= ЭКСПЕРИМЕНТАЛЬНЫЕ РАБОТЫ
УДК 577
PROTECTIVE EFFECTS OF THE PROLINE-RICH PEPTIDES (PRP-1, PRP-3) ON THE ACTIVITY OF THE SPINAL CORD NEURONS UNDER THE SNAKE VENOMS NAJA NAJA OXIANA AND VIPERA LEBETINA OBTUSA INTOXICATION
© 2007 r. A. A. Galoyan1, J. S. Sarkissian1' 2*, V. A. Chavushyan1' 2, Z. E. Avakyan2, I. B. Meliksetyan2, A. V. Voskanyan2, H. H. Mkrtchian2, M. V. Poghosyan2, A. J. Gevorkyan2, Z. A. Avetisyan2, D. O. Abrahamyan3
1Buniatian Institute of Biochemistry NAS RA, Yerevan, Republic of Armenia 2Orbeli Institute of Physiology NAS RA, Yerevan, Republic of Armenia 3Sargsyan Military Institute, Yerevan, Republic of Armenia
Poisoning by Elapidae and Viperidae snake venoms (SV) leads to acute impairment of the blood and circulatory systems with severe hypotension, toxic neurodegeneration and other fatal consequences due to the hypoxia and toxic metabolites, resulting in lethal outcome. The purpose of the present study was to investigate the protective action of two main members of hypothalamic proline-rich peptides family (pRP-1 and PRP-3) in Central Asian cobra (Naja naja oxiana, NOX) and lebetina viper (Vipera lebetina obtusa, VLO) SV envenomation of rats. In acute experiments, we recorded the flow of evoked spike activity of single lumbar inter- and motoneu-rons on bilateral stimulation of flexor and extensor nerves of the hind limbs after the poisoning by NOX and VLO venoms with preliminary and subsequent systemic administration of PRP-1 and PRP-3. Stabilized recovery of spinal neurons' activity without hypoxic features was shown under the protective action of those neuropeptides. In semichronic experiments, we also performed the subsequent control of autopsy material. The preliminary (a day before) administration of PRP-1 and PRP-3 with subsequent injection of VLO venom (1, 2 LD50) resulted in more survival, and the survived rats were investigated in acute-electrophysiological test on days 4-12. We noted significant delaying of the lethal outcome after the poisoning by high doses of the venoms in semi-chronic experiments. The main reasons of animals' death were curariform block of synaptic transmission in the respiratory musculature (NOX) and painful haemorrhagic oedema with subsequent necrosis in the site of injection (VLO). Thus, the protective action of PRP-1 and PRP-3 against NOX and VLO envenoming reflected in the prevention of the development of lesions in the nervous system, as well as in the blood and in parenchyma-tous organs.
Key words: proline-rich peptides, hypothalamus, snake venoms, protective effect, spinal cord inter- and motoneurons, neuronal flow activity.
Envenoming from poisonous snakebites is an important public health hazard, particularly in tropical and subtropical regions. Several million cases of bites occur worldwide each year, with hundreds of thousands of amputations, severe neurological disorders and deaths [1-3]. Children and agricultural workers are the most common victims. Unfortunately, public health authorities give little attention to this problem, relegating snakebite envenomation by WHO to the category of a major neglected disease of the XXI century [4].
The clinical presentation of venomous serpents' bites envenomation is called ophitoxaemia [5]. Snake venom (SV) is a highly modified, toxic saliva containing more than 20 types of enzymes (phospholipases
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(PL -A2, -B, -C, -D), acetylcholinesterase (AChE), phosphatases, hyaluronidase, proteases, nucleases, phosphodiesterase, etc.); toxic polypeptides - neurotoxins (a-, P-, K-toxins, dendrotoxins, etc.), heamotox-ins, cardiotoxins, myotoxins and others; biologically active proteins (e.g. nerve growth factor (NGF) from cobra venom); low-molecular organic compounds (amino acids, nucleotides, cholesterol, lecithin, pigments, etc.); glucoproteins; as well as non-organic components (Ca, Zn, Mg, P, Si, Fe, Al, Cu, etc.) [6-11].
Different species have differing proportions of the above mixtures - this is why poisonous species are formerly classified as neurotoxic, haematotoxic or myo-toxic [5]. Most severe cases of ophitoxaemia are inflicted by species of the neurotoxic family Elapidae (cobras, sea snakes, etc.) and haematotoxic Viperidae (vipers, rattlesnakes, etc.) [4]. Elapid SV cause progressive descending paralysis by pre- and/or postsyn-
Table 1. Series NOX
N. of rats "Pre-treatment" Envenomation by NOX, timing "Treatment" N. of IN/MN
1 - LD50 = 1 mg/kg i/p PRP-1, 10 |g/100g i/m, 1 h 1/2
1 PRP-1, 10 |g/100g i/m LD50 = 1 mg/kg i/p, 2 h - 0/3
1 PRP-1, 10 |g/100g i/m LD50 = 1 mg/kg i/p, x5 - 24, 25.5, 26.3, 26.8 and 28.3 h - 1/2
1 PRP-1, 20 |g/100g i/m LD50 = 1 mg/kg i/p, x2 - 1.5 and 3 h - 0/3
1 PRP-3, 10 |g/100g i/m LD50 = 1 mg/kg i/p, x2 - 3 and 4 h - 0/5
aptic action of neurotoxins and AChE. Some elapid SV also induce local necrosis and rhabdomyolysis due to the action of PLA2 and Zn-dependent metalloproteinas-es [4]. Viperid ophitoxaemia induces prominent local tissue damaging effects, such as swelling, pain, haemorrhage, blistering and necrosis of skeletal muscle, often accompanied by secondary infection. Systemic viperid envenoming is characterized by a complex pathophysiological profile including spontaneous haemorrhage (cerebral haemorrhage being the most serious manifestation), coagulopathy (defibrinogenation, disseminated intravascular coagulation - DIC syndrome); hypovol-aemia, vasodilation and cardiotoxic effects with secondary cardiovascular shock; acute renal failure and acute respiratory distress syndrome [4-16]. Some viperid SV also display primary neurotoxic effects [4]. Snakebite survivors may have major chronic physical and neurological disability [5].
None of the enzymes is responsible for acute toxicity [16]. They are mainly responsible for local capillary injury and tissue necrosis, different pro- and anti coagulant actions, acute hypotension and pain, resulted from liberation of vasoactive peptides by kininogenas-es [17-20].
The influence of SV, especially of elapid neurotoxins, on synaptic transmission is intensively studied. For example, cobrotoxin blocks the neuro-muscular cholin-ergic transmission (N-blocker), which is almost 40 times more potent than tubocurarine [5, 21]. a-Bungarotoxin (a-BuTX) acts as a reversible inhibitor of ACh-evoked increase of Cl- conduction [22]. P-BuTX and PLA2 inhibit the 3H-choline uptake, which is necessary for ACh synthesis [23].
The investigation of interaction of hypothalamohy-pophysial hormones and SV has shown that elapid den-drotoxins intensify more than twofold the secretion of vasopressin and oxytocin [24]. The role of hypothalam-ic-pituitary-adrenal axis have been revealed in the hy-perglycaemic effect of neurotoxic fraction F3 from
banded cobra (Naja haje) venom, which stimulates the liberation of glucocorticoids from adrenocortical cells, modulating the insulin and glucose turnover for hyper-glycaemia maintenance during the period of stress [25].
Antisera (equine antivenoms) represent the only therapy for the treatment of envenomation [26]. However, most manufacturers in the developed world have abandoned production of snake antivenoms due to reduced clinical needs in industrialized countries. Most remaining producers are located in developing countries, where the application of quality and safety standards needs to be improved [2, 26]. Moreover, the antivenins not even registered in many developing countries because of their short shelf life and lowered profitability.
The present work is devoted to comparative study of protective action of two main specimens of hypotha-lamic proline-rich peptides PRP-1 and PRP-3 in ophitoxaemia, caused by systemic administration of Central Asian cobra (Naja naja oxiana - NOX) or lebetina viper (gyurza, Vipera lebetina obtusa - VLO) venom.
METHODS
Fifty-two adult male and female Albino rats (200-300g) were used in conditions of acute and semichronic experiments.
Acute experiments. Acute electrophysiological experiments (28 rats) were carried out on 24 rats at the first hours of SV envenomation (before or after PRP administration), and on four of them on survival days 412 (after PRP 'pre-treatment' and VLO envenomation) from semichronic experiments (see below).
Series NOX. The mode and timing of PRP and NOX administration, as well as the number of investigated single spinal neurons provided on Table 1.
Series VLO. The mode and timing of PRP and VLO administration, as well as the number of investigated single spinal neurons provided on Table 2.
Table 2. Series VLO.
N. of rats "Pre-treatment" Envenomation by VLO, timing "Treatment" N. of IN/MN
5 - LD50 = 1.9 mg/kg i/p PRP-3, 10 |g/100g i/p, 6-20 min 3/16
4 PRP-3, 10 |g/100g i/m LD50 = 1.9 mg/kg i/p, 30-90 min - 3/7
3 PRP-3, 10 |g/100g i/m LD50 = 1.9 mg/kg i/p, 24 h and 2 LD50 = 3.8 mg/kg i/p, 24.5 h - 2/5
4 PRP-1, 20 |g/100g i/m LD50 = 1.9 mg/kg i/p, 4 h - 1/5
2 PRP-1, 20 |g/100g i/m LD50 = 1.9 mg/kg i/p, 24 h - 2/4
1 PRP-1, 10 |g/100g i/m 3 LD50 = 5.7 mg/kg i/p, 24 h - 1/1
2* PRP-3, 20 |g/100g i/m LD50 = 1.9 mg/kg i/p, 24 h - 6th and 9th days of survival - 5/6
1* PRP-1, 20 |g/100g i/m LD50 = 1.9 mg/kg i/p, 24 h - 12th day of survival - 8/5
1* PRP-1, 20 |g/100g i/m 2 LD50 = 3.8 mg/kg i/p, 24 h - 4th day of survival - 3/5
* Animals from semichronic experiments.
Electrophysiological recording. In acute experiments, the animals immobilized by 1% dithylinum (25mg/kg i/p) and artificially ventilated. Under the local anaesthesia (Novocain), the spinal cord (SC) cut with an ultrasonic knife at T2-T3 level. After fixation of the lumbosacral region in a stereotaxic apparatus, the laminectomy is performed on L3-L4 level. Then the flexor (n. gastrocnemius - G) and extensor (n. peroneus communis - P) branches of n. ischiadicus of ipsi (i) - and contra (c) - lateral hind limbs were separated.
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