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Page 2 of 14 Darbinyan et al. Neuroimmunol Neuroinflammation 2018;5:41 I http://dx.doi.org/10.20517/2347-8659.2018.33

INTRODUCTION
The Vipers are the lone family of venomous snakes found in Armenia. The main action of viper venoms is
related to protein-degrading proteases, which cause local necrosis, systemic cardiovascular damage, and
[1]
disruption of the blood-clotting system . The whole venom of Caucasian Levantine viper [syn.: blunt-
nosed viper, Macrovipera lebetina obtusa (MLO)] contains many enzymatically active proteins and different
polypeptides [Figure 1] with a variety of effects on cells and organs, including pronounced hemorrhagic
[4,5]
[2,3]
effect . Macrovipera lebetina species venom contains snake venom metalloproteinases (SVMP) which
2+
2+
are Zn -containing, Ca -dependent enzymes that differ in domain structure: some of them contain just
metalloproteinase domain, others contain metalloproteinase domain and non-catalytic domains. MLO
[2]
venom contains only two out of four types of SVMPs: P-I and P-III . Metalloproteinase P-I has only
[6,7]
catalytic domain and consitutes more than one-quarter of the whole MLO venom.
[6,8]
The P-III is characterized by proteolytic enzyme property, high ability to bind to the substrate and
[9]
high hemorrhagic activity (with rare exceptions ). Together PI and PIII are making up approximately
one-third of the entire contents of the MLO venom. SVMPs, which are secretory proteinases, cause
hemorrhagic injuries, splitting the components of the basement membrane and disrupting their interaction
[10]
with endothelial cells . In addition, they induce muscle necrosis, skin damage, and inflammation. Some
[11]
SVMPs induce apoptosis in human umbilical vein endothelial cells . Moreover, SVMPs interact with
matrix metalloproteinases (MMPs) of a prey. MMPs are carrying out tissue remodeling, angiogenesis, cell
[12]
proliferation, migration, differentiation, apoptosis, and in curbing the growth of tumors . They are involved
in the cleavage of membrane receptors, ligand release, as well as in the activation/deactivation of the
[13]
chemokines and cytokines . SVMP P-I and P-III can trigger secretion of prey’s MMP-2 and MMP-9, which
[4]
together with venom MPs may destroy extracellular matrix or VE-cadherins . These and other cadherins (β
[11]
and γ) are denatured leading to increased intracellular permeability . During such damage of brain tissue,
microglia plays a critical role in the protection of brain from the action of harmful factors of the venom [14-16] .

Mechanisms by which different doses of snake venom, and particularly, SVMPs affect or disrupt the
microvasculature of brain and cause hemorrhage have not been fully elucidated. The goal of our investigation
was to assess hemorrhagic effects of MLO venom on rat brain given at different doses and for different
periods when administered via intraperitoneal (IP) route.

METHODS
Animals
This study was conducted in accordance with “Principles of Laboratory Animal Care” and as carried out in
accordance with the European Communities Council Directive of September 22 2010 (2010/63/EU). Sixty-
two adult Wistar albino male rats were used in this study.

Reagents
MLO venom was milked and dried in the Laboratory of Physiologically Active Substances Purification,
Certification and Standardization (PAS PCS) of Orbeli Institute of Physiology of National Academy of
Sciences of Armenia. Snakes were supplied by the licensed catcher “GEBEVSS” Ltd., Armenia. Other
reagents were purchased from “Sigma-Aldrich”, “Merck” “Reanal” and “H. Lundbeck A/S” companies as
indicated. All other chemicals were of analytical or sequencing grade.

Verification of venom hemorrhagic activity in vivo
[17]
The hemorrhagic activity of SVMPs was tested in vivo by a modified method of Kondo et al. . In brief,
2
2 rats (around 250 g) were anesthetized with Nembutal (pentobarbital, at a dose of 40 mg/kg) and 4 cm
of dorsal skin was chemically depilated (“Veet”, Reckitt and Benckizer, France). 0.1 mL aliquots of a