Cardiopulmonary complications induced by Iranian Mesobuthus eupeus scorpion venom in anesthetized rabbits

Scorpion envenomation is a life-threatening condition, especially in children and elderly individuals affected by respiratory and cardiovascular diseases. In this study, the toxic effects of median lethal dose (LD50) injections of Mesobuthus eupeus (Me) venom on the heart and lungs of anesthetized rabbits were investigated. Six rabbits were selected and alterations in their electrocardiogram, heart rate, respiration and blood pressure before and after venom injection were recorded. Cardiac troponin T (cTnT), creatinine kinase muscle-brain fraction (CK-MB) and lactate dehydrogenase (LDH) were measured at 0, 1 and 3 hours after envenomation and pathology studies were carried out postmortem. All the animals showed signs and symptoms of envenomation within 40 minutes and died 3 to 3.5 hours after venom injection. Pathology studies revealed alveolar edema in 100% of the rabbits and myocardial infarction in 16%. The main histopathological changes were myocytolysis, coagulation necrosis, focal hemorrhage, thrombus formation both in myocardium and on endocardial surfaces as well as inflammatory infiltrates in the heart and hemorrhage, vascular thrombus and interstitial inflammation in the lungs. ECG monitoring of rabbits showed ST elevation, ST depression and inverted T and Q waves. In addition, although cTnT levels increased in 16% of the animals and serum LDH was also augmented, none of these changes was statistically significant. The enzyme CK-MB also did not show any change after Me venom injection. In conclusion, the results of this study showed that Me venom killed animals in less than 3.5 hours through severe pulmonary damage and it appears that the deaths could not be attributed to cardiovascular lesions. Therefore, Me venom effects on the lungs are so important that they appear to be independent of heart damage.


INTRODUCTION
Scorpion envenomation is a life-threatening condition, especially in children and older individuals who suffer from respiratory and cardiovascular disease (1).Mesobuthus eupeus is one of the major scorpions in Iran and its stings constitute a public health problem.The majority of stings that occur in Iran are attributed to the Buthidae family, which includes Mesobuthus eupeus, Androctonus crassicauda, Odontobuthus odonturus and Apistobuthus pertigus (2).In addition, Mesobuthus eupeus shows high geographic distribution in Turkey, Iraq, Turkestan, Afghanistan and Pakistan.
Radmanesh (2) showed that 45% of all cases of scorpion stings were due to Mesobuthus eupeus in Iran.

Experimental Protocol
Six male New Zealand white rabbits with an average weight of 2 kg were used in this study.All animals were housed under conditions of controlled light (12-

Electrocardiogram, Blood Pressure and Arterial Oxygen Saturation Recording
Electrocardiogram was recorded at regular intervals using 12 electrocardiographic leads (Cardimax FX-2111®, Fukudadenshi, Japan).Blood pressure was measured until death using the neonatal blood pressure cuff from a Spacelabs medical device (Spacelabs Medical Incorporation, USA).Arterial oxygen saturation was measured using a Spacelabs sensor.

Assay of Myocardial Markers
Troponin T (TnT) was measured using an electrochemiluminescence immunoassay (Elecsys 2010® analyzer and Troponin T STAT® kit, Roche, Germany) according to the manufacturer's instructions.Lactate dehydrogenase (LDH) and creatinine kinasemuscle brain (CK-MB) were assayed in the sera using the electrochemiluminescence immunoassay (Elecsys 2010® analyzer and CK-MB STAT® kit, Roche, Germany) according to the manufacturer's instructions.The enzyme values were expressed in international units (µg/L).

Histological Analysis
After the animals died the heart and lungs were removed carefully and immersed in 10% buffered formalin at room temperature and then sectioned transversely into 3-4 μm slices.Specimens were dehydrated in a graded series of alcohol and xylene and embedded in paraffin.Multiple slices were made and stained by hematoxilin and eosin stains.Sections were viewed and photographed using a Nikon E200® light microscope (Japan).

Statistical Analysis
All results were expressed as mean ± SD.The statistical significance of differences among groups was analyzed by the Student's t-test.Data were considered statistically significant if p-values were < 0.05.

Histopathological Evaluation
Histopathological evaluation of the heart showed extensive coagulation necrosis in one rabbit, myocytolysis in two animals, focal hemorrhage and thrombus formation in myocardium and endocardial surfaces in two rabbits and lymphomononuclear inflammatory infiltration in three rabbits.Evaluation of the lungs showed alveolar edema in all animals, thrombus formation in two rabbits, hemorrhage in three rabbits, vascular congestion in five rabbits and lymphomononuclear interstitial inflammation in three rabbits (Figure 1).

Tissue Necrosis Marker Assays after Envenomation
Tissue necrosis marker assays showed a non-significant increase in the level of TnT activities in the serum of all envenomated rabbits.However, the TnT level in one rabbit that showed considerable ECG alterations (infarct-like patterns) and severe histopathological alterations (coagulation necrosis, myocytolysis) was considerably higher than in the others.Serum LDH displayed non-significant increase after venom injection.CK-MB measurement did not show significant changes (Table 1).

DISCUSSION
In the present study all the animals died within 3 to 3.5 hours after venom injection.
The dose of injected venom was in accordance with the LD50 level (4.5 mg/kg) determined in mice.The LD50 dose of Me venom in mice appears to be the lethal dose in rabbits.This venom dose was selected because we wanted to see the acute effect of venom within a limited time.
Electrocardiographic changes such as infarct-like pattern and arrhythmia, induced by injecting Me venom into rabbits, appeared to be the result of myocardial ischemia.
Ismail et al. (16) reported myocardial damage and cardiac arrhythmia following scorpion envenomation in experimental animals and Gueron et al. (9) reported such manifestations in humans.ST elevation or depression in limb and precordial leads, and the inversion of T and Q waves configure an acute myocardial infarction-like pattern.These electrocardiographic abnormalities have been observed following scorpion envenomation (1,17).This study showed myocytolysis, coagulation necrosis, focal hemorrhage and thrombus formation in myocardium and on endocardial surfaces as well as inflammation in the heart after Me venom in rabbits, The lungs showed alveolar edema, hemorrhage, vascular congestion thrombus and interstitial inflammation.Cardiac dysfunction, pulmonary edema and hemorrhage with inflammatory infiltrates in the heart and lung were previously described (18)(19)(20).
Release of cholinergic and adrenergic neurotransmitters from sympathetic and parasympathetic nerve terminals after the binding of scorpion toxins to sodium channels of axonal membranes has been demonstrated (21).In addition, scorpion venoms may have a direct effect on cardiac muscle fibers while their toxins may also inactivate sodium channels of cardiac cells (22).Cardiac troponin T elevation in one rabbit coupled with LDH elevation in serum of all animals is one of the interesting results of this study.Although the average levels of troponin and LDH after venom injection increased by almost two fold, neither alteration was statistically significant.
These variations may be explained by the different physiological response of each individual.Cardiac troponins, cardiac troponin T and cardiac troponin C comprise the gold standard for the detection of myocardial necrosis (23).Elevation of LDH in blood serum usually implies tissue injury (18).Although the ECG changes reveal myocardial damage in some of the envenomed rabbits, the important heart necrotic marker CK-MB did not show a significant elevation, perhaps due to the time required for serum elevation of this marker.
The other important finding was severe lung edema in all animals.Pulmonary edema is one of the most important complications following scorpion poisoning (18).A case report presented lung evidence compatible with adult respiratory distress syndrome and electron microscopic findings consistent with acute lung injury and increased alveolocapillary membrane permeability after Tityus serrulatus scorpion sting (24).
Two factors can induce pulmonary edema: a cardiogenic factor due to dysfunction of the left ventricle and a non-cardiogenic factor related to inflammatory response (13,14).In this study, 40-60 minutes after Me venom injection, all the rabbits showed signs resembling acute respiratory distress syndrome (ARDS).Animals presented decreases in respiratory rate, apnea, heavy open-mouth wheezing, bronchial secretions and severe decline in oxygen arterial saturation.This syndrome has been observed in ram lungs after Tityus discrepans scorpion envenomation (25).
Additionally D'Suze et al. (26) suggested that scorpion venom respiratory distress syndrome (SVRDS) should be implicated in place of pulmonary edema.Although only one animal in the present study showed myocardial infarction, all the specimens showed severe alveolar edema and respiratory distress which indicates that the pulmonary changes due to Me venom are non-cardiogenic in origin.Thrombi observed in rabbit lungs could be explained by venom damage of vascular endothelial cells or by actions exerted on the coagulation system.The consequence of thrombosis depends on the site of vessel occlusion, and can result in death (27).
Therefore, Me venom may be toxic to the vessel wall.Endothelial cells represent the first cellular barrier to blood-borne toxins that reach the deeper vessel wall layers.
These cells are particularly susceptible to toxic insult.Toxins that reach the subendothelial space may cause injury to medial smooth muscle cells and/or adventitial fibroblasts (27).Hence, Me venom may cause injury to endothelial cells and/or medial smooth muscle cells and/or adventitial fibroblasts.Furthermore, Me venom produced direct inflammatory reaction in the lungs and heart.Tityus discrepans venom also produced inflammatory reaction in different tissues and fibrin deposition (25).Our finding also agrees with another study that found a highmolecular-weight (100 kDa) toxin (T3) which, having been isolated from Mesobuthus tamulus venom, exhibited toxicity in cardiopulmonary parameters.It was called "pulmonary edema-producing toxin (PoTx)" and was identified as the toxic principle for the manifestation of pulmonary edema seen after Mesobuthus tamulus envenomation (15).
In conclusion, the present data show that Me venom injection in rabbits evoked severe pulmonary edema and death.Heart changes were also induced but can be excluded as the cause of death due to their lower severity and prevalence.
Therefore, pulmonary damage following envenomation may result from a direct effect of Me venom on endothelial cells of pulmonary vessels, whereas pulmonary edema following Me envenomation is not cardiogenic in origin.Understanding the cardiopulmonary complications that follow Me envenomation could lead us to a better therapeutic strategy.
Mesobuthus eupeus venom was obtained by electric shock at the scorpion telson in the Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute of Iran.It was lyophilized and stored at 4°C until use.

Figure 2 .Figure 3 .
Figure 2. (A) ST elevation in I, II and AVF, (B) ST depression in V1, V2 and V3, (C) Q waves in I, II and AVF at 20, 30 and 180 minutes after Me venom injection, respectively.

Table 1 .
Tissue necrosis marker changes in anesthetized rabbits before and one and three hours after Me venom injection Baseline: before venom injection; TnT: troponin T, CK-MB: creatinine kinase muscle-brain; LDH: lactate dehydrogenase; NS: not significant (p < 0.05).

of Venom on ECG, Blood Pressure, Heart Rate, Respiratory Rate and Arterial Oxygen Saturation In
one rabbit that showed higher TnT elevation in the serum and histopathological complications, as compared to other rabbits, the ECG showed an infarct-like pattern (ST elevation in I, II and AVF, ST depression in AVF, V1, V2 and V3, Q waves in I, II and AVF) at 20, 30 and 180 minutes after Me venom injection, respectively (Figure2).The electrocardiograph of another rabbit showed inverted T waves in I, II, AVL, and finally respiratory arrest.Blood pressure progressively decreased until rabbits died at 180 ± 30 minutes.Furthermore, the injection of Me venom caused no significant decrease in heart rate until death (Table2).