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Darbinyan et al. Neuroimmunol Neuroinflammation 2018;5:41 I http://dx.doi.org/10.20517/2347-8659.2018.33 Page 3 of 14

%

2+
Zn -metalloproteinase PIII 4.3
2+
Zn -metalloproteinase PI 27.8
L-amino acid oxidase 1.7
C-type lectin-like 14.8
Serine proteinase 14.9
PLA2 14.6
CRISP 2.6
DC-fragment 1.7
Dimeric disintegrin 14
Short disintegrin 2.8
BPP/C-NP 5.3
0 5 10 15 20 25 30

Figure 1. Percentile of venom of Macrovipera lebetina obtusa components . (BPP/C-NP: bradykinin potentiating/C-natriuretic peptide; CRISP:
[2]
cysteine-rich secretory protein; PLA2: phospholipase A 2 )
physiological solution containing 2.0 mg venom were injected subcutaneously (SC). After 2 h the animals
were euthanized and dorsal skin was removed. Hemorrhagic spots were identified and counted on the inner
surface of the skin.

Verification of venom toxicity in vivo
Experiments were done on 30 adult male rats weighing 220-250 g to determine MLO venom toxicity LD (IP
50
injection). Five doses were chosen for determination of LD starting from 100% vitality to 100% mortality.
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In our study for estimation of LD of venom, 5 doses were given intraperitoneally to 5 groups of rats, 6 in
50
each group. Each rat in the first group was injected IP with MLO venom at a dose of 1 mg/kg. In the second
group rats were injected with 2 mg/kg, the third: 3 mg/kg, fourth: 4 mg/kg and fifth: 5 mg/kg respectively.
The number of dead animals was recorded at 24 h and the percent of mortality in each group was calculated.
Percentage mortalities were transformed to probits and corrected formula for 0% mortality [100(0.2/N)]
and 100% mortality [100(N-0.25/N)] was used. Approximate standard error was calculated from following
formula:

1/2
Approx. SE of LD = (LogLD - LogLD )/(2N) ,
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84
50
where N is number of animals in each group.
LD , LD and final data were calculated using table of probits for Behrens and Miller-Tainter methods .
[18]
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Histochemical studies
Thirty rats were used for histochemical studies. The animals were kept in the nursery for laboratory
animals of Institute of Physiology NAS of Armenia. Daylight and diet were conditioned in accordance
with the Animal Protocol of the Institute. Venom was injected in at 2.0, 3.0 and 5.0 mg/kg IP, which was
approximately equal to 1.0, 1.5 and 2.5 LD for MLO venom. Animals were divided into 5 groups with 6
50
animals in each: Group #1 was a control group with intact rats that have only been anesthetized before they
were sacrificed. The other 4 experimental groups were set as follows: Group #2, #3 and #4 were injected with
different doses of MLO venom (1.0 LD , 1.5 LD , and 2.5 LD respectively) followed by extraction of the
50
50
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brain 2 h post-injection. Group #5 was injected with a dose of MLO venom (2.5 LD ), however, brains were
50
isolated 1 h post-injection.