies from Brazil (Freire-Maia et al., 1994), Mexico (Dehesa-Davila and Possani, 1994), Saudi Arabia (Ismail, 1994, 1995) and other places, advocate antivenom administration. A recent study from Belo-Horizonte, Brazil (Rezende et al., 1995), strongly supported antivenom therapy by documenting the presence of free toxins in the blood stream of envenomated human victims on arrival to the hospital, about 50±240 min after the sting, and the clearance of these toxins with antivenom therapy. While clearance of the toxins led to clinical improvement, it has been observed that in patients with heart failure and pulmonary edema the hemodynamic disturbances persisted for 24 h. These important data may ®t the hypothesis that heart failure and cardiogenic shock are mostly related to substances released by the toxins rather than the toxins itself, and accordingly do not response to antivenom (Gueron et al., 1992; Sofer et al., 1994). We therefore decided to test the eects of the antivenom on hemodynamics in a dog model, adapted to study hemodynamic and respiratory changes after scorpion venom injection (Tarasiuk and Scharf, 1994; Tarasiuk et al., 1994; Tarasiuk et al.,
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1997). In previous studies we described the two-stage eect of the venom (L. quinquestriatus) on hemodynamics. Within a few minutes after venom injection there was a signi®cant increase in systemic and pulmonary blood pressure and cardiac output that declined below baseline values at 60 min. In addition, all dogs developed respiratory and metabolic acidosis, bradycardia and elevation of hemoglobin (Tarasiuk et al., 1994; Tarasiuk et al., 1997). In this study, we looked for changes in respiratory and circulatory systems following injection of venom and antivenom given at dierent stages of envenomation. The following hypotheses were tested: (1) antivenom given before venom injection may eliminate all respiratory and circulatory eects of the venom; (2) antivenom given after the inotropic stage of envenomation cannot reverse cardiac output decline since these eects are caused mainly by secondary mediators unresponsive to antiserum. MATERIALS AND METHODS Conditions for preoperative care, surgery, and euthanasia were consistent with the guidelines of the USA National Institutes of Health and were approved by the Local Institutional Review Board for Animal Studies. Euthanasia was applied at the conclusion of the experiments by intravenous injection of 10 ml 3 M KCl in 5 g pentobarbital. A dog model was used for all experiments. Anesthesia was induced with intravenous (IV) injection of sodium pentobarbital at a dose of 25 mg/kg. Additional IV injections of 30 to 60 mg pentobarbital were given every 1 to 3 h as required to maintain an adequate depth of anesthesia, de®ned as the presence of slight corneal re¯ex. Surgical preparation A group of 25 dogs of mixed breed, either sex, weighing 18±25 kg, were fasted for 12 h prior to surgery. After anesthesia, animals were intubated and mechanically ventilated (tidal volume, 10 ml/kg; respiratory rate adjusted so as to maintain arterial PCO2 at 35±45 Torr, FiO2 21%). A large bore 7F catheter was passed through the left femoral artery for monitoring arterial blood pressure and withdrawing blood for measurements of arterial blood gases (PO2, PCO2, pH and HCOÿ 3 ) and hemoglobin (Nova Biomedical). A femoral venous catheter was used for administration of ¯uids, venom and antivenom. A balloon and thermistor-tipped 7F pulmonary artery catheter (Baxtar, USA) was passed via the right external jugular vein into the pulmonary artery to measure pulmonary artery pressure, pulmonary artery occlusion pressure and measurements of cardiac output. Cardiac output was measured using the thermodilution technique by injecting 5 ml iced saline into the right atrium and sampling the pulmonary artery. Calculations were performed on a previously calibrated cardiac output computer (Baxter, USA). All vascular pressures were zero referenced to the level of the right atrium. Scorpion venom Dry crude scorpion venom obtained from the yellow scorpion Leiurus quinquestriatus (provided by the Israeli Society for Venom Research) was used. The venom was diluted to 1 mg/5 ml in saline and stored at 48C. 0.1 mg/kg venom was given IV in all dogs. Antivenom Antivenom, prepared in donkeys, speci®c to the venom of the yellow scorpion Leiurus quinquestriatus (processed and supplied by the Central Laboratories, Israeli Ministry of Health, Jerusalem) was used. According to the manufacturer, 1 ml antivenom neutralizes 100 LD50 of the venom in 20 g mice. Our preliminary studies showed that 6 ml antivenom injected simultaneously with 0.1 mg/kg of the venom prevented all circulatory eects of the venom and even prevented all hemodynamic eects when additional amounts of the venom were given at the end of 3 h experiments. Protocol After surgery animals were allowed to stabilize for 60 min before any experimental procedures were done. Measurements of arterial blood gases, hemoglobin, heart rate, mean arterial pressure, pulmonary artery pressure, pulmonary artery occlusion pressure and cardiac output were taken before venom injection (baseline) and at 5, 15, 30, 60, 90, 120 and 180 min after venom injection. The dogs were randomly divided into 4 groups: (1)
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5 dogs were given venom alone (control group). (2) 6 dogs were given 6 ml antivenom, one minute before venom injection. (3) 8 dogs were given 6 ml antivenom, 20 minutes after venom injection (immediately after highest inotropic eect). (4) 6 dogs were given 6 ml of antivenom, 60 min after venom injection. Data analysis Data were acquired in 90 s epochs at a digitizing rate of 100 Hz using a commercially available software package (Acq 4600, Gould Instruments, Cleveland, OH) on a microcomputer (PC 386) and downloaded into a hard disk for later analysis (View II, Gould Instruments, Cleveland, OH). Mean data were taken over 2±3 respiratory cycles. Data were compiled and tested for normal distribution (Kolmagorov±Smirnov test) and expressed as mean2SD. Dierences within a group were determined using one-way analysis of variance (Anova-I) for repeated or non-repeated measurements followed by a post-hoc analysis (Newman±Keuls) to determine the source of signi®cance. Two-way analysis of variance (Anova II) was performed for comparison between groups. The null hypotheses was rejected at the 5% level.
RESULTS
Scorpion venom caused severe combined hemodynamic and respiratory alterations. Antivenom given prior to venom injection prevented all hemodynamic and respiratory changes caused by the venom. Mean heart rate, cardiac output, systemic and pulmonary blood pressures, and arterial blood gases did not change from baseline to the end of the 180 min experiments. In all other groups injection of scorpion venom caused immediate and prominent eects characterized by elevation of ca 100% in cardiac output within 5 min (Fig. 1). This inotropic eect lasted for about 10 min, cardiac output then gradually declined and ended 48% of baseline values (p < 0.01) in dogs treated with venom alone, and 56% (p < 0.001) and 27% (p < 0.001) of baseline values in dogs treated with antivenom 20 and 60 min after venom injection, respectively. Mean systemic blood pressure (Table 1) increased from 128.6 221.9 to 184.0 2 22.6 mm Hg at 15 min measurement (p < 0.001) in dogs treated with venom alone, and from 119.4 2 20.8 to 200.2 236.0 mm Hg (p < 0.001) and 129.2 2 24.6 to 200.8 2 37.8 mm Hg (p < 0.001) in
Fig. 1. Eects of scorpion venom on cardiac output. Broken lines represent mean baseline values. Values are mean 2SD.
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Table 1. Systemic and pulmonary blood pressure following scorpion venom administration Variable Blood pressure (Torr)
Pulmonary artery pressure (Torr)
Groups
Baseline
15 min
60 min
120 min
180 min
control pre. antivenom antivenom20 min antivenom60 min
128.62 21.9 128.42 12.8
184.0222.6# 126.92 7.6
107.82 26.3 121.02 12.5
69.12 15.3# 114.82 14.4
45.9 24.2# 121.12 16.8
119.42 20.8
200.2236.0#
106.82 29.8
86.42 27.2#
86.42 27.2#
129.22 24.6
200.8237.8#
124.02 32.6
56.42 18.1#
39.72 15.7#
control pre. antivenom antivenom20 min antivenom60 min
15.32 1.1 16.02 4.7
40.7 25.7# 17.02 4.7
21.82 5.3 17.12 3.9
15.02 6.0 15.72 2.7
15.02 5.9 15.12 2.9
13.02 2.8
40.6 28.3#
18.62 4.2
17.72 2.3
17.42 3.3
16.22 3.9
35.62 11.8#
16.92 4.7
11.2 20.9#
10.8 22.8#
For comparison with baseline (Anova-I): #p < 0.001.
dogs which received antivenom 20 and 60 min after venom injection, respectively. Blood pressure declined later on and ended below baseline values in dogs treated without antivenom, 45.9 24.2 mm Hg (p < 0.001) and in dogs treated with antivenom at 20 and 60 min after venom injection, 86.4 227.2 mm Hg (p < 0.01) and 39.7 215.7 mm Hg (p < 0.001), respectively. Pulmonary artery pressure (Table 1) showed similar pattern of initial elevation (about 200% from baseline values) in all 3 groups with a gradually decline to values not signi®cantly dierent from baseline at the end of the experiment. During the ®rst inotropic phase, mean pulmonary artery occlusion pressure increased
Fig. 2. Eects of scorpion venom on heart rate. Broken lines represent mean baseline values. Values are mean2 SD, *p < 0.01.
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from 5.6 to 24.9 mm Hg (p < 0.001) but returned to baseline values when cardiac output declined. The eects of the venom on heart rate are shown in Fig. 2. In all 3 groups the venom caused a rapid but short episode of tachycardia which then reverted to bradycardia. Heart rate then reached a plateau and ended 64% below baseline in untreated dogs (p < 0.001). Antivenom given at 20 and 60 min after venom injection reversed heart rate decline. Recorded heart rate in these 2 groups at the end of the experiments were not dierent from heart rate recorded on baseline measurements. Scorpion venom caused also a decrease in arterial PO2, from 91.7 2 10.5 mm Hg at baseline measurements to 74.1 221.3 mm Hg (p < 0.05) at the end of the experiments, as well as a decrease in pH from 7.36 2 0.03 to 7.16 20.02 units (p < 0.01) and a decrease in HCOÿ 3 from 22.5 21 to 16.7 22 mM (p < 0.05). PCO2 increased from 40.6 2 4.1 to 46.6 25.0 mm Hg (p < 0.05) at the end of the experiment (Fig. 3). Antivenom given at 20 and 60 min after venom injection reversed PO2 decline and improved pH values. PO2 in these groups ended signi®cantly above PO2 of untreated dogs and did not dier from baseline PO2. PCO2 in these 2 groups ended signi®cantly lower than PCO2 of untreated dogs and did not dier from baseline PCO2. Recorded pH values in dogs treated at 20 min, were signi®cantly higher (7.312 0.04 units) at the end of the experiments than pH of untreated dogs (p < 0.05), while in dogs treated at 60 min, pH at the end of the experiments (7.21 20.05 units) did not reach statistical signi®cance compared with pH of untreated dogs. Improvement of acidosis was secondary only to the improvement in respiration since HCOÿ 3 decline was not corrected following antivenom administration and was eventually lower (12.3 2 2.7 mM) in the group of dogs where antivenom was given 60 min after venom injection (p < 0.05).
Fig. 3. Eects of scorpion venom on arterial blood gases. Open circle-control; Open triangles, antivenom at 20 min; Closed triangles, antivenom at 60 min. Values are mean 2SD, *p < 0.01.
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969
DISCUSSION
These series of experiments on canine model did not precisely mimic nature envenomation in terms of dosage (1.8±2.5 mg dry venom per dog, while L. quinquestriatus scorpion injects ca 1±2 mg dry venom per sting) and route of administration (IV instead of subcutaneous). Notwithstanding, we believe that our results are signi®cant and useful. Dosage and route of administration in this study were chosen in accordance with results of similar studies on dogs and pigs describing the hemodynamic features of the model following injection of L. quinquestriatus venom (Tarasiuk et al., 1994; Tarasiuk et al., 1997; Sofer et al., 1997) and they enabled us to observe cardiovascular changes within reasonable timetable. The experiments were dose dependent, and since antivenom given before venom injection completely prevented all venom eects, it was important to notice whether antivenom immunoglobulines were able to reverse pharmacological eects on heart and circulation after their exertion by the venom. Indeed, administration of serum immunoglobulines against scorpion L. quinquestriatus venom given at dierent intervals following venom injection, resulted in signi®cant improvements of some parameters. The main positive eects were on the respiratory system which showed improved oxygenation (PO2) and ventilation (PCO2). These ®ndings were surprising and unexpected since all dogs were anesthetized, intubated and mechanically ventilated (constant minute ventilation), thus excluding the possibilities that deterioration was secondary to upper airway obstruction, hypopnea, or apneic episodes. Other explanations for respiratory changes included cardiogenic pulmonary edema (Sofer and Gueron, 1988). This possibility was recently ruled out in this model (Tarasiuk et al., 1994) and was excluded in current experiments with observation of normal pulmonary artery occlusion pressures during periods of decreased cardiac output. Non cardiogenic pulmonary edema (increased capillary permeability) was not anticipated since it is most likely not caused by the venom itself but is secondary to activation of proin¯ammatory substances released by the venom (Amaral et al., 1993; Freire-Maia and DeMatos, 1993; Sofer, 1996; Amaral and Rezende, 1997). It is therefore unlikely that it would respond to antivenom immunoglobulines. Other explanations for respiratory disturbances following venom injection imply bronchoconstriction due to the direct eects of venom toxins on Ca2+ activated K+ channels of the airways smooth muscles (Rogers, 1996) or secondary to cholinergic eects of the venom. It should be mentioned that the dog's small airways are especially sensitive to cholinergic stimulants and therefore are used as a model for research of hyperresponsive airways (Becker et al., 1989). The deleterious eects of the venom on small airways may be augmented by the eects of the venom on other skeletal muscles (Couraud and Jover, 1984) resulting in thoracic muscle rigidity with disturbances in gas exchange. Antivenom may obviously reverse all these disturbances by neutralizing free toxins from circulation and/or tissues. In this regard, it should be mentioned that reversion of bradycardia, a known cholinergic eect of the venom (Freire-Maia et al., 1974; El-Asmar, 1984), may resulted from reversion of cholinergic activity by clearance of free toxins from circulation. Antivenom did not change cardiac output decline. These ®ndings are in agreement with clinical ®ndings of Rezende et al. (1995) who found no correlation between clearance of toxins from circulation and persistence of cardiac symptoms in human victims of scorpion stings. The lack of improvement in cardiac output in our study implies that the decrease in cardiac output is either secondary to the eects of toxins which rapidly aect cardiac tissue and circulation, while the antivenom immunoglobulines given after
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venom injection is diused slowly into these organs. Alternatively it is possible that the decrease in cardiac output is secondary to other substances released by the venom (proin¯ammatory cytokines or humoral transmitters) rather than the venom itself (Gueron et al., 1992; Sofer et al., 1994; Sofer, 1995). In this case clearance of serum toxins by the antivenom would not improve cardiac output. We previously demonstrated in dogs that the decline in cardiac output following scorpion venom injection was not related to myocardial ischemia or bradycardia (Tarasiuk et al., 1997), but was associated with signi®cant changes in the resistance to venous return. The decrease in cardiac output during the second stage of intoxication may be explained by persistence of venoconstriction and redistribution of blood ¯ow. This eect is most likely secondary to massive discharge of catecholamines (Sofer et al., 1997). Another important ®nding in our study involves the eects of the venom and antivenom on serum bicarbonate and pH. While antivenom signi®cantly elevated falling pH, this eect was secondary to the eect of the antivenom on elevated PCO2. Conversely, bicarbonate gradually decreased and was not aected by the antivenom. Metabolic acidosis and decreased bicarbonate levels following human scorpion sting is not an uncommon event (Freire-Maia and Campos, 1989; Sofer et al., 1994) and was consistently found in animal studies (Tarasiuk et al., 1994; Tarasiuk et al., 1997; Sofer et al., 1997). Besides a decrease in cardiac output, which may result in decreased tissue perfusion and metabolic acidosis, hemodynamic studies on pigs have shown gradual decrease in bicarbonate levels and development of lactic acidosis starting during the ®rst inotropic stage of envenomation, when cardiac output and total oxygen delivery to the periphery was increased or well maintained (Sofer et al., 1997). It has been shown that the massive discharge of catecholamines that starts within a few minutes after venom injection is associated with gastrointestinal hypoperfusion resulting in local and general lactic acidosis. Antivenom given at 20 or 60 min after venom injection could not aect catecholamines discharge or cardiac output decline.
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(1997) Scorpion venom leads to gastrointestinal ischemia despite increased oxygen delivery in pigs. Crit. Care. Med. 25, 834±840. Tarasiuk, A., Sofer, S., Huberfeld, S. I. and Scharf, S. M. (1994) Hemodynamic eects following injection of venom from the scorpion Leiurus quinquestriatus. J. Crit. Care 9, 134±140. Tarasiuk, A. and Scharf, S. M. (1994) Cardiovascular eects of periodic obstructive and central apneas in dogs. Am. J. Resp. Crit. Care. Med. 150, 83±89. Tarasiuk, A., Janco, J. and Sofer, S. (1997) Eects of scorpion venom on central and peripheral circulatory response in opened chest dog model. Acta Physiol. Scand. 161, 141±149.
Toxicon, Vol. 36, No. 7, pp. 963±971, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0041-0101/98 $19.00 + 0.00
EFFECTS OF ANTIVENOM SEROTHERAPY ON HEMODYNAMIC PATHOPHYSIOLOGY IN DOGS INJECTED WITH L. QUINQUESTRIATUS SCORPION VENOM ARIEL TARASIUK,* SOFIA KHVATSKIN and SHAUL SOFER Pediatric Intensive Care Unit, Division of Pediatrics, Soroka Medical Center and Department of Physiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84101 Beer-Sheva, Israel (Received 22 September 1997; accepted 5 January 1998)
A. Tarasiuk, S. Khvatskin and S. Sofer. Eects of antivenom serotherapy on hemodynamic pathophysiology in dogs injected with L. quinquestriatus scorpion venom. Toxicon 36, 963±971, 1998.ÐIn dogs, scorpion venom causes an immediate increase in cardiac output that declines below baseline values within one hour. We tested the hypotheses that antivenom given before venom injection may prevent changes in cardiac output, while antivenom given after the inotropic stage of envenomation cannot reverse cardiac output decline. Twenty-®ve anesthetized, mechanically ventilated dogs were given 0.1 mg/kg IV venom of the scorpion Leiurus quinquestriatus. The dogs were randomized into 4 groups: 5 dogs were given venom alone (control group) and 6 dogs were given 6 ml of antivenom one minute before venom injection while 8 and 6 dogs were given 6 ml of antivenom 20 and 60 min after venom injection, respectively. Parameters re¯ecting respiratory and circulatory functions were measured for 180 min after venom injection. Scorpion venom caused a gradual decrease in heart rate, an initial elevation of systemic and pulmonary blood pressure and cardiac output followed by a decline in these parameters. PO2, pH and HCOÿ 3 gradually decreased, while PCO2 gradually increased from baseline. Antivenom given before venom injection prevented all the eects induced by the venom. Antivenom given at 20 and 60 min after venom injection had no eect on cardiac output and HCOÿ 3 decline, but caused an increase in heart rate, PO2 and pH and a decrease in PCO2. We assume that antivenom clears free toxins from the circulation, and since cardiac output and HCOÿ 3 did not improve after this clearance, we conclude that following intravenous venom injection, heart and circulation are rapidly aected by the toxins or by other substances released by the venom which do not respond to antivenom. Improvements in respiration and heart rate with antivenom given after venom injection may be secondary to reversion of cholinergic eects of the venom. Improvement in respiration may be also explained by reversion of the toxic eects on Ca2+ activated K+ channels of bronchial smooth muscle. All these eects may be * Author to whom correspondence should be addressed. 963
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secondary to clearance of toxins by the antivenom. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
Scorpion envenomation is a common medical hazard in various parts of the world. Severe intoxication may include cardiac and respiratory dysfunction leading to multisystem organ failure and death. The pathogenesis of respiratory and heart failure are as yet not completely elucidated but may involve a direct eect of the toxins on heart and lung tissues and/or secondary eects of neurotransmitters and other substances released by the venom (Gueron and Yarom, 1970; Freire-Maia et al., 1973; Freire-Maia et al., 1974; Gueron et al., 1980; Sofer and Gueron, 1988; Gueron et al., 1990; Sofer et al., 1991; Abroug et al., 1995; Sofer, 1995). Most researchers and clinicians dealing with victims of scorpion sting advocate administration of speci®c serum immunoglobulines as the mainstay of therapy (FreireMaia and Campos, 1987, 1989; Ismail, 1994, 1995; Dehesa-Davila and Possani, 1994; Freire-Maia et al., 1994; Gateau et al., 1994). The eectiveness of antiserum in the treatment of envenomation has been questioned over the last two decades for several reasons (Gueron and Ovsyshcher, 1987; Gueron et al., 1992; El-Amin, 1992; Bawaskar and Bawaskar, 1994; Sofer et al., 1994): (1) Most patients presenting to a medical facility after having been stung exhibit signs and symptoms of systemic intoxication, and it is assumed that at this stage of envenomation symptoms are mostly related to neurotransmitters released by the venom rather than to the venom itself. (2) No single control study has been published which documents the eectiveness of antivenom above placebo in a prospective clinical trial. (3) Although some ``rescued'' animal studies showed ecacy of antiserum given after venom injection (Ismail et al., 1992; Ismail and AbdElsalam, 1996; Kri® et al., 1996; Revelo et al., 1996), most animal studies that document the eectiveness of antiserum, used antivenom concomitantly with venom or before injection of venom. (4) It has been shown that following venom injection, low molecular weight toxins of venom rapidly enter the blood stream and organs, while the heavy chain antivenom molecules are diused slowly into ``shallow'' and ``deep'' compartments (Ismail et al., 1983). Furthermore, antivenom contains animal proteins which may cause allergic reactions, anaphylaxis, and serum sickness (Cupo et al., 1991; Dudin et al., 1991; Bond, 1992). In spite of these reservations, retrospective epidemiological studies from Brazil (Freire-Maia et al., 1994), Mexico (Dehesa-Davila and Possani, 1994), Saudi Arabia (Ismail, 1994, 1995) and other places, advocate antivenom administration. A recent study from Belo-Horizonte, Brazil (Rezende et al., 1995), strongly supported antivenom therapy by documenting the presence of free toxins in the blood stream of envenomated human victims on arrival to the hospital, about 50±240 min after the sting, and the clearance of these toxins with antivenom therapy. While clearance of the toxins led to clinical improvement, it has been observed that in patients with heart failure and pulmonary edema the hemodynamic disturbances persisted for 24 h. These important data may ®t the hypothesis that heart failure and cardiogenic shock are mostly related to substances released by the toxins rather than the toxins itself, and accordingly do not response to antivenom (Gueron et al., 1992; Sofer et al., 1994). We therefore decided to test the eects of the antivenom on hemodynamics in a dog model, adapted to study hemodynamic and respiratory changes after scorpion venom injection (Tarasiuk and Scharf, 1994; Tarasiuk et al., 1994; Tarasiuk et al.,
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1997). In previous studies we described the two-stage eect of the venom (L. quinquestriatus) on hemodynamics. Within a few minutes after venom injection there was a signi®cant increase in systemic and pulmonary blood pressure and cardiac output that declined below baseline values at 60 min. In addition, all dogs developed respiratory and metabolic acidosis, bradycardia and elevation of hemoglobin (Tarasiuk et al., 1994; Tarasiuk et al., 1997). In this study, we looked for changes in respiratory and circulatory systems following injection of venom and antivenom given at dierent stages of envenomation. The following hypotheses were tested: (1) antivenom given before venom injection may eliminate all respiratory and circulatory eects of the venom; (2) antivenom given after the inotropic stage of envenomation cannot reverse cardiac output decline since these eects are caused mainly by secondary mediators unresponsive to antiserum. MATERIALS AND METHODS Conditions for preoperative care, surgery, and euthanasia were consistent with the guidelines of the USA National Institutes of Health and were approved by the Local Institutional Review Board for Animal Studies. Euthanasia was applied at the conclusion of the experiments by intravenous injection of 10 ml 3 M KCl in 5 g pentobarbital. A dog model was used for all experiments. Anesthesia was induced with intravenous (IV) injection of sodium pentobarbital at a dose of 25 mg/kg. Additional IV injections of 30 to 60 mg pentobarbital were given every 1 to 3 h as required to maintain an adequate depth of anesthesia, de®ned as the presence of slight corneal re¯ex. Surgical preparation A group of 25 dogs of mixed breed, either sex, weighing 18±25 kg, were fasted for 12 h prior to surgery. After anesthesia, animals were intubated and mechanically ventilated (tidal volume, 10 ml/kg; respiratory rate adjusted so as to maintain arterial PCO2 at 35±45 Torr, FiO2 21%). A large bore 7F catheter was passed through the left femoral artery for monitoring arterial blood pressure and withdrawing blood for measurements of arterial blood gases (PO2, PCO2, pH and HCOÿ 3 ) and hemoglobin (Nova Biomedical). A femoral venous catheter was used for administration of ¯uids, venom and antivenom. A balloon and thermistor-tipped 7F pulmonary artery catheter (Baxtar, USA) was passed via the right external jugular vein into the pulmonary artery to measure pulmonary artery pressure, pulmonary artery occlusion pressure and measurements of cardiac output. Cardiac output was measured using the thermodilution technique by injecting 5 ml iced saline into the right atrium and sampling the pulmonary artery. Calculations were performed on a previously calibrated cardiac output computer (Baxter, USA). All vascular pressures were zero referenced to the level of the right atrium. Scorpion venom Dry crude scorpion venom obtained from the yellow scorpion Leiurus quinquestriatus (provided by the Israeli Society for Venom Research) was used. The venom was diluted to 1 mg/5 ml in saline and stored at 48C. 0.1 mg/kg venom was given IV in all dogs. Antivenom Antivenom, prepared in donkeys, speci®c to the venom of the yellow scorpion Leiurus quinquestriatus (processed and supplied by the Central Laboratories, Israeli Ministry of Health, Jerusalem) was used. According to the manufacturer, 1 ml antivenom neutralizes 100 LD50 of the venom in 20 g mice. Our preliminary studies showed that 6 ml antivenom injected simultaneously with 0.1 mg/kg of the venom prevented all circulatory eects of the venom and even prevented all hemodynamic eects when additional amounts of the venom were given at the end of 3 h experiments. Protocol After surgery animals were allowed to stabilize for 60 min before any experimental procedures were done. Measurements of arterial blood gases, hemoglobin, heart rate, mean arterial pressure, pulmonary artery pressure, pulmonary artery occlusion pressure and cardiac output were taken before venom injection (baseline) and at 5, 15, 30, 60, 90, 120 and 180 min after venom injection. The dogs were randomly divided into 4 groups: (1)
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5 dogs were given venom alone (control group). (2) 6 dogs were given 6 ml antivenom, one minute before venom injection. (3) 8 dogs were given 6 ml antivenom, 20 minutes after venom injection (immediately after highest inotropic eect). (4) 6 dogs were given 6 ml of antivenom, 60 min after venom injection. Data analysis Data were acquired in 90 s epochs at a digitizing rate of 100 Hz using a commercially available software package (Acq 4600, Gould Instruments, Cleveland, OH) on a microcomputer (PC 386) and downloaded into a hard disk for later analysis (View II, Gould Instruments, Cleveland, OH). Mean data were taken over 2±3 respiratory cycles. Data were compiled and tested for normal distribution (Kolmagorov±Smirnov test) and expressed as mean2SD. Dierences within a group were determined using one-way analysis of variance (Anova-I) for repeated or non-repeated measurements followed by a post-hoc analysis (Newman±Keuls) to determine the source of signi®cance. Two-way analysis of variance (Anova II) was performed for comparison between groups. The null hypotheses was rejected at the 5% level.
RESULTS
Scorpion venom caused severe combined hemodynamic and respiratory alterations. Antivenom given prior to venom injection prevented all hemodynamic and respiratory changes caused by the venom. Mean heart rate, cardiac output, systemic and pulmonary blood pressures, and arterial blood gases did not change from baseline to the end of the 180 min experiments. In all other groups injection of scorpion venom caused immediate and prominent eects characterized by elevation of ca 100% in cardiac output within 5 min (Fig. 1). This inotropic eect lasted for about 10 min, cardiac output then gradually declined and ended 48% of baseline values (p < 0.01) in dogs treated with venom alone, and 56% (p < 0.001) and 27% (p < 0.001) of baseline values in dogs treated with antivenom 20 and 60 min after venom injection, respectively. Mean systemic blood pressure (Table 1) increased from 128.6 221.9 to 184.0 2 22.6 mm Hg at 15 min measurement (p < 0.001) in dogs treated with venom alone, and from 119.4 2 20.8 to 200.2 236.0 mm Hg (p < 0.001) and 129.2 2 24.6 to 200.8 2 37.8 mm Hg (p < 0.001) in
Fig. 1. Eects of scorpion venom on cardiac output. Broken lines represent mean baseline values. Values are mean 2SD.
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Table 1. Systemic and pulmonary blood pressure following scorpion venom administration Variable Blood pressure (Torr)
Pulmonary artery pressure (Torr)
Groups
Baseline
15 min
60 min
120 min
180 min
control pre. antivenom antivenom20 min antivenom60 min
128.62 21.9 128.42 12.8
184.0222.6# 126.92 7.6
107.82 26.3 121.02 12.5
69.12 15.3# 114.82 14.4
45.9 24.2# 121.12 16.8
119.42 20.8
200.2236.0#
106.82 29.8
86.42 27.2#
86.42 27.2#
129.22 24.6
200.8237.8#
124.02 32.6
56.42 18.1#
39.72 15.7#
control pre. antivenom antivenom20 min antivenom60 min
15.32 1.1 16.02 4.7
40.7 25.7# 17.02 4.7
21.82 5.3 17.12 3.9
15.02 6.0 15.72 2.7
15.02 5.9 15.12 2.9
13.02 2.8
40.6 28.3#
18.62 4.2
17.72 2.3
17.42 3.3
16.22 3.9
35.62 11.8#
16.92 4.7
11.2 20.9#
10.8 22.8#
For comparison with baseline (Anova-I): #p < 0.001.
dogs which received antivenom 20 and 60 min after venom injection, respectively. Blood pressure declined later on and ended below baseline values in dogs treated without antivenom, 45.9 24.2 mm Hg (p < 0.001) and in dogs treated with antivenom at 20 and 60 min after venom injection, 86.4 227.2 mm Hg (p < 0.01) and 39.7 215.7 mm Hg (p < 0.001), respectively. Pulmonary artery pressure (Table 1) showed similar pattern of initial elevation (about 200% from baseline values) in all 3 groups with a gradually decline to values not signi®cantly dierent from baseline at the end of the experiment. During the ®rst inotropic phase, mean pulmonary artery occlusion pressure increased
Fig. 2. Eects of scorpion venom on heart rate. Broken lines represent mean baseline values. Values are mean2 SD, *p < 0.01.
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from 5.6 to 24.9 mm Hg (p < 0.001) but returned to baseline values when cardiac output declined. The eects of the venom on heart rate are shown in Fig. 2. In all 3 groups the venom caused a rapid but short episode of tachycardia which then reverted to bradycardia. Heart rate then reached a plateau and ended 64% below baseline in untreated dogs (p < 0.001). Antivenom given at 20 and 60 min after venom injection reversed heart rate decline. Recorded heart rate in these 2 groups at the end of the experiments were not dierent from heart rate recorded on baseline measurements. Scorpion venom caused also a decrease in arterial PO2, from 91.7 2 10.5 mm Hg at baseline measurements to 74.1 221.3 mm Hg (p < 0.05) at the end of the experiments, as well as a decrease in pH from 7.36 2 0.03 to 7.16 20.02 units (p < 0.01) and a decrease in HCOÿ 3 from 22.5 21 to 16.7 22 mM (p < 0.05). PCO2 increased from 40.6 2 4.1 to 46.6 25.0 mm Hg (p < 0.05) at the end of the experiment (Fig. 3). Antivenom given at 20 and 60 min after venom injection reversed PO2 decline and improved pH values. PO2 in these groups ended signi®cantly above PO2 of untreated dogs and did not dier from baseline PO2. PCO2 in these 2 groups ended signi®cantly lower than PCO2 of untreated dogs and did not dier from baseline PCO2. Recorded pH values in dogs treated at 20 min, were signi®cantly higher (7.312 0.04 units) at the end of the experiments than pH of untreated dogs (p < 0.05), while in dogs treated at 60 min, pH at the end of the experiments (7.21 20.05 units) did not reach statistical signi®cance compared with pH of untreated dogs. Improvement of acidosis was secondary only to the improvement in respiration since HCOÿ 3 decline was not corrected following antivenom administration and was eventually lower (12.3 2 2.7 mM) in the group of dogs where antivenom was given 60 min after venom injection (p < 0.05).
Fig. 3. Eects of scorpion venom on arterial blood gases. Open circle-control; Open triangles, antivenom at 20 min; Closed triangles, antivenom at 60 min. Values are mean 2SD, *p < 0.01.
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969
DISCUSSION
These series of experiments on canine model did not precisely mimic nature envenomation in terms of dosage (1.8±2.5 mg dry venom per dog, while L. quinquestriatus scorpion injects ca 1±2 mg dry venom per sting) and route of administration (IV instead of subcutaneous). Notwithstanding, we believe that our results are signi®cant and useful. Dosage and route of administration in this study were chosen in accordance with results of similar studies on dogs and pigs describing the hemodynamic features of the model following injection of L. quinquestriatus venom (Tarasiuk et al., 1994; Tarasiuk et al., 1997; Sofer et al., 1997) and they enabled us to observe cardiovascular changes within reasonable timetable. The experiments were dose dependent, and since antivenom given before venom injection completely prevented all venom eects, it was important to notice whether antivenom immunoglobulines were able to reverse pharmacological eects on heart and circulation after their exertion by the venom. Indeed, administration of serum immunoglobulines against scorpion L. quinquestriatus venom given at dierent intervals following venom injection, resulted in signi®cant improvements of some parameters. The main positive eects were on the respiratory system which showed improved oxygenation (PO2) and ventilation (PCO2). These ®ndings were surprising and unexpected since all dogs were anesthetized, intubated and mechanically ventilated (constant minute ventilation), thus excluding the possibilities that deterioration was secondary to upper airway obstruction, hypopnea, or apneic episodes. Other explanations for respiratory changes included cardiogenic pulmonary edema (Sofer and Gueron, 1988). This possibility was recently ruled out in this model (Tarasiuk et al., 1994) and was excluded in current experiments with observation of normal pulmonary artery occlusion pressures during periods of decreased cardiac output. Non cardiogenic pulmonary edema (increased capillary permeability) was not anticipated since it is most likely not caused by the venom itself but is secondary to activation of proin¯ammatory substances released by the venom (Amaral et al., 1993; Freire-Maia and DeMatos, 1993; Sofer, 1996; Amaral and Rezende, 1997). It is therefore unlikely that it would respond to antivenom immunoglobulines. Other explanations for respiratory disturbances following venom injection imply bronchoconstriction due to the direct eects of venom toxins on Ca2+ activated K+ channels of the airways smooth muscles (Rogers, 1996) or secondary to cholinergic eects of the venom. It should be mentioned that the dog's small airways are especially sensitive to cholinergic stimulants and therefore are used as a model for research of hyperresponsive airways (Becker et al., 1989). The deleterious eects of the venom on small airways may be augmented by the eects of the venom on other skeletal muscles (Couraud and Jover, 1984) resulting in thoracic muscle rigidity with disturbances in gas exchange. Antivenom may obviously reverse all these disturbances by neutralizing free toxins from circulation and/or tissues. In this regard, it should be mentioned that reversion of bradycardia, a known cholinergic eect of the venom (Freire-Maia et al., 1974; El-Asmar, 1984), may resulted from reversion of cholinergic activity by clearance of free toxins from circulation. Antivenom did not change cardiac output decline. These ®ndings are in agreement with clinical ®ndings of Rezende et al. (1995) who found no correlation between clearance of toxins from circulation and persistence of cardiac symptoms in human victims of scorpion stings. The lack of improvement in cardiac output in our study implies that the decrease in cardiac output is either secondary to the eects of toxins which rapidly aect cardiac tissue and circulation, while the antivenom immunoglobulines given after
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venom injection is diused slowly into these organs. Alternatively it is possible that the decrease in cardiac output is secondary to other substances released by the venom (proin¯ammatory cytokines or humoral transmitters) rather than the venom itself (Gueron et al., 1992; Sofer et al., 1994; Sofer, 1995). In this case clearance of serum toxins by the antivenom would not improve cardiac output. We previously demonstrated in dogs that the decline in cardiac output following scorpion venom injection was not related to myocardial ischemia or bradycardia (Tarasiuk et al., 1997), but was associated with signi®cant changes in the resistance to venous return. The decrease in cardiac output during the second stage of intoxication may be explained by persistence of venoconstriction and redistribution of blood ¯ow. This eect is most likely secondary to massive discharge of catecholamines (Sofer et al., 1997). Another important ®nding in our study involves the eects of the venom and antivenom on serum bicarbonate and pH. While antivenom signi®cantly elevated falling pH, this eect was secondary to the eect of the antivenom on elevated PCO2. Conversely, bicarbonate gradually decreased and was not aected by the antivenom. Metabolic acidosis and decreased bicarbonate levels following human scorpion sting is not an uncommon event (Freire-Maia and Campos, 1989; Sofer et al., 1994) and was consistently found in animal studies (Tarasiuk et al., 1994; Tarasiuk et al., 1997; Sofer et al., 1997). Besides a decrease in cardiac output, which may result in decreased tissue perfusion and metabolic acidosis, hemodynamic studies on pigs have shown gradual decrease in bicarbonate levels and development of lactic acidosis starting during the ®rst inotropic stage of envenomation, when cardiac output and total oxygen delivery to the periphery was increased or well maintained (Sofer et al., 1997). It has been shown that the massive discharge of catecholamines that starts within a few minutes after venom injection is associated with gastrointestinal hypoperfusion resulting in local and general lactic acidosis. Antivenom given at 20 or 60 min after venom injection could not aect catecholamines discharge or cardiac output decline.
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