histopathological eff ects of montivipera xanthina...

H. TOPYILDIZ, S. HAYRETDAĞ

517

Turk J Zool

2012; 36(4): 517-525

© TÜBİTAK

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

Research Article

* E-mail: [email protected]


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Introduction

Venom in snakes is produced, stored, and secreted in venom glands (Duvernoy’s glands). Th e venom consists of organic ingredients such as proteins, glycoprotein, and phospholipids, as well as inorganic ingredients such as Ca, Cu, Fe, K, Mg, Mn, Na, P, Co, and Zn. Components of the venom vary between snake species and that variation produces diff erent reactions in living organisms (Bjarnason and Fox, 1989).

Th e eff ects of venom can be classifi ed into 2 separate groups: neurotoxins and haemolytic toxins. Some of the main biological eff ects of snake venoms are neurotoxic and cause coagulation, cytotoxic, haemolysis, haemorrhagic activity, hypotension, and necrosis (Bjarnason and Fox, 1989; Warrell, 1989).

Leonardi et al. (2001) established that Vipera ammodytes haemorrhagin protein 1, which is present in the venom of Vipera ammodytes, causes coagulation in the blood. Another relevant study suggests that Macrovipera lebetina venom causes coagulation as well (Samel et al., 2002). In another study carried out on albino rats and guinea pigs, Vipera ammodytes venom was the cause of gangrene found in muscular tissue (Balija et al., 2005). A study executed by Aznaurian and Amiryan (2006) focused on the damage caused by Montivipera raddei venom to rabbit tissues including the liver, heart, kidneys, lungs, spleen, and adrenal gland. Results of the study showed that the venom causes serious tissue damage such as kidney failure due to an accumulation of blood in the glomerulus as a result of nephrotoxic eff ects and cell fragmentation-based gangrene in lung tissue.

Research has established that 42 snake species inhabit Turkey, and 13 of these species are venomous. Among the venomous species, 1 belongs to Elapidae, 10 are from Viperidae, and the remaining 2 are semivenomous (Başoğlu and Baran, 1980; Baran, 2005). Th e endemic snake M. xanthina is one of the most dangerous among these species (Baran and Atatür, 1998). A set of studies executed by Arıkan et al. (2003) gives information about the electrophoretic protein structures of venoms from a number of venomous snakes, including M. xanthina. However, there are no histological studies available on the damage caused by M. xanthina venom on tissues. Th e

aim of this study was to histopathologically examine

the eff ects of intramuscularly injected M. xanthina

venom on rat tissues such as skin, skeletal muscle,

and liver tissues.

Materials and methods

Montivipera xanthina and venom

Venom was extracted from adolescent M. xanthina

individuals from the Aegean Region in the western

part of Turkey. Th e snakes were kept in a terrarium

in the laboratory. When the venom was extracted,

the snakes were ‘secured’ on the ground by pushing

on the spot between their heads and necks with an

L-shaped stick, and their heads were held with bare

hands. Th e snakes’ venom teeth were sunk into a

50-mL beaker that was prepared beforehand and

covered with Parafi lm. Th e venom in the beaker was

transferred into sterilised tubes. Th e venom in the

tubes was immediately centrifuged at 2850 rpm for

5 min through a cooling centrifuge (Arıkan et al.,

2003). Aft er precipitating tissues and other particles

to the bottom of the tubes, the supernatant was

transferred to a sterilised Eppendorf tube. Th e clean

venom was extracted, lyophilised, and stored at −20

°C (Acosta et al., 2003). Before application to the rats,

the venom was dissolved in 10 μL of physiological

saline solution according to dose.

Dosage

Th e dose given to the rats was based on a ratio of

200 mg of venom with a value of LD50

for a human

body weighing 70 kg (Başoğlu and Baran, 1980). In

accordance with this formula, individual doses were

calculated for the rats; their weights varied between

200 and 400 g. Th e venom doses calculated for each

rat were dissolved in 10 μL of physiological saline

solution and intramuscularly injected into the right

gastrocnemius muscle of the rats.

Animals and venom application

Th e 25 male rats (albino Wistar; Rattus rattus) used

in this study were purchased from the Experimental

Animals Laboratory of the Çapa Faculty of Medicine

of İstanbul University. Th e 25 male rats were divided

into 5 groups (n = 5 animals per group). Th ese

groups were used as the control (groups 1 and 2)

and assay (groups 3, 4, and 5). No application was


519

made to the fi rst control group (control 1). Since the

venom was diluted in physiological saline solution,

10 μL of physiological saline solution (0.9% NaCl)

was injected into the right gastrocnemius muscle

of the second control group (control 2). Aft er the

intramuscular venom injection, the rats from the

fi rst, second, and third assay groups were dissected 1,

3, and 6 h later, respectively.

Histopathological examinations

Th e rats were anaesthetised with ether and dissected

through a cervical dislocation to take samples of

their skin, liver, and skeletal muscle tissues 1, 3, and

6 h aft er the venom injection. Aft er being fi xed in

Bouin solution and following routine histological

procedures, the tissues were embedded in paraffi n

blocks. Sections 5 μm thick were taken from these

blocks and stained with haematoxylin and eosin

(McManus and Moury, 1964). Stained preparations

were examined under an Olympus CX21 light

microscope; images were taken using an Olympus

BX51 light microscope and Olympus Analysis LS

soft ware.

Results

Skin

Th e skin samples taken from the control 1 and

control 2 groups conformed to set norms (Figure

1). Th e epidermis layers from the experimental

groups at 1, 3, and 6 h showed no signs of change. In contrast, however, superfi cial dermis oedema and collagen degeneration was observed on the dermis layer from all application groups (Figures 2-4). Th e eff ect was 100% on the skins of the 1-h application group and 80% on the skins of the 3-h and 6-h application groups (Table). Rare infl ammatory cells on the dermis and large haemorrhagic areas on the hypodermis appeared on all samples (Figures 2-3). Also evident in the same areas were infl ammatory cell infi ltration and infl ammation, which had an intense mixed character, especially around the veins (Figure 5).

Liver

Th e sections taken from control group rat liver tissues had a normal appearance (Figures 6a and 6b). Congestion and hepatocellular degeneration were detected within liver sinusoidal cells in all of the groups that were subjected to venom. Th is eff ect applied to 100% of the application groups (Table) (Figures 7-9).

Muscle

Th e muscular tissues from the control groups were of normal appearance (Figures 10a and 10b). In the samples of rat muscle tissues taken 1 h aft er the venom application, haemorrhage, myonecrosis, and mixed character cell infi ltration were generally detected in the perimysium and endomysium (Figures 11a and 11b). Th e muscular tissues taken 3 h aft er the

Figure 1. Skin section of rats in control group: epidermis (E),

dermis (D). Stained with haemotoxylin and eosin

(H&E).

Figure 2. Rare infl ammatory cells (➡), oedema and collagen

degeneration (▲), fat necrosis (asterisk), and local

haemorrhage (➞) in the skin section of 1-h application

group. H&E.

100 μm100 μm


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venom application showed that the formation of haemorrhage and myonecrosis increased, the mixed character cell infi ltration in the perimysium and endomysium became more intense, and inclusions in the myofi brils became evident (Figures 12a and

12b). Th e muscular tissues taken aft er 6 h showed large areas of haemorrhage and myonecrosis, mixed character strong cell infi ltration in the perimysium and endomysium, and formations of inclusions in the myofi brils (Table) (Figures 13a, 13b, 14).

Table. Incidence of observed lesions in the skin, liver, and muscle of male rats

administered venom.

LesionsTime of venom application (h)

0 1 3 6

Skin

Haemorrhage 0 5 5 4

Oedema 0 5 5 5

Infl ammatory cell 0 5 5 5

Fat necrosis 0 5 5 5

Collagen degeneration 0 5 5 5

Liver

Haemorrhage 0 5 5 5

Hepatocellular degeneration 0 5 5 5

Muscle

Haemorrhage 0 5 5 5

Mixed character infl ammatory cell inf. 0 5 5 5

Myonecrosis 0 5 5 5

Formation of inclusions in myofi brils 0 5 5 5

Numbers indicate the number of animals observed with lesions in their tissues; n = 5.


degeneration (▲), and local haemorrhage (➞) in the

skin section of 3-h application group. H&E.


degeneration (▲), fat necrosis (asterisk), and local

haemorrhage (➞) in the skin section of 6-h application

group. H&E.

100 μm100 μm


521

Figure 6a. Liver section from rat in control group: central vein

(cv) and portal area (pa). H&E.

Figure 5. Intense mixed character infl ammatory cell infi ltration

(➯) and infl ammation, especially around the capillary

vessel, in the hypodermis of 6-h application group.

H&E.

Figure 7. Sinusoidal congestion (➡) and hepatocellular

degeneration (white arrow) in the liver of 1-h

application group. H&E.

20 μm100 μm

Figure 8. Sinusoidal haemorrhage (➡) and hepatocellular



100 μm

50 μm

Figure 9. Sinusoidal haemorrhage (➡) and hepatocellular



Figure 6b. Liver section from rat in control group: hepatocyte

(H) and central vein (cv). H&E.

50 μm50 μm


522

Figure 10a. Longitudinal section of muscle from rat in control

group: myocyte ({ ). H&E.

Figure 10b. Transversal section of muscle from rat in control

group: perimysium (➡), myofi bril (➞). H&E.

50 μm 100 μm

Figure 11a. Haemorrhage (➡), rare mixed character

infl ammatory cell infi ltration (➯), and myonecrosis

(▲) in the longitudinal section of muscle of 1-h


Figure 11b. Haemorrhage (➡) and myonecrosis (▲) in the

transversal section of muscle of 1-h application

group. H&E.

50 μm 100 μm

Figure 12a. Mild mixed character infl ammatory cell infi ltration

(➯) and myonecrosis (▲) in the transversal section

of muscle of 3-h application group. H&E.

Figure 12b. Haemorrhage (➡), mixed character infl ammatory cell infi ltration (➯), and myonecrosis (▲) in the transversal section of muscle of 3-h application group. H&E.

50 μm 100 μm


523

Discussion

Although there are no specifi c data on the number of venomous snakebite cases in Turkey, it is estimated that a few people die from snakebite every year (Okur et al., 2001). Venomous snakebites are especially common in rural areas. For this reason, the clarifi cation of snake venom structure and its eff ects on organisms is very important in terms of administrating medication. In this study, rats were intramuscularly injected with M. xanthina venom on an LD

50 dose basis, and very serious damage

was detected in their skin, liver, and skeletal muscle tissues 1, 3, and 6 h aft er the injection intervals.

Many pathological symptoms in skin tissue were detected in previous studies carried out using a variety of diff erent snake venoms. In a study by Jimenez et al. (2008), Bothrops asper venom was injected into the skin around the ear area of rats. Th e authors observed that oedema and haemorrhage were present at 1- and 6-h intervals, respectively. In addition, 24 h aft er the samples were taken from the application group, collected data included loss of epidermis tissue,

Figure 13a. Intense mixed character infl ammatory cell infi ltration

(➯) and myonecrosis (▲) in the longitudinal section

of muscle of 6-h application group. H&E.

Figure 13b. Haemorrhage (➡), intense mixed character

infl ammatory cell infi ltration (➯), and myonecrosis

(▲) in the transversal section of muscle of 6-h


100 μm 100 μm

Figure 14. Haemorrhage (➡), mixed character infl ammatory

cell infi ltration (➯), and formations of inclusion in

myofi brils (⇔) in the transversal section of muscle of

6-h application group. H&E.

20 μm


524

cell infi ltration, the presence of protein liquid, and formation of scabs on the skin. Granule formation and increasing epithelial cells were observed in the 72-h application group, and in the 7- and 14-day application groups, the skin returned to its normal condition. In another study carried out with the same species, 3 h aft er intramuscular injection of venom, the epidermis and dermis layers slightly separated from each other with a low level of haemorrhage and cell infi ltration (Rucavado et al., 1998). A study using Naja nigricollis venom showed that the venom caused necrosis in the application area; infl ammation occurred around the veins and mixed character cell infi ltration had taken place (Iddon et al., 1987). Contrary to the results found by Jimenez et al. (2008), in this study, no damage to the epidermis was detected following an intramuscular injection of M. xanthina venom. However, similar to previous studies, oedema on the dermis and collagen degeneration were detected. Th is degeneration was widespread in tissues from the 1-h application group, whereas collagen degeneration was generally local in the 3- and 6-h application groups. In all of the application groups, there was large-scale haemorrhage in the hypodermis, fat necrosis, strong cell infi ltration, focal bleedings on the dermis, rare infl ammatory cells, and cell infl ammation around veins. During histopathological research, it was established that the venom caused serious damage to connective tissues in rats 1 h aft er injection and that this damage was partially restored in the 3- and 6-h application groups. Elements in the M. xanthina venom structure cause bleeding by tearing vein walls as a result of haemolytic activity. Skin oedema, cell infi ltration, and infl ammatory reactions can be target symptoms for reducing the damaging eff ects of venom on tissues. In addition, local necrotic areas can be the result of the lytic activity of one or more venom elements.

In analyses of the eff ects of Hydrophis cyanocinctus sea snake venom on liver tissue, Ali et al. (2000) detected hepatocyte degeneration, infl ammation, necrosis, fi brosis, regeneration, and cell infi ltration. Teibler et al. (1999) observed that Bothrops alternatus snake venom caused no damage to liver tissue. In a study on venom from Agkistrodon halys, a Pallas-type venomous snake, the fi brotic activity of rat livers was examined and hepatocellular damage was detected (Chang et al., 2005). In another study that included pathological symptoms from the venom of Cerastes

cerastes of the family Viperidae, the results detected were as follows: hyperaemia around the central vein, focal mononuclear infl ammation of white blood cells, bleeding in sinusoids around the portal area, and hepatocyte necrosis tissue on the liver (Hanafy et al., 1999). Th e study found sinusoidal bleeding and hepatocellular degeneration in the liver tissues of rats in all 3 application groups. Unlike previous studies, however, it did not fi nd the formation of cell infi ltration, infl ammation, or necrosis.

Since venom is generally transferred by the snake intramuscularly, the histopathological evaluations of muscular tissue are of special importance. Ali et al. (2000) did some case studies on the venom of Hydrophis cyanocinctus (sea snakes) and examined the myotoxic eff ect of venom, which established the onset of myonecrosis and cell infi ltration. Th e eff ect of Philodryas patagoniensis snake venom was analysed using albino rats. Th e results showed evidence of haemorrhage and myonecrosis in the gastrocnemius muscle in the 1-h application group and the addition of intense infi ltration in the 3-h application group. All of these symptoms were more prominent in the 24-h application group (Acosta et al., 2003). Similarly, Tu et al. (1969) observed that venom of Vipera russelli siamensis causes myonecrosis despite the fact that it does not cause haemorrhage in muscular tissues. Th e venom of Vipera apis, on the other hand, causes both haemorrhage and myonecrosis in muscular tissue. It was established that the venoms of Bitis gabonica and Bitis nasicornis cause haemorrhage, but not myonecrosis, in muscular tissue. Th e venom of Bitis arietans brought about both haemorrhage and myonecrosis in muscular tissue. Similar to previous studies, there was haemorrhage seen in muscular tissues at the point of injection and evidence of mixed character cell infi ltration. Th ere was myonecrosis in all 3 application groups. When comparing application groups to each other, these symptoms were not very intense in the 1-h application group; they were intense in the 3-h application group and much more intense in the 6-h application group. In the 3-h application group, there were also inclusions within myofi brils, which were not detected in the 1-h application group; these became serious in the 6-h application group. Some researchers have pointed out that the proteolytic enzymes available in snake venom bring about haemorrhage and necrosis in muscular tissues (Flowers, 1963; Goucher and Flowers, 1964).


525

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As illustrated in this study, M. xanthina venom is the cause of many pathological eff ects in diff erent tissues. Due to the occurrence of these eff ects shortly aft er injection, it is very important to give snakebite victims immediate medical attention. Hence, this study may enlighten practitioners/professionals as to the eff ects of snake poisons and serve as a guide for related medical treatment methods.

Acknowledgements

Th is study was supported by the Çanakkale Onsekiz Mart University Scientifi c Research Project Commission (2007/15 project).

Th is research is a part of an MSc thesis. Th e authors wish to thank Professor Dr C. Varol Tok for his assistance with the Montivipera xanthina description and the venom used in this study.

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