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UDC 612.13:616-005.4
STATE OF THE HEMOSTATIC SYSTEM IN RATS BY ONE-TIME AND MULTIPLE EXPOSURE TO HYPOXIC HYPOXIA OF HIGH INTENSITY
1 Altai State Medical University, Barnaul
2 Scientific-Research Institute of Physiology & Basic Medicine, Novosibirsk
S.V. Moskalenko1, I.I. Shakhmatov12, V.I. Kiselev12, Yu.A. Bondarchuk12, O.M. Ulitina12, O.V. Alekseyeva12
The aim of the study was to analyze the state of the reaction of the hemostasis system in rats to a single and multiple exposure to hypoxic hypoxia of high intensity. Materials and methods: male rats (40 individuals) of the Wistar line were used in the work. Animals were subjected to a single/daily multiple hypoxic hypoxia by "lifting" in a pressure chamber for 1 hour. Hypoxic hypoxia was modeled with the aid of a pressure chamber of the extract and exhaust type. The air discharge created in the pressure chamber in the training mode corresponded to a rise to a "height" of 7000 m above sea level (41.105 kPa, 308.3 mm Hg, hypoxia of strong intensity). Results and its discussion. A single exposure to a strong intensity of hypoxic hypoxia is characterized by a state of thrombotic readiness (based on the revealed hypercoagulation and high level of markers of intravascular coagulation). After completion of the 30-day cycle of severe intensity hypoxia, activation of the platelet system of the hemostasis system was noted, hypercoagulation along the internal clotting path with the disappearance of signs of thrombinemia. In addition, an increase in the antithrombin reserve of blood plasma was recorded in the group of experimental animals. Key words: hypoxic hypoxia, hemostasis.
The study of the effect of prolonged hypoxia on the human and animal organism until now is one of the most important problems of physiology and medicine. This problem acquires a particular urgency in connection with the fact that the state of hypoxia, in addition to a number of diseases, has a place both in the physiology of sports and also in the process of exploration of different habitats [1].
Adaptation to hypoxia is a very controversial and multifaceted process in which all organs and body systems are involved, and in the first place the blood system [2, 3]. It is established that in response to acute hypoxia phase changes occur in the system of hemostasis. The primary reaction of the coagulation system of the hemostasis system is hypercoagulability. However, a longer stay at the height leads to the development of hy-pocoagulation, which is caused by consumption of the clotting factors [4].
Earlier, the effect of hypoxic hypoxia (HH) on the blood system at low (4000 m), medium (5000 m) and moderately strong (6000 m) intensity was studied [5]. However, the data for high (7000 m) and severe (8000 m) intensity of hypoxia in the literature are mainly presented by blood hemorheol-ogy [6, 7], without affecting, in particular, the he-mostasis system.
In connection with the foregoing, the purpose of this work was to study the state of the hemosta-sis system in response to a single and daily multiple exposure to hypoxic hypoxia (HH) of high intensity.
Materials and methods
The studies were performed in 40 mature male Wistar rats with an average mass of 254.0±36.0 g. All experimental animals were divided into four groups: two control (n=10x2) and two experimental groups (n=10x2).
The first experimantal group was subjected to a single exposure to HH in a pressure-and-ex-haust chamber for 1 hour at a pressure of 41.105 kPa (308.3 mm Hg), which corresponded to an elevation of 7000 m, with the first control group staying for the same time in a pressure chamber under conditions of ordinary atmospheric pressure; the second experimental group was exposed to a 30-fold daily exposure to HH for 1 hour at a height of 7000 m, while the second control group was daily kept for 1 hour for 30 days in a pressure chamber at ambient atmospheric pressure.
The mode of hypoxic exposure was based on the literature data: according to N.I. Mamadalieva et al. (2014), the modeling of the ascent to a height of 4000 m and 5000 m corresponds to low and medium intensity hypoxia; "At an altitude of 6,000 m and 7,000 m, conditions of hypoxia of moderately strong and high intensity are created, and a simulation of a rise to a height of 8,000 m corresponds to severe hypoxia [8].
Before the experiment was started, during a week of adaptation to the conditions of the vivarium, all the rats were in standard conditions of detention. The use of rats in experiments was carried out in accordance with the European Convention for the Protection of Vertebrate Animals used in the experiment and Directives 86/609/EEC. Anesthesia and killing of animals was carried out
in accordance with the "Rules for conducting work using experimental animals" [9].
The blood for study of experimental animals was taken immediately after the end of a single (the first test group) or the 30th experiment (the second experimental group). In the rats of the control groups, the blood was taken immediately after the completion of a single/multiple exposure in a pressure chamber under conditions of normal atmospheric pressure.
The blood for the study was taken under ether anesthesia from the hepatic sinus in a volume of 5 ml. All blood samples were stabilized with a 3.8% sodium citrate solution at a 9:1 ratio [10].
A set of techniques allowing to assess the state of the hemostasis system included a study of the aggregation capacity of platelets, the coagulation unit of hemostasis, as well as anticoagulant and fibrinolytic activity of the blood. Diagnostic kits of the firm "Technology-Standard" (Russia) with the use of the coagulometer "Minilab" (Russia), "Trombostat-2" (Germany) were chosen as reagents for evaluation of the hemostatic system. Calculation of the number of platelets of peripheral blood was carried out using the hematological analyzer Drew3 - PAC (UK). The determination of platelet aggregation activity was carried out using the "Biola" aggregometer, (Russia). The determination of the level of antithrombin III was carried out using a spectrophotometer SF-46 (Russia).
All digital data obtained during the study were subjected to statistical processing. The results of the studies are presented in the form m [25% - 75%], where m is the median in the sample population, [25% - 75%] - the 25th and 75th percentile. The reliability of the differences was estimated using the nonparametric U Mann-Whitney criterion. Differences were considered reliable at a level of statistical significance p <0.05. To process and store the obtained experimental material, databases were created using the Microsoft Excel 2010 spreadsheet editor. Statistical processing of the results was carried out using mathematical statistics programs Jmp Statistical Discovery v 6.1.2 and Biostat 5.03 on a personal computer.
Results and discussion
From the results obtained, it follows that after a single exposure to HH, in the blood plasma, activation of the vascular-platelet unit of the hemostasis system was detected, characterized by an increase in ADP-induced platelet aggregation by 43.8% (p <0.001). In the coagulation unit of hemostasis, hypercoagulation along the internal and external pathways was noted, which was manifested in the shortening of activated partial thromboplas-tin time (APTT) by 7.7% (p <0.01) and prothrombin time (PV) by 6.0% (p <0 , 01). At the final stage of blood coagulation, hypercoagulability was also recorded, which was characterized by a shortening
of the polymerization time of fibrin-monomer complexes (PCPM) by 15.4% (p <0.001).
Furthermore, by reducing the amount of fibrinogen by 20.7% (p <0.001) the increase of SFMC by 50% (p <0,001) was recorded, as well as reducing the blood plasma antithrombin reserve by 16.2% (p <0.001).
Analysis of the experimental results showed that the hemostatic system reaction in response to a single HH exposure of high intensity is expressed by activation of vascular-platelet and coagulation components of hemostasis, accompanied by signs of the state of thrombotic readiness (based on the identified hypercoagulable and high intra-vascular coagulation markers and reducing an anticoagulant reserve of blood plasma [10]).
Thus, on the part of the hemostasis system, in response to a single overall effect of HH of high intensity, a marked shift of the hemostatic potential towards thrombinemia was noted, which can be regarded as a distress response.
The state of the hemostasis system, registered after a 30-fold exposure to HH, was characterized by activation of the vascular-platelet link, which was manifested by an increase in platelet aggregation activity by 20.3% (p <0.001). On the part of the plasma hemostasis, hypercoagulation along the internal pathway (shortening of APTT by 8.8% (p <0.001)) was noted, in addition, an increase in the fibrinogen concentration by 23.3% (p <0.01) was recorded.
As can be seen from the same table, the heparin cofactor activity of antithrombin III in the throm-bin-heparin test (ARP-antithrombin reserve of plasma) increased by 8.7% (p <0.001). The level of anticoagulant antithrombin III increased by 10.8% (p <0.01), and fibrinolytic activity, estimated by spontaneous lysis of euglobulins, increased by 24.4% (p <0.05).
In contrast to the response of the hemostasis system in response to a single exposure to HH, the state of the hemostatic system recorded after the end of the 30-day cycle of exposure to HH of high intensity is characterized by a less pronounced activation of the vascular-platelet unit with persistent hypercoagulation along the internal clotting pathway. At the same time, the hyper-coagulation registered at this stage along the internal blood clotting pathway was compensated by the pronounced activation of the fibrinolytic blood system. It should also be taken into account that after 30-fold exposure to HH there is no increase in the marker of intravascular coagulation -SFC in blood plasma. In addition, against the background of less pronounced hypercoagulable shifts compared with a single exposure, there was an increase in the anticoagulant activity of blood plasma.
Proceeding from the above, it can be concluded that by the 30th day of hypoxic effects, virtually all
signs of thrombotic state were registered, recorded immediately after the end of a single HH of high intensity. The analysis of the obtained data allows to characterize the state of the hemostasis system after 30-fold daily HH training as a manifestation of long-term adaptation to this type of stressor.
Conclusion
It is revealed that the reaction of the hemostasis system to a single exposure to HH of high intensity is the activation of the vascular-platelet and coagulation units of hemostasis, accompanied by signs of the development of thrombotic state.
References
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2. Makarenko A.N., Karandeeva Yu.K. Adaptation to hypoxia as a protective mechanism in pathological conditions. Bulletin of the problems of biology and medicine. 2013; 2 (100): 27-33.
It has been established that during the course of daily hypoxic training, the signs of thrombotic readiness, recorded with a single exposure, disappear, the growth of fibrinolytic and anticoagulant activity of blood is recorded.
The obtained data allow to draw a conclusion that the daily application of the training regime of HH of high intensity within 30 days contributes to the increase of resistance of the organism to oxygen deficiency and reduces the risk of development of thrombus formation.
3. Rachkov A.G., Kurmanbekova G.T., Aidarov Z.A. High-altitude thrombohemorrhagic syndrome, prognosis and ways of correction. Materials of the International Conference. Bishkek, 1996.
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Indicators of the blood plasma hemostasis system of rats after a single and multiple exposure to hypoxic hypoxia(m [25 %^75 %]) iauie i
Indicators One-time exposure to HH (1 h - 7000 m) 30-time exposure to HH (1 h - 7000 m)
Control 1 Experiment 1 Control 2 Experiment 2
Platelets, xl09/l 473,0 [464,5-489,8] 492,5 [456,8-507,5] 495,0 [487,0-498,0] 508,5 [502,5-515,0]
Induced ADP-aggregation of platelets, max. value 27,4 [23,9-28,9] 39,4*** [37,6-40,5] 21,7 [21,3-22,9] 26,1*** [24,8-27,4]
APTT, s 18,1 [17,7-18,8] 16,7** [15,5-17,2] 16,0 [15,7-16,4] 14,6*** [12,6-15,3]
Prothrombin time, s 26,0 [24,7-27,1] 24,4** [23,7-24,8] 21,7 [20,4-23,5] 21,2 [19,9-22,0]
Thrombin time, s 46,7 [45,2-48,0] 43,1 [40,7-45,7] 42,7 [39,8-43,6] 41,9 [39,8-44,0]
CTSFC, s 59,1 [56,9-61,0] 50,0 *** [49,2-52,0] 62,5 [60,8-63,1] 61,9 [60,3-64,7]
Fibrinogen, g/l 2,9 [2,8-2,9] 2,3*** [2,3-2,3] 3,0 [2,8-3,2] 3,7** [3,5-3,9]
SFC, mg/100 ml 3,0 [3,0-3,1] 4,5*** [4,0-5,4] 3,0 [3,0-3,0] 3,0 [3,0-3,1]
Antithrombin III, % 94,5 [88,8-97,3] 89,0 [84,0-90,7] 102,5 [99,8-104,1] 113,6** [110,9-115,5]
Antithrombin reserve of plasma, % 87,9 [84,4-92,4] 73,7*** [71,9-83,0] 84,3 [82,0-87,5] 91,6*** [89,4-96,4]
Spontaneous euglobulin fibrinolysis, min 600,0 [570,0-630,0] 540,0 [532,5-637,5] 615,0 [570,0-660,0] 465,0* [450,0-480,0]
Note: Statistically significant differences from the corresponding indicators of the control group are indicated: * - for p <0.05; ** - for p <0.01; *** - for p <0.001. APTT - activated partial thromboplastin time; CTSFC - curing time of soluble fibrin complexes; SFC -soluble fibrin complexes. CT - coagulation time; a is the angular constant; MCF is the maximum amplitude of the TEG; CFT - clot formation time; ML - maximum lysis.
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Contacts
Corresponding author: Moskalenko Svetlana
Valeryevna, Lecturer of the Department of Normal
Physiology of the Altai State Medical University
University, Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: (3852) 566928.
Email: sunrisemsv@gmail.com
Shakhmatov Igor Ilyich, Doctor of Medical Sciences, Head of the Department of Normal Physiology of the Altai State Medical University, Barnaul. 656038, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566928. Email: iish59@yandex.ru
Kiselev Valery Ivanovich, Doctor of Medical Sciences, Professor, Professor of the Department of Normal Physiology of the Altai State Medical University, Barnaul. 656038, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566928. Email: vik@mail.ru
Bondarchuk Yulia Alekseevna, Doctor of Medical
Sciences, Associate Professor of the Department
of Normal Physiology of the Altai State Medical
University, Barnaul.
656038, Barnaul, ul. Papanintsev, 126.
Tel.: (3852) 566928.
Email: bondarchuk2606@yandex.ru
Ulitina Oksana Mikhailovna, Doctor of Medical Sciences, Associate Professor of the Department of Normal Physiology, Altai State Medical University, Barnaul.
656038, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566928. Email: oulitina@mail.ru
Alekseeva Olga Vasilyievna, Candidate of Medical Science, Associate Professor of the Altai State Medical University, Barnaul. 656038, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566928. Email: alekseeva0506@mail.ru
