J Arthropod-Borne Dis, March 2017, 11(1): 42–49 E Valikhanfard-Zanjani et al.: Antivenom Efficacy … 42 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Antivenom Efficacy in Neutralizing Histopathological Complications Following Latrodectus dahli Envenomation *Elham Valikhanfard-Zanjani 1, Abbas Zare-Mirakabadi 2, Ehsan Zayerzadeh 3 1Department of Biology, Faculty of Sciences, Kharazmi University, Tehran, Iran 2Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute, Karaj, Iran 3Department of Biology, Faculty of Food Industry and Agriculture, Standard Research Institute, Karaj, Iran (Received 17 Sep 2015; accepted 17 Nov 2015) Abstract Background: Nowadays use of specific antivenin for latrodectism is considered as the most effective treatment in the world. This study was undertaken to investigate the efficacy of specific antivenom against histopathological complications caused by Latrodectus dahli venom on liver, heart and kidneys tissues within 72h. Methods: Two groups were selected, each one contained 6 male New Zealand rabbits weighing 2±0.5kg. The ani- mals were anesthetized with 0.5ml ketamine and 0.5ml xylazine by intramuscular route. The L. dahli venom (0.5mg/kg) was injected subcutaneously to both the groups. The second group of rabbits 24h after the venom injec- tion received specific antivenom by intravenous route. Seventy-two hours after the venom and antivenom injections, the rabbits were dissected to obtain heart, liver and kidney tissues. The tissues were stained by hematoxylin and eo- sin stains and histopathological studies were examined by optical microscope. Results: In group one, the venom induced myocytolysis, myocarditis, coagulation necrosis in the heart tissue and the liver tissue showed central vein congestion, congested vessels, dilated sinusoids and inflammation. However, no sig- nificant histopathological complications were observed in kidney tissues. In the second group, antivenom injection greatly prevented escalation of the complications on foresaid tissues. Conclusion: Latrodectus dahli venom induces histopathological complications on vital organs. Specific antivenom injection, 24h after the venom injection, could protect the tissues from incidence and intensification of histopatho- logical complications. Future studies in human beings should be conducted to assess the protection against the spe- cific-Latrodectus antivenin. Keywords: Latrodectus, Antivenom, Histopathological complication Introduction Black widow spider envenomation, because of systemic complications, sometimes fatal, has become as a candidate for the most dan- gerous arachnids in the world (Vetter and Isbister 2007, Rey et al. 2011). Among more than 40 species that are identified in all over the world, only 4 species were reported as inhabitant of Iran which including Latrodec- tus tredecimgottatus, L. dahli, L. geometricus and L. pallidus (Rafinejad et al. 2007, Afshari et al. 2009, Shahi et al. 2011). The Latrodectus spp. are predominantly distributed in the North East and the South of the country. Latrodectism, syndrome caused by Latrodec- tus spp., generally is characterized by pain, muscular weakness, nausea, vomiting, pain- ful abdominal cramping, perspiration and car- diovascular complications (Marzan 1955, Ma- retic 1983, Prior and Park 2004). The most reports of latrodectism in Iran return to the 1994, so that nearly 190 cases of latrodectism were reported only in the Khorasan Province (Mirshamsi kakhaki 2005, Afshari et al. 2009). Black widow spider venom contains a neu- *Corresponding authors: Miss Elham Valikhanfard- Zanjani, E-mail: evalikhanfard@yahoo.com J Arthropod-Borne Dis, March 2017, 11(1): 42–49 E Valikhanfard-Zanjani et al.: Antivenom Efficacy … 43 http://jad.tums.ac.ir Published Online: March 14, 2017 rotropic protein termed α- Latrotoxin which is thought to be responsible for the most clin- ical effects in human (Nelson et al. 2011, Nentwig and Nentwig 2013). α-LTX exerts its destructive effects through interaction with specific receptors termed neurexin and latro- philin, so that leads to neurotransmitter deple- tion of both cholinergic and adrenergic ter- minals. Given that the highest expression of the receptors are in neurons of nervous sys- tem, so it seems that nervous system is the primary target of black widow spider venom (Henkel and Sankaranarayanan 1999, Ush- karyov et al. 2008, Silva and Ushkaryov 2010). Duration of latrodectism in untreated cases can be different in ranges, from a few hours to several days (Malley et al. 1999, Vetter and Isbister 2007). In the past, in order to treat symptoms caused by latrodectism, vari- ous medications were used, like calcium glu- conate and benzodiazepines. The success rates of these drugs are different but all of them were used just to relieve pain and other symp- toms caused by envenomation (Allen and Norris 1991, Clark et al. 1992). Nowadays usage of antibodies and anti- body fragments are considered as the most effective treatment in latrodectism. However, there is a substantial controversy about the route and time of black widow spider anti- venom injection, so that previously black wid- ow specific antivenom was injected intra- muscularly, but according to the published data in recent decades, intramuscular route is not efficient, so intravenous route was pro- posed. However, determination of antivenom ability to reverse complications of black wid- ow envenomation needs more investigations (Isbister 2002, Brown et al. 2007, Ahmed and Bushra 2008). So far, most of clinical studies performed on latrodectism, were concerning on the ven- om effects on organs functions. Considera- bly less attention has been paid to venom effects on organs tissues. Expressions of the certain alpha latrotoxin receptors on some of the mammalian tissues, makes the direct effects of venom expectable (Herberth et al. 2005). Therefore the present study was under- taken to investigate effects of L. dahli venom on liver, heart and kidneys tissues within 72h. Besides, we evaluated efficacy of anti- venom to inhibit lethality and neutralizing potency on toxicity effects of L. dahli venom within 72h. The findings of the present in- vestigation can be an important step to im- prove guidelines for an optimal immunother- apeutic treatment of spider envenomation. Materials and Methods Venom and Antivenom Crude spider venom and specific antivenom were provided by Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute, Ka- raj, Iran. In order to obtain the crude venom, L. dahli spiders were dissected out, and a pair of glands was collected into ice cold phosphate buffered saline (PBS). The glands were washed in PBS in order to remove pos- sible contaminants, and venom was harvest- ed in PBS by gentle compressing of the glands. The suspension was clarified by cen- trifugation at 8000rpm, and the venom was stored at −20 °C until use. Experimental Protocols Two groups of New Zealand white rab- bits weighing 2±0.5kg (6 animals in each group) were selected. Before the experiment all animals were maintained for at least 3 d, under conditions of controlled light (12h light, 12h dark), temperature (18–22 °C) and humidity (55±5%), with standard diet and water available ad libitum. At first rabbits were anaesthetized with intramuscular injec- tion of 0.5ml ketamine and 0.5ml xylazine in ratio 1:1 respectively. In order to study the hepatotoxicity, myotoxicity and nephrotoxici- ty of L. dahli venom, the first group received only L. dahli venom (0.5mg/kg) through sub- J Arthropod-Borne Dis, March 2017, 11(1): 42–49 E Valikhanfard-Zanjani et al.: Antivenom Efficacy … 44 http://jad.tums.ac.ir Published Online: March 14, 2017 cutaneous route, while the second group 24h after the venom injection, received specific antivenom (2.5ml with neutralization capaci- ty of 500 LD50/ml) by IV route. Seventy- two h after the venom and antivenom injec- tion, experiment ended by scarifying the rab- bits for obtaining the heart, liver and kidney tissues throughout the surgery. Histological Analysis After the animals died, the heart, liver and kidneys were removed carefully and immersed in 10% formaldehyde at room temperature and then sectioned transversely into 5μm slices. Specimens were dehydrated in a graded se- ries of alcohol and xylene and embedded in paraffin. Multiple slices (15 fields for each slide) were made and stained by hematoxilin and eosin stains. Sections were viewed and were photographed using a Nikon E200® light microscope (Japan). This study was approved by Ethics Com- mittee of this institute. Razi Vaccine and Se- rum Research Institute. Results The signs and symptoms of envenomation appeared within first few hours by redness, mild swelling and muscle cramps at injection site. Twenty-four h after the venom injection difficulty in respiration was observed in most of the animals. Following 72h after the venom (0.5mg/ kg, S.C) and the antivenom (2.5ml, I.V) in- jection, histological changes were investigat- ed in the rabbit’s heart, liver and kidney tis- sues in each group. Figure 1 shows the histo- pathological evaluation of the first group of animals, which venom injection induced com- plications such as myocytolysis, myocarditis, coagulation necrosis, myocardial edema, hem- orrhage and inflammation in the heart tissues (Fig.1). On the other hand, evaluation of the liver tissues, showed central vein congestion, congested vessels in portal areas, dilated si- nusoids and inflammation, however no sig- nificant histopathological complications were observed in kidney tissue within 72 h after the venom injection (Fig. 1). In the second group of the animals, im- munotherapy prevented severe myocytolysis, edema and hemorrhage in the heart of treated animals. However, in this group, mild my- ocytolysis and mild hemorrhage in the heart were still observed in the experimented ani- mals (Fig. 2). Antivenom injection also pre- vented severe central vein congestion and in- flammation in the liver. However, mild con- gestion and mild inflammation were observed in the livers of this group (Fig. 2). Fig. 1. Histopathological changes of heart (A, B), liver (C, D) and kidney (E, F) tissues induced by Latrodectus dahli venom (0.5mg/kg, S.C). Within 72h after the venom injection myocytolysis, lympho- mononuclear inflammatory infiltrate on the myocar- dium (A), hemorrhage, myocarditis and myocytolysis (B) are presented. The central vein congestion (C and D) in the liver tissues are presented. Following the venom injection no changes were observed in the kidney tissue (E and F) (Original) J Arthropod-Borne Dis, March 2017, 11(1): 42–49 E Valikhanfard-Zanjani et al.: Antivenom Efficacy … 45 http://jad.tums.ac.ir Published Online: March 14, 2017 Fig. 2. The histopathological changes of heart (A, B), liver (C, D) and kidney (E, F) tissues following the antivenom injection (2.5ml, I.V) 24h after the venom injection. The antivenom prevented severe myocytol- ysis, edema and hemorrhage in the heart tissue (A), mild myocytolysis and mild hemorrhage are observed in one of the rabbit’s heart (B). The antivenom pre- vention of the severe central vein congestion and in- flammation in the liver are presented (C), mild in- flammation were observed in one of the rabbit’s livers in this group (D). No changes were observed in the kidney tissues within 72h after the antivenom injec- tion (E and F) (Original) Discussion In the present study, the toxic effects of L. dahli venom were investigated within 72h on vital organs including the liver, heart and kidney tissues. The findings showed that, the venom injection induced severe cardiovas- cular complications such as myocytolysis, my- ocarditis and myocardial hemorrhage in heart. In addition, the venom injection evoked cen- tral vein congestion and dilated sinusoids in liver. The spider genus Latrodectus, because of the extreme potency of their neurotoxic com- pound termed α-latrotoxin, is one of the most notorious spiders in all over the world (Garb et al. 2004). Clinical manifestations of latrodectism, syndrome caused by Latrodectus, indicated that the nervous system is the pri- mary target of α –latrotoxin (α-LTX), which follows affecting whole body of organism (Vetter and Isbister 2007). According to our study, it seems that heart is one of the main goals of the venom. Besides, the venom in- jection induced some histopathological com- plications such as myocytolysis, myocarditis, coagulation necrosis, myocardial edema and hemorrhage and tissue inflammation. Ap- proximately, in all performed studies on latrodectism, cardiovascular complications have been reported mostly manifested by arrhyth- mia, tachycardia, bradycardia and ECG chang- es (Maretic and Stani 1954, Pulignano et al. 1998, Akyildiz et al. 2009). According to the same reports, the changes in ECG waves af- ter black widow spider venom injection were observed clearly, so that ST waves changes and widening of QRS waves indicated my- ocardial ischemia and myocardial damage, respectively. On the other hand, cardiomyo- pathy and myocarditis were reported earlier (Akyildiz et al. 2009, Rajabi 2014), detected by reduced height of R and P waves respec- tively. Some previous studies (Pulignano et al. 1998, Akyildiz et al. 2009, Rajabi 2014) have reported increasing in cardiovascular enzy- matic markers such as CPK, CK-MB and LDH after black widow spider venom injec- tion, which are the indicators of myocardial injury, heart muscle inflammation and ar- rhythmia. Increasing in foresaid enzymes, are quite compatible to the results of the present study. On the other hand, the histological find- ings confirmed the incidence of myocarditis, hemorrhage and inflammation in heart tis- sues after black widow envenomation (Clark et al. 1992, Pulignano et al. 1998, Akyildiz et al. 2009). In addition, occurrence of myo- cytolysis, and myocardial edema in Marzan and Maretic studies after Latrodectus enven- EE E J Arthropod-Borne Dis, March 2017, 11(1): 42–49 E Valikhanfard-Zanjani et al.: Antivenom Efficacy … 46 http://jad.tums.ac.ir Published Online: March 14, 2017 omation are another confirmation for the re- sults of the present study (Maretic and Stani 1954, Marzan 1955). The liver and the kid- neys because of their extensive blood supply network are as one of the most vulnerable organs to toxin injury (Sitprija and Sitprija 2012). Histological examination performed on the liver tissues showed that within 72h the venom caused central vein congestion, congested vessels in portal areas, dilated si- nusoids and inflammation. Marzan and Ma- retic investigations confirm the pathological effects of the black widow spider venom on the liver tissue. According to their observa- tions, after 30min to 6h of the venom injec- tion, the hepatic cells swollen and gradually massive hyperaemia appears, pericapillary ede- ma after 10 h; necrosis and lobular necrosis were seen respectively after 12 and 24 h of the venom injection (Maretic 1953, Marzan 1955). Following the venom injection, sig- nificant increasing in AST and ALT levels are good indicators for cytotoxic effects of the venom on the liver tissue. So that the AST to ALT ratio within 24h after the ven- om injection was more than 1 (< 1), which from clinical point of view, it is a symptom of impaired liver function (Pulignano et al. 1998, Valikhanfard et al. 2014). These re- sults further confirm our findings in the pre- sent study. The kidney tissues, within 72h after the venom injection, unexpectedly showed no sig- nificant histopathological complications. While other histological studies (Maretic 1953, Mar- zan 1955) have shown that after 10–20h of black widow spider venom injection, degen- eration of tubular epithelium with necrosis (within 24 hours) was seen in kidney tissue. Significant increasing in creatinine, bilirubin and urea, as important kidney health parame- ters, reported within 24h after Latrodectus venom injection, represents toxic effects of the venom on kidney function (Maretic 1953, Marzan 1955, Valikhanfard et al. 2014). Alt- hough the recent performed studies demon- strate toxic effects of black widow spider venom on kidneys, however we did not ob- serve any significant toxic effects on kidney tissues in our survey. Other purpose of this study was to evalu- ate the efficacy of specific antivenom in neu- tralizing and prevent escalation of the com- plications on vital organs. According to the obtained results, in the second group of rab- bits, the antivenom 24h after the venom in- jection could protect the heart tissue from severe myocytolysis, edema and hemorrhage. However, mild myocytolysis and mild hem- orrhage were observed in some rabbits of this group. In addition, the antivenom prevented histopathological effects on liver tissues, so that except mild congestion and mild inflam- mation observed in some rabbits; antivenom could protected liver from severe central vein congestion, dilated sinusoids and sever inflam- mation. Hence it seems that the antivenom injection even 24h after the venom, greatly could prevent incidence and intensification of histopathological complications caused by toxic effects of the venom. However, the mild histopathological effects observed in rabbit tissues may be due to the late (24h after ven- om injection) injection of antivenom. The obtained results of the antivenom efficacy in the present study corresponded with some studies performed on reducing concentrations in foresaid organs serum enzymes follow in- travenous injection of antivenom (Rajabi 2014, Valikhanfard et al. 2014). Based on same stud- ies, 72h after the antivenom injection, sig- nificant decreasing was observed in AST, ALT, Urea and Bilirubin, so that the AST to ALT ratio in this period decreased to phys- iological state, that is less than 1 (< 1). Ac- cording to the same reports, significant de- creasing were observed in CPK, CK-MB and LDH, as predominant heart health markers, also ECG waves returned to its initial state (before venom injection) (Rajabi 2014, Vali- khanfard et al. 2014). Black widow spider venom exerts its destructive effects by both J Arthropod-Borne Dis, March 2017, 11(1): 42–49 E Valikhanfard-Zanjani et al.: Antivenom Efficacy … 47 http://jad.tums.ac.ir Published Online: March 14, 2017 direct and indirect pathways. Alpha latrotox- in, the neurotropic venom compound, by in- teraction with specific receptors on nerve cells, results in releasing of huge amount of cate- cholamines (Henkel and Sankaranarayanan 1999, Ushkaryov et al. 2008, Silva and Ush- karyov 2010). Hence it seems catecholamines, are the main mediator of the venom effects on organs (Clark et al. 1992). The α-LTX af- ter entering the circulation, through activat- ing L-type calcium channels, which are abun- dant on vascular smooth muscle cells, results in calcium influx, which leads to vasocon- striction and hypertension (Sitprija and Sit- prija 2012). Catecholamines, by interaction with beta adrenoceptors on cardiovascular system re- sult in activation of L- type calcium channel. Influx of calcium leads to vasoconstriction and hypertension in smooth muscle cells of vascular system, on the heart tissue, activa- tion of L- type calcium channel causes an in- crease in cytosolic calcium in myocytes, which subsequently induces the cells death (Opie et al. 1985, Van der Heyden et al. 2005). On the other hand, catecholamines by stimulating cat- echolamine- sensitive lipase in myocardium lead to an increasing in catabolism of free fat- ty acids. Free fatty acids increasing in myo- cardium by disrupt the process of oxidative phosphorylation, lead to reduced ATP syn- thesis, which is other way to induce myo- cytes death (Opie 1975). Beside all foresaid mechanisms, accord- ing to immunological studies of Herberth in 2005 some latrophilin receptor (α- Latrotoxin independent calcium receptor) genes are ex- pressed in various mammalian tissues, so that the highest expression of latrophilin II can be found in placenta, lungs, liver and mammary glands tissues, respectively. However, some expressions of this gene have also been demon- strated in the heart and kidney tissues (Her- berth et al. 2005). Hence, expressions of the latrophilin II, on some of the mammalian tis- sues, makes the direct effects of the venom expectable, so that latrophilin II by activa- tion of PLC and DAG causes huge released internal calcium from endoplasmic reticulum (Henkel and Sankaranarayanan 1999, Ush- karyov et al. 2008). It can be predicted that black widow neu- rotropic venom through interaction with latro- philin receptors expressed on various mam- malian organs, induce its cytotoxic effects directly. Conclusion Latrodectus dahli venom injection in rab- bits, within 72 h, evoked severe cardiovas- cular complications such as myocytolysis, my- ocarditis, myocardial edema, hemorrhage and inflammation in heart. In addition, the venom injection induced central vein congestion, con- gested vessels in portal areas, dilated sinus- oids and inflammation in liver. 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