Histopathological Effects of Montivipera Xanthina Venom on Rats
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H. TOPYILDIZ, S. HAYRETDAĞ Turk J Zool 2012; 36(4): 517-525 © TÜBİTAK Research Article doi:10.3906/zoo-1007-23 Histopathological eff ects of Montivipera xanthina venom on rats Hüseyin TOPYILDIZ, Sibel HAYRETDAĞ* Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, 17100 Çanakkale - TURKEY Received: 16.07.2010 Abstract: In this study, the histopathological changes caused by Montivipera xanthina venom to rat skin, skeletal muscle, and liver tissue were analysed by light microscope. Th e venom (approximately 2.85 mg/kg) was injected intramuscularly into the gastrocnemius muscle of the rats. Th e animals were dissected 1, 3, and 6 h aft er the injection time, and tissue samples were taken. Sections 5 μm thick were taken from paraffi n blocks of tissue samples, and the sections were stained with haematoxylin and eosin in order to facilitate examination by light microscope. Examinations showed no damage to the epidermis layer of skin. However, damage to the dermis layer, including oedema, haemorrhage, local bleeding, rare infl ammatory cells, strong cell infi ltration with mixed characteristics, infl ammation around the veins, and fat necrosis, was detected. Th e damage observed in muscular tissue was myonecrosis, mixed character cell infi ltration, haemorrhage, and the formation of inclusion on myofi brils. Th e liver tissue revealed sinusoidal congestion and hepatocellular degeneration. Key words: Montivipera xanthina, histopathology, venom, skin, skeletal muscle, liver Sıçanlarda Montivipera xanthina venomunun histopatolojik etkileri Özet: Bu çalışmada Montivipera xanthina zehirinin, sıçanların deri, iskelet kası ve karaciğer dokularında meydana getirdiği histopatolojik değişiklikler ışık mikroskobu ile incelenmiştir. Venom (yaklaşık 2,85 mg/kg) sıçanların gastrocnemius kası içerisine enjekte edilmiştir. Enjeksiyondan 1, 3 ve 6 saat sonra hayvanlar disekte edilmiş ve doku örnekleri alınmıştır. Bu örneklerden hazırlanan parafi n bloklardan 5 mikron kalınlığında alınan kesitler hematoksilen ve eosin ile boyanarak ışık mikroskobunda incelenmiştir. İncelemeler sonucunda derinin epidermis kısmında bir hasara rastlanmamıştır. Dermis kısmında ise ödem, hemoraji, lokal kanlanmalar, seyrek iltihabi hücreler, karışık karakterli şiddetli hücre infi ltrasyonu, damar etrafı yangı ve yağ nekrozu tespit edilmiştir. Kas dokusunda miyonekroz, karışık karakterli hücre infi ltrasyonu, hemoraji, miyofi brillerde inklüzyon oluşumu gözlenen hasarlardır. Karaciğer dokusu incelendiğinde sinüzoidal kanlanma ve hepatoselüler dejenerasyon tespit edilmiştir. Anahtar sözcükler: Montivipera xanthina, histopatoloji, venom, deri, iskelet kası, karaciğer * E-mail: [email protected] 517 Histopathological eff ects of Montivipera xanthina venom on rats Introduction aim of this study was to histopathologically examine Venom in snakes is produced, stored, and secreted the eff ects of intramuscularly injected M. xanthina in venom glands (Duvernoy’s glands). Th e venom venom on rat tissues such as skin, skeletal muscle, consists of organic ingredients such as proteins, and liver tissues. glycoprotein, and phospholipids, as well as inorganic ingredients such as Ca, Cu, Fe, K, Mg, Mn, Na, P, Co, Materials and methods and Zn. Components of the venom vary between snake species and that variation produces diff erent Montivipera xanthina and venom reactions in living organisms (Bjarnason and Fox, Venom was extracted from adolescent M. xanthina 1989). individuals from the Aegean Region in the western Th e eff ects of venom can be classifi ed into 2 part of Turkey. Th e snakes were kept in a terrarium separate groups: neurotoxins and haemolytic toxins. in the laboratory. When the venom was extracted, Some of the main biological eff ects of snake venoms the snakes were ‘secured’ on the ground by pushing are neurotoxic and cause coagulation, cytotoxic, on the spot between their heads and necks with an haemolysis, haemorrhagic activity, hypotension, and L-shaped stick, and their heads were held with bare necrosis (Bjarnason and Fox, 1989; Warrell, 1989). hands. Th e snakes’ venom teeth were sunk into a 50-mL beaker that was prepared beforehand and Leonardi et al. (2001) established that Vipera ammodytes haemorrhagin protein 1, which is covered with Parafi lm. Th e venom in the beaker was present in the venom of Vipera ammodytes, causes transferred into sterilised tubes. Th e venom in the coagulation in the blood. Another relevant study tubes was immediately centrifuged at 2850 rpm for suggests that Macrovipera lebetina venom causes 5 min through a cooling centrifuge (Arıkan et al., coagulation as well (Samel et al., 2002). In another 2003). Aft er precipitating tissues and other particles study carried out on albino rats and guinea pigs, to the bottom of the tubes, the supernatant was Vipera ammodytes venom was the cause of gangrene transferred to a sterilised Eppendorf tube. Th e clean found in muscular tissue (Balija et al., 2005). A study venom was extracted, lyophilised, and stored at −20 executed by Aznaurian and Amiryan (2006) focused °C (Acosta et al., 2003). Before application to the rats, on the damage caused by Montivipera raddei venom the venom was dissolved in 10 μL of physiological to rabbit tissues including the liver, heart, kidneys, saline solution according to dose. lungs, spleen, and adrenal gland. Results of the study Dosage showed that the venom causes serious tissue damage Th e dose given to the rats was based on a ratio of such as kidney failure due to an accumulation of 200 mg of venom with a value of LD for a human blood in the glomerulus as a result of nephrotoxic 50 body weighing 70 kg (Başoğlu and Baran, 1980). In eff ects and cell fragmentation-based gangrene in accordance with this formula, individual doses were lung tissue. calculated for the rats; their weights varied between Research has established that 42 snake species 200 and 400 g. Th e venom doses calculated for each inhabit Turkey, and 13 of these species are venomous. rat were dissolved in 10 μL of physiological saline Among the venomous species, 1 belongs to Elapidae, solution and intramuscularly injected into the right 10 are from Viperidae, and the remaining 2 are gastrocnemius muscle of the rats. semivenomous (Başoğlu and Baran, 1980; Baran, Animals and venom application 2005). Th e endemic snake M. xanthina is one of the most dangerous among these species (Baran and Th e 25 male rats (albino Wistar; Rattus rattus) used Atatür, 1998). A set of studies executed by Arıkan et in this study were purchased from the Experimental al. (2003) gives information about the electrophoretic Animals Laboratory of the Çapa Faculty of Medicine protein structures of venoms from a number of of İstanbul University. Th e 25 male rats were divided venomous snakes, including M. xanthina. However, into 5 groups (n = 5 animals per group). Th ese there are no histological studies available on the groups were used as the control (groups 1 and 2) damage caused by M. xanthina venom on tissues. Th e and assay (groups 3, 4, and 5). No application was 518 H. TOPYILDIZ, S. HAYRETDAĞ made to the fi rst control group (control 1). Since the groups at 1, 3, and 6 h showed no signs of change. venom was diluted in physiological saline solution, In contrast, however, superfi cial dermis oedema and 10 μL of physiological saline solution (0.9% NaCl) collagen degeneration was observed on the dermis was injected into the right gastrocnemius muscle layer from all application groups (Figures 2-4). Th e of the second control group (control 2). Aft er the eff ect was 100% on the skins of the 1-h application intramuscular venom injection, the rats from the group and 80% on the skins of the 3-h and 6-h fi rst, second, and third assay groups were dissected 1, application groups (Table). Rare infl ammatory cells 3, and 6 h later, respectively. on the dermis and large haemorrhagic areas on the hypodermis appeared on all samples (Figures 2-3). Histopathological examinations Also evident in the same areas were infl ammatory Th e rats were anaesthetised with ether and dissected cell infi ltration and infl ammation, which had an through a cervical dislocation to take samples of intense mixed character, especially around the veins their skin, liver, and skeletal muscle tissues 1, 3, and (Figure 5). 6 h aft er the venom injection. Aft er being fi xed in Liver Bouin solution and following routine histological procedures, the tissues were embedded in paraffi n Th e sections taken from control group rat liver blocks. Sections 5 μm thick were taken from these tissues had a normal appearance (Figures 6a and blocks and stained with haematoxylin and eosin 6b). Congestion and hepatocellular degeneration (McManus and Moury, 1964). Stained preparations were detected within liver sinusoidal cells in all of were examined under an Olympus CX21 light the groups that were subjected to venom. Th is eff ect microscope; images were taken using an Olympus applied to 100% of the application groups (Table) BX51 light microscope and Olympus Analysis LS (Figures 7-9). soft ware. Muscle Th e muscular tissues from the control groups were Results of normal appearance (Figures 10a and 10b). In the samples of rat muscle tissues taken 1 h aft er the venom Skin application, haemorrhage, myonecrosis, and mixed Th e skin samples taken from the control 1 and character cell infi ltration were generally detected control 2 groups conformed to set norms (Figure in the perimysium and endomysium (Figures 11a 1). Th e epidermis layers from the experimental and 11b). Th e muscular tissues taken 3 h aft er the 100 μm 100 μm Figure 1. Skin section of rats in control group: epidermis (E), Figure 2. Rare infl ammatory cells (➡), oedema and collagen dermis (D). Stained with haemotoxylin and eosin degeneration (▲), fat necrosis (asterisk), and local (H&E). haemorrhage (➞) in the skin section of 1-h application group. H&E. 519 Histopathological eff ects of Montivipera xanthina venom on rats 100 μm 100 μm Figure 3.