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12IMPACT OF MAKROVIPERA LEBETINA OBTUSA VENOM ON CARDIOVASCULAR AND NEURONAL REACTIONS IN ADULT RATS
ELMIRA ALEXANDER AVETISYAN PhD in Biology, senior researcher of the scientific and educational center of L.A. Orbeli Institute of
Physiology, NAS RA, Yerevan, Armenia
ANAIDA AGHVAN PETROSYAN
PhD in Biology, senior researcher of the scientific and educational center of L.A. Orbeli Institute of
Physiology, NAS RA, Yerevan, Armenia
VAGHINAK HAYKAZ SARGSYAN Doctor of Sciences in Biology, Head of Laboratory of Sensorimotor Integration of L.A. Orbeli Institute of Physiology, NAS RA, Yerevan, Armenia
NARINE ANATOLI SAHAKYAN
Doctor of Sciences in Biology, Associate Professor, Head of the Chair of First Aid, Emergency Situations and Civil Defense of ASPU after Khachatur Abovyan, Yerevan, Armenia
SIRINE ARMEN SHOGHERYAN
PhD in Biology, Associate Professor, Head of the Chair of Ecology and Sustainable Development of ASPU after Khachatur Abovyan, Yerevan, Armenia
Annotation. We investigated the influence of high-frequency tetanic stimulation of the hypothalamic paraventricular nucleus on the activity of the vagosensitive neurons of the solitary tract nucleus and on heart rate variability in norm andfollowing injection of macrovipera lebetina obtuse venom. We studied the most important indices: heart rate, sympatho-vagal balance and the tension index of regulatory systems. Significant reactivity of solitary tract neurons (70.3%) to descending paraventricular inputs was revealed. The tetanic stimulation of paraventricular nucleus resulted in pronounced tetanic depression with post-tetanic potentiation. The analysis of some functionally significant heart rate variability parameters before and after injection of the venom showed a strong stress-like effect following venom (LD50) injection. Results suggest that with strong intoxication, in order to avoid irreversible changes in the body, it is necessary to quickly restore homeostasis not only by antidote, but also by factors preventing development of the stress pathology.
Keywords: paraventricular nucleus of the hypothalamus, solitary tract nucleus, tetanic potentiation and depression, venom, extracellular recording.
INTRODUCTION
One of the main properties of animal toxins is their effect on the basic integrating systems of the body: the nervous system, blood and the cardiovascular system, causing an imbalance state. It is well known that when severe intoxication with large doses of poison a collapse state occurs, in which the vital structures of the medulla oblongata (vasomotor, respiratory centers) are affected primarily, causing a disturbance in the activity of the heart and paresis of capillaries, etc.
It is known that bee venom and cobra venom violate subordinate intracentral relationships, which is probably related on blocking of poisons that regulate the effects on the spinal cord from suprasegmental structures [1]. The blocking effect of the used poisons was explained by the researchers with ganglion-blocking ability and strong cholinolytic effect of their components in relation to the corresponding cholinergic systems [2, 3]. Currently there are a large number of studies
on the effects of toxins, in particular, venoms of various species of poisonous snakes, [4, 5, 6, 7, 8]. A number of studies have shown cardiotoxicity when bitten by poisonous snakes [4, 9, 5]. Cases of not only cardiac, but also other systemic effects such as koagulopathy, acute renal failure, etc., are described [10, 11, 12]. So, a number of authors have shown that the venom of Makrovipera lebetina obtusa contains 38 types of proteins, the presence of which can cause a wide range of disorders and thereby a sharp imbalance in the coordinated activity of the body [13]. Significant lesions with the introduction of lethal doses of Makrovipera lebetina obtusa venom were detected in various brain structures of rats. In a normal brain, microglia control the functional state of synapses in vivo and determine the fate of terminal ischemia [14, 15]. The same malfunction in brain activity can be observed in acute and chronic stress inducing a violation of microglial plasticity, phenotype, and function [16.17]. Histochemical studies of the rat brain with intraperitoneal injection of the Makrovipera lebetina obtusa LD 50 venom showed significant morphological changes in microglia in the subcortical structures of the brain and cerebellum [18].
In our previous studies on the effect of hypothalamic structures (paraventricular nucleus - PVN and supraoptical nucleus - SON) on nucleus of solitary tract (NST) neurons and HRV under psychoemotional stress, we showed significant shifts in sympatho-parasympathetic homeostasis, causing changes in both the neuronal and cardiovascular spheres [20, 21].
The aim of this study was to study changes in the spike activity of NST neurons during tetanic stimulation of PVN and the reaction of PVN neurons during tetanic stimulation of NST to detect the feedback interdependence of these structures in the mechanisms of homeostasis regulation, identification of the effect of Makrovipera lebetina obtusa venom on the background and induced activity of NST neurons and HRV during tetanic stimulation of PVN. In this work, we present data of the mathematical analysis (MA) of electrocardiographic indices of HRV and neural reactions of the above structures with deep intoxication.
MATERIALS AND METHODS:
Chemicals
Urethane was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Other chemicals and Makrovipera lebetina obtusa venom were provided by local commercial sources.
Experimental animals
All of the experimental protocols were approved by the Committee of Ethics of the Yerevan State Medical University (YSMU) (Yerevan, Armenia). All animal procedures were carried out in accordance with the European Communities Council Directive 2010/63/UE and the local Animal Care Committee. Adult male Wistar albino rats weighing 300 ± 30 g were purchased from the experimental center of Orbeli Institute of Physiology NAS RA. The experiments were performed at the same time period of the day (09:00-12:00 h) and during the light period of the light-dark cycle. The animals were maintained at 25 ± 2 °C, 12 h light - dark cycle and lights on 07:00-19:00 h. Food and water ad libitum was provided to the animals. All experiments were carried out in separate and isolated laboratories, which have the same environmental conditions as the colony room.
The experiments were performed on rats, anesthetized with urethane (1.2 g / kg intraperitoneally) and immobilized with dithylin. After appropriate surgical intervention, bipolar stimulating electrodes with tip diameter of 100 p,m and an interpole distance of 50 p,m were alternately inserted into the anteromedial region of the hypothalamus or into the NST using the coordinates of D.Paxinos atlas (19). Extracellular registration of neuron activity was carried out by glass microelectrodes filled with a 2M solution of potassium citrate, which were introduced either into the PVN or into the medial region of the NST at a level or caudal to the obex. On-line registration of impulse and evoked activity of neurons was carried out according to the program analysis method developed by V.S.Kamenetskiy. We used low and high frequency burst stimulation (HFS) (20, 50, 100 Hz). The recording of the background impulse activity lasted for 10 s., the duration of the tetanic
stimulus was 1 s., and post-stimulus changes in the behavior of neurons of both structures were recorded for 10 s. in 200 or 400ms / bin. According to the program used in these experiments, raster peristimulus histograms were constructed with the allocation of the frequency spectrum of neuron activity before, during and after tetanization. ECG recording was performed in the 2nd standard registration. Analysis of ECG indicators was carried out by the method of MA HRV. The most significant indices studied were: heart rate (HR), index of tension of the regulatory systems (ITRS) and sympathovagal balance (SVB). The data were processed using a Microsoft Excel spreadsheet, the level of statistical reliability was determined using t-student test. Intraperitoneal injection of venom about 2.5 pl of LD-50 was carried out after recording an ECG or activity of NST neurons in the norm and then every 5 min after injection for 60-80min.
RESULTS
In the first series of experiments, the character of the influence of tetanic high frequency stimulation (HFS) (100 Hz) of PVN on the viscero-sensory neurons of the mediobasal region of NST (n=60) was studied. We have revealed a significant reactivity of the studied units (70.3%, n=-44) to hypothalamofugal messages. When studying the nature of the reactions of NST neurons to HFS of PVN, the most striking effects were detected in neurons with a background spikes of 20-25 sp / s (n=21). Stimulation of PVN led to a four-fold increase in spike activity (110-120 sp / s) with a long post-effect up to 20 seconds (Fig. 1A). Background-active units with a pulse frequency of 50-60 sp / s (n=-16) were sharply depressed during stimulation with some post-stimulation decrease or increase in spontaneous rhythm (irregular changes) (Fig. 1B). Figure 1C shows a group of mostly burst -discharging neurons (n=7) that are reactive to HFS (100 Hz) of PVN. Program analysis of peristimulus histograms showed that the NST neurons are susceptible to significant PVN effects. To identify the effect of afferent impulse ascending to the hypothalamus from the viscerosensory region of NST, we investigated the reactions of PVN neurons to tetanic stimulation of the above structure with a frequency of 20, 50, 100 Hz. A high degree of reactivity of background-active neurons of PVN was revealed. Of the 55 units studied, 78.25% (n =43) were reactive.
Fig. 1-
A-C - peristimulus histograms of the sum of spikes (from the top ) of pre- and post-stimulus manifestations of spike activity of single solitary tract nucleus (NST) neurons before and after the burst high frequency stimulation (HFS) (100 Hz) of paraventricular nucleus (PVN). MBE- before stimulation (10 sec.), MTT- stimulation time (1 sec.), MPE- after tetanization (10 sec.), BE- before event , TT -time tetanization, PE- postevent, TP - tetanic potentiation, TD - tetanic depression.
It should be noted that most of the PVN neurons exhibited inhibitory effects during tetanization even at a frequency of 20 Hz (Fig. 2 A, B) with an increase in frequency to 50 and 100 Hz. A similar pattern was observed in (Fig. 2. C, D). Figure 2 shows examples. of increased spontaneous rhythm (Fig. 2 B) and activation (Fig. 2 E) of a silent neuron. Program analysis showed the prevalence of tetanic inhibition and post-tetanic potentiation of a significant part of reactive units of PVN. Such mutual influence indicates the presence of feedback interaction between two key structures (NST and PVN), which play an important role in the activity of the visceral sphere of the body to support homeostasis. When studying the effect of intoxication on the reactivity of NST neurons, we found that lethal doses of Macrovipera lebitina obtusa venom immediately after administration cause a violation of the responsiveness of background -active units.
Fig. 2.
Peristimulus histograms of the sum of spikes (from the top ) of pre- and post-stimulus manifestations of spike activity of paraventricular nucleus (PVN) neurons before, during, and after
burst high frequency stimulation (HFS) (20, 50, 100 Hz) of solitary tract nucleus (NST). Other designations as in Fig. 1.
Figure 3 shows an example of one such neuron in the norm (Fig. 3, A), immediately after the injection of venom (Fig. 3B) and every 5, 30, 45, 60 minutes (Fig. 3. C, D, E, F, G,). Most of the neurons we studied (n=40) did not show clear effects on PVN stimulation, reacting mainly with an increase in frequency without significant shifts during tetanization (100 Hz). At the 60th, 80th minute, the irregular rhythm was regrouped into the burst rhythm, and as a rule, then the activity was reduced to zero and the cells died. In a series of experiments, we studied electrocardiographic parameters with MA of HRV during LD 50 venom injection and a comparative analysis of the results obtained with previous ECG data of indicators obtained by us when studying the development of psychoemotional stress in rats (20, 21), we revealed a striking similarity of the results.
Fig. 3 -
Changes in the activity of solitary tract nucleus (NST) neurons with intraperitoneal injection of Ld 50 venom. A - normal, B-G in the early stages after injection, with stimulation of paraventricular nuvleus (PVN).
So, in heart rate indices there is a sharp increase in the frequency of heart contractions with a further decline of 60 minutes after the introduction of the venom (Fig.4), as well as a sharp centralization of regulatory processes (ITRS) (Fig.5) and a violation of the sympathovagal balance (SVB) (Fig.6), similar to that with prolonged immobilization.
HR
400
Fig. 4-
Comparative electrocardiogram (ECG) indices of heart rate (HR) during the injection of LD50 venom ( on the right) and after prolonged immobilization (on the left).
In HRV indices, a 2.5-fold increase in ITRS was revealed in comparison with the control data (Fig. 5, on the right). By the 60th minute after injection, partial decentralization occurs due to the deterioration of the animal.
Fig. 5.
Diagrams of calculations of centralization of regulatory processes (ITRS) in heart rate variability (HRV) indices in the early stages of venom injection (on the right) and after prolonged immobilization of rats (on the left).
Calculations of the sympathovagal balance / SVB / showed that in the first 15 minutes after injection there is a shift of processes towards sympatric, which is followed by a secondary decline in sympatric-parasympathetic equilibrium with prevailing parasympathetic effects (Fig. 6, on the right).
SVB
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Fig. 6. -
Sympatho-vagal balance (SVB) indices by mathematical analysis (MA) of heart rate variability (HRV) calculations under the influence of the LD-50 venom (on the right) and with long-term immobilization of rats (on the left).
DISCUSSION
In a comparative analysis of these series of experiments with the results obtained in the study of psychoemotional stress, we found a strong stress-like effect of a lethal dose of venom on HRV indices.
An increase in heart rate in the first minutes after injection is replaced to bradycardia, by the end of the first hour. MA HRV showed a sharp centralization of regulatory processes, the decline of which is observed at the end of the first hour. Such decentralization is apparently associated with the destruction of neurons of overlying regulatory systems under the influence of venom toxins. It should be noted that along with the centralization of regulatory processes in the first minutes of injection, there is a sharp violation of the sympato-vagal balance with the prevalence of the high-frequency (HF) spectrum in the HRV rhythm gram, followed by a decrease in the sympathetic and the predominance of the low-frequency (LF) wave spectrum in HRV; a similar restructuring occurs in the reactivity of neurons, which again indicates a violation of homeostasis in the activity of both the neuronal and cardiovascular spheres of the body when all external and internal stress inducing factors. A deep intoxication contributes to abrupt disturbance of the balanced activity of vital body systems.
Thus, a comparative analysis of disorders, after psychoemotional stress and when intoxication showed a striking similarity of body reactions, on the basis of which we may suggest that with strong intoxication, in order to avoid irreversible changes in the body, it is necessary to quickly restore homeostasis not only by antidote, but also by factors preventing development of the stress pathology.
Acknowledgements
The authors would like to acknowledge for the outstanding special computer software analysis of data, developed by V.S.Kamenetski. This study was supported by the National Academy of Sciences of Armenia and the State Science Committee Armenia.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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