J Arthropod-Borne Dis, March 2018, 12(1): 31–40 Kh Pourkhalili et al.: Hemodynamic Changes in … 31 http://jad.tums.ac.ir Published Online: March 18, 2018 Original Article Hemodynamic Changes in Experimentally Envenomed Anaesthetized Rats by Intravenous Injection of Hemiscorpius lepturus Venom Khalil Pourkhalili 1, Hossein Fatemikia 2, Euikyung Kim 3, Navid Reza Mashayekhy 4, Naser Mohammadpour Dounighi 5, Abdollah Hajivandi 6, Yaghoob Hassan 7, *Ramin Seyedian 8 1Department of Physiology, Faculty of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran 2Department of Physiology, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 3College of Veterinary Medicine, Gyeongsang National University, Jinju, South Korea 4Department of Cardiology, Amir Kabir Hospital, Arak University of Medical Sciences, Arak, Iran 5Department of Human Vaccine and Serum, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran 6Department of Biostatistics, Faculty of Health, Bushehr University of Medical Sciences, Bushehr, Iran 7Student Research Committee, Bushehr University of Medical Science, Bushehr, Iran 8Department of Pharmacology, Bushehr University of Medical Sciences, Bushehr, Iran (Received 14 Jan 2017; accepted 13 Feb 2018) Abstract Background: We investigated the hemodynamic changes (Inotropic, chronotropic and arrhythmogenic) in intrave- nously envenomed anesthetized rats with Hemiscorpius lepturus venom. The neutralizing potencies of different drugs and commercial antivenom were assessed simultaneously. Methods: Different doses of the crude venom (100, 200 and 400μg/rat) were injected during five minutes via the femoral vein and cardiovascular changes were recorded in rats in Razi Institute Corporation, Karaj, Iran in 2017. The drugs (Atropine, lidocaine, propranolol and prazosin) were injected before the venom for determination of the coun- teracting effects. Different volumes (100, 500 and 1000µl) of the antivenom were pre envenomed to neutralize cardi- ovascular changes. Results: Temporary hypertension and bradycardia with no arrhythmogenic effects were depicted within twenty minutes. There was a difference in arterial pressure between the venom (400μg/rat) and the vehicle at 8 minutes (114.68±5.1mmHg versus 70.2±4.3mmHg). Elevation of the mean arterial pressure was inhibited by propranolol (2 mg/kg) and neutralized by prazosin (1mg/kg) while lidocaine (4mg/kg) and atropine (1mg/kg) had no effects. Pre- medication with Iranian commercial antivenom (1000μl) produced surprisingly temporary hypertension compared to the vehicle (140.84±4.5 versus 84.3±3.2). It had no neutralizing properties on blood pressure variation before the venom injection. Volume-expanded hypertension phenomenon was ruled out in a parallel study. Conclusion: This venom has vasoconstrictive effects in rats probably due to the presence of norepinephrine like ma- terials in its content or liberated from adrenal gland inhibited by prazosin premedication. The neutralizing effects of antivenom on venom-induced hypertension are questionable. Keywords: Hemiscorpius lepturus, Vasoconstriction, Polyvalent antivenom Introduction More than one million scorpion stings are reported in the world annually (1, 2). The re- sultant mortality is less than snake enven- omation but the physicians encounter mor- bidity sequels especially in infants (3, 4). Scor- pion bite is a prevalent problem in tropical areas of the world including southwestern part of Iran in particular Khuzestan Province (5, 6). The scorpion Hemiscorpius lepturus, a member of Hemiscorpiodie family, belongs to Iranian rich fauna from 17 genera (7). This scorpion is a member of 7 dangerous species *Corresponding author: Dr Ramin Seyedian, E-mail: raminseyedian@gmail.com http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 31–40 Kh Pourkhalili et al.: Hemodynamic Changes in … 32 http://jad.tums.ac.ir Published Online: March 18, 2018 living in Iran causing medically implications in envenomed patients (7, 8). The clinical observations of H. lepturus sting show diverse pathological forms including bloody urine due to hemoglobinuria or hemolysis, der- monecrotic reactions, cardiovascular effects, and in a few cases disseminated intravascu- lar coagulation in infants (9, 10). Although there is no comprehensive agree- ment about the efficacy of scorpion antiven- om in envenomed patients, close monitoring and injection of pepsin-digested Razi Institute polyvalent raised against 6 common Iranian scorpions (Androctonus crassicauda, Buthotus saulcyi, Buthus schach, Odontobuthus doriae, Mesobuthus eupeus and H. lepturus) is used in this country (6, 11, 12). There is no short-term efficacy of old world scorpion antivenom in the literature (13). Our former report on isolated rat hearts with Langendorff apparatus showed negative inotropic and late arrhythmogenic effects of this venom like loxosceles enven- omation (14, 15). The aim of this study was to evaluate the cardiotoxic properties of different doses of the scorpion venom by intravenous injection in rats. Due to the role of α1-adrenoceptors in the peripheral vasculature and β1 receptors in the heart maintaining blood pressure (16), their possible impacts on the cardiovascular activity were investigated. The diverse cardio- vascular effects induced by intravenous H. lepturus injection in anesthetized rats due to pharmacologically distinct causes. Materials and Methods Scorpion venom and antivenom The crude venom was obtained by apply- ing mild electrical shock (20mV). It was ex- tracted with normal saline and pooled, ly- ophilized and stored at -20 ℃ following cen- trifugation at 10000rpm for 15min. The mul- tivalent antivenom (5ml ampoules, stored at 2–8 ℃) was a pepsin-digested and concen- trated preparation obtained from equine hy- perimmune serum in Razi Institute Corpora- tion, Karaj, Iran in 2017. The protein content of this product was 3.6 mg/ml with a neutral- izing potency of 26 LD50/ml. Chemicals and drugs Atropine sulfate, prazosin hydrochloride, lidocaine hydrochloride and propranolol hy- drochloride were purchased from the Sigma Company (Germany). All the chemicals used were of the purest grade available. Anaesthetized rats Male Wistar rats (250–300gr) were placed in polycarbonate cages with free access to water and normal laboratory chow in the ani- mal house of Bushehr University of Medical Sciences. Three animals in each cage. All the animals were kept at 20±2 ℃ and maintained at 12h light-dark cycle starting at 7AM. Rats were anesthetized with ketamine (100mg/kg, IP) and xylazine (10mg/kg, IP), placed su- pine on a heated surgical table to keep the animal warm at 37±1 ℃, monitored through a rectal probe connected to a thermistor (Physitemp BAT-12, Texas Scientific In- struments, San Antonio, Texas, USA). A cannula was inserted into the right fem- oral vein for administration of venom and drugs. Another one was placed into the right femoral artery and connected to a pressure transducer (MLT844, AD Instruments, Aus- tralia) for continuous recording of the arterial pressure by means of a Power Lab/4SP data acquisition system (AD Instruments). Animals were allowed to become stable for 20min prior to administration of the venom or any drugs. Effects of Hemiscorpius lepturus venom on hemodynamic parameters Rats were divided into four groups (n= 5) and baseline hemodynamic status was record- ed for twenty minutes before venom injection. The first group was injected with normal sa- line (200μl) via femoral vein as the negative control. Each dose of the venom (100, 200, and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 31–40 Kh Pourkhalili et al.: Hemodynamic Changes in … 33 http://jad.tums.ac.ir Published Online: March 18, 2018 400μg/rat) dissolved in normal saline (200μl) was injected into experimental animals. Mean arterial pressure, heart rate and arrhythmogenic properties were evaluated compared with nega- tive control. Experimental protocol Cardiovascular changes provoked by the venom (400μg/ 200μl) were measured in the presence or absence of various chemicals. In this experiment, prazosin (1mg/kg), the α1 receptor antagonist was injected (n=5) via femoral vein before venom installation. This study was performed with propranolol (2mg/ kg), atropine (1mg/kg) and finally lidocaine (4mg/kg) in similar examinations. The hemo- dynamic effects were analyzed in the absence or the presence of each testing reagent. Antivenom premedication effects Rats were randomly divided into four ex- perimental groups (n=5). Escalating doses of the antivenom (100, 500, and 1000μl) dissolved in normal saline with same volume (1ml) were injected in 5min and venom instillation via femoral vein was performed after returning of arterial pressure to the premedication state. Neutralizing effects of this remedy on hemo- dynamic changes were compared with the con- trol groups. Data analysis Statistical analysis was performed using SPSS version 16.0 (Chicago, IL, USA). Re- peated measurement ANOVA was used to detect any differences among means in dif- ferent time intervals. Data were expressed as mean±SD. The level of statistical significance was P< 0.05 Animal ethics Ethical approval of all animal experiments was obtained from the Bushehr University of Medical Sciences Animal Ethics Committee (IR.BPUMS.REC.1396.90). Results Hemodynamic effects of Hemiscorpius lep- turus venom Transient blood pressure elevation and bradycardia induced with different doses (100, 200, and 400μg) of the venom were depicted in Table 1 and Fig. 1, respectively. Results represent mean ± SD of five independent experiments. Intravenous injection of venom- induced an elevation in mean arterial blood pressure (positive inotropic) and the decrease in heart rate (negative chronotropic) for 20min before ending the experiment an hour later. The minimum effective dose that dramatically evoked this effect (400μg/per rat) was used for further studies. No arrhythmogenic effects in- duced by the venom injection were seen com- pared with control (Fig. 2). No mortality was observed during the time following this study in each group. Existing data on controls re- vealed that the stress and surgical operation had no significant effects on hemodynamic changes and arrhythmogenic states in our experiments. Effects of pharmacological antagonists upon Hemiscorpius lepturus induced blood pres- sure alterations Pretreatment with lidocaine (4mg/kg) as a sodium channel blocker had no effects on blood pressure elevation (n= 5) (Fig. 3D). Atropine (1mg/kg) as an anticholinergic drug could not inhibit this phenomenon (Fig. 3C) but counteracted bradycardia (data not shown). Pretreatment with propranolol (2mg/kg), a nonselective beta antagonist, suppressed rising of blood pressure (Fig. 3B) and finally prazosin, a selective alpha-1adrenergic blocker, almost completely neutralized the responses induced with H. lepturus venom in rats (Fig. 3A). Effect of prophylactic treatment with an- tivenom on mean arterial pressure A large increase in blood pressure recorded http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 31–40 Kh Pourkhalili et al.: Hemodynamic Changes in … 34 http://jad.tums.ac.ir Published Online: March 18, 2018 following injection of Razi Institute antiven- om (100, 500 and 1000μl) in anesthetized rats via femoral vein in 5min. Antivenom injec- tion had no neutralizing effects on raising arterial pressure induced by the venom in all experiments as shown in Fig. 4 (A, B, C). Injection of physiological saline (1ml) had no effects on the mean arterial pressure in treated rats ruling out volume-expanded hy- pertension (Fig. 5). Pretreatment with these remedies did not induce the arrhythmia in anesthetized rats (not shown). Table 1. Mean arterial pressure in treated rats with Hemiscorpius lepturus venom injection Time (min) Control Dose(µg/rat) 100 200 400 0 70.1±3.4 66.7±4.3 74.3±5.1 60.9±5.6 4 69.4±2.8 77.7±3.6 83.1±4.3 95.3±4.9* 8 70.2±4.3 79.9±3.6 103.2±5.4* 114.7±5.1* 12 67.8±3.8 73.1±4.1 92.2±3.6* 102.4±5.5* 16 63.3±3.4 79.9±4.1 84.3±3.2 92.6±4.5* 20 61.2±3.8 73.5±3.6 71.2±4.2 78.6±4.1 Values are the mean ± SD of the mean arterial pressure of the five animals before and after intravenous injection of Hemiscorpius lepturus venom. *Significant difference from control group with ANOVA test, P< 0.05 Fig. 1. Heart rate changes with intravenous injection of Hemiscorpius lepturus in rats. Values are means ± SD of five rats/group Fig. 2. Arrhythmogenic analsis of Hemiscorpius lepturus venom upon intravenous injection in rats http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 31–40 Kh Pourkhalili et al.: Hemodynamic Changes in … 35 http://jad.tums.ac.ir Published Online: March 18, 2018 Fig. 3. Neutralizing effects of different drugs (Prazosin, Propranolol, Atropine, and Lidocaine) upon blood pressure elevation evoked by Hemiscorpius lepturus injection. The trace represents a single rat receiving a single dose of ven- om (representing measurements of five rats). Fig. 4. Intravenous pretreatment with different doses (100, 500, 1000μl) of multivalent antivenom diluted with nor- mal saline, evoked an increase in blood pressure in anesthetized rats. This remedy could not neutralize temporary changes in blood pressure evoked by Hemiscorpius lepturus venom http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 31–40 Kh Pourkhalili et al.: Hemodynamic Changes in … 36 http://jad.tums.ac.ir Published Online: March 18, 2018 Fig. 5. Effects of Iranian commercial multivalent antivenom diluted with normal saline on the mean arterial pressure of anesthetized rats. All remedies have equal volume (1ml). Data represent mean ± SD (n= 5) Discussion Scorpions belonging to class Arachnidae are relatively different from other arthropods including Insects, Crustacea and Myriapods (17). A large number of deadly scorpions be- long to the family of Buthidae while some species are from at least two other families Hemiscorpiidae and Scorpionidae posing a threat to humans (18). Most cases of scorpi- on stings (95%) only cause local discomfort including pain while in minority of enven- omed people devastating manifestations can be observed, involving nervous system, au- tonomic system and finally respiratory and heart failure leading to death especially in infants (19). Hemiscorpius lepturus and Androctonus crassicauda are the two most important haz- ardous scorpions in Iran (20). Very few ob- servations explain the complex cardiovascu- lar alterations and hemodynamic effects in- duced by Iranian scorpions like H. lepturus in animals (21, 22). Escalating doses of H. lepturus venom were selected in our study on the basis of arterial pressure modulations regardless of their LD50 via subcutaneous injection in rats (3.22mg/ kg) (23). There was no mortality following intravenous injection of the venom (400μg/ rat) up to an hour indicating that its strength in creating the cardiovascular response is much less than other venomous animals especially jellyfish and other scorpions (24-26). Some scorpions like Leiurus quinques- tratus could produce cardiovascular effects including hypertension, arrhythmia, tachycar- dia and enzymatic changes in envenomed pa- tients showing myocardial damage (27). Wid- ened QRS complexes, ST depression or ele- vation and the inversion of T and Q waves were not seen in our experiment up to 20min ruling out acute myocardial ischemia and in- farction-like patterns as other scorpions (28- 30) (Fig. 2). Venom-induced bradycardia was accord- ant with the previous study performed by sub- cutaneous injection (1500μg/kg) in rabbits (31). The inotropic potency, arrhythmogenic prop- erties and neutralization efficacy of pre and post treatment with Iranian antivenom in our study were completely different. Intravascu- lar instillation of H. lepturus provoked ex- treme elevation of mean arterial pressure af- ter 8min compared to normal saline (114.7±5.1 mmHg versus 70.2±4.3mmHg) probably due to release of catecholamines like substances including epinephrine and especially norepi- nephrine from the sympathetic nervous sys- tem and adrenal glands (32). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 31–40 Kh Pourkhalili et al.: Hemodynamic Changes in … 37 http://jad.tums.ac.ir Published Online: March 18, 2018 Another possibility was the presence of norepinephrine like materials in its own ven- om like other venomous creatures (33). Pre- medication with lidocaine (4mg/kg) as a so- dium channel blocker and atropine (2mg/kg) as an anticholinergic drug had no neutraliz- ing or summative effects on blood pressure alterations respectively. It was accordant with previous experiments carried on overwhelm- ing hemodynamic effects induced by marine venoms in rats (34, 35). This experiment is ruling out the activation of voltage-sensitive sodium channels in this phenomenon with our venom (36). Propranolol treatment as a non-selective beta blocker (2mg/kg) suppressed arterial pres- sure alterations in our study signifying the role of adrenergic receptors in blood pressure chang- es (37). Prazosin premedication as a selective alpha-adrenergic blocker (1mg/kg) neutralized almost completely undesired effects on the vas- culature (38). Venom-induced bradycardia might be due to cholinergic and adrenergic activities in rats (Fig. 2). Atropine premedication as an anti- cholinergic drug neutralized induced brady- cardia totally while propranolol as a nonse- lective beta blocker enhanced it (data not shown). Intravenous pretreatment with different doses of commercial Iranian antivenom (100, 500, 1000μl) with the same volume had no effects on hypertension-induced with H. lep- turus injection as shown in Fig. 4 (A, B, C). The mean arterial pressure was elevated ab- ruptly by injection of this remedy (1000μl) in five minutes while normal saline with the same volume had no effects (140.8±4.5 versus 84.3±3.2). More evaluation must be carried out to find the impurities in this product re- sponsible for this undesired phenomenon. The hypertensive potencies of this ven- om are less than other scorpions like Leiurus quinquestriatus (1000μg/kg versus 350μg/kg) (39). This may raise concerns about the lib- eration of neurotransmitters like norepineph- rine responsible for the elevation of blood pressure and bradycardia by releasing of ace- tylcholine from vagal ganglia. It requires per- forming this experiment in adrenalectomized rats treated with guanethidine in our further studies to reveal the exact mechanism of this phenomenon (32). Neutralization of hemody- namic changes with prazosin as a selective alpha-1 blocker supports this possibility. Our results on blood pressure were not compatible with the previous study on iso- lated rat hearts (15) possibly due to cardio- toxicity induced by enzymes like sphingo- myelinase-D presenting in both Loxosceles intermedia and H. lepturus venoms with sim- ilar clinical manifestations (40, 41). There is no consensus on using scorpion antivenom for treatment of undesirable cardiovascular chang- es in a human being after envenomation espe- cially for old world scorpions (42). Even in the United States, using antivenom should be limited to severe envenomations due to its questionable effects and high costs (43). Prazosin is used for decreasing of periph- eral vascular resistance in envenomation es- pecially in India while its effectiveness in re- duction of mortality and morbidity is approved only in a small controlled trial (44). Special evaluation must be carried out for finding spe- cific cardiac receptors to evaluate the cardio- vascular events thoroughly. Conclusion Scorpion envenomed patients are usually treated with symptomatic therapy and com- mercial antivenom infusion. Unfortunately, Iranian antivenom premedication and post medication (data not shown) either increase the blood pressure or had no neutralizing ef- fects in transient hemodynamic changes in- duced by H. lepturus injection in rats. Little is known about the role of other mediators (serotonergic, prostaglandins and nitric ox- ide inhibitors) to increase blood pressure in http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 31–40 Kh Pourkhalili et al.: Hemodynamic Changes in … 38 http://jad.tums.ac.ir Published Online: March 18, 2018 envenomed rats with this venom. Nonethe- less, more investigations must be performed to find the simplest way for counteracting ven- om-induced undesirable cardiovascular al- terations in rats. Acknowledgements The authors would like to thank the au- thorities of Bushehr University of Medical Sciences for providing facilities. The authors declare that there is no conflict of interests. References 1. Isbister GK, Bawaskar HS (2014) Scorpion envenomation. N Engl J Med. 371 (5): 457–463. 2. Chippaux JP, Goyffon M (2008) Epidemi- ology of scorpionism: a global ap- praisal. Acta Trop. 107(2): 71–79. 3. 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