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UDC 612.112.5:612.225-092.4
STATE OF THE HEMOSTASIS SYSTEM IN RATS ON THE BACKGROUND OF DAILY COOLING TO ULTRA-HIGH DEGREE OF HYPOTHERMIA
Altai State Medical University, Barnaul
N.A. Lycheva, D.A. Makushkina, A.V. Sedov, I.I. Shakhmatov, V.I. Kiselev
The aim was to study the state of hemostasis system parameters in rats in response to daily cooling up to an ultra-high degree of hypothermia. Materials and methods: the study involved male Wistar rats (140 animals). Hypothermia was modeled by daily placing the animals in water at a temperature of 5°C for 30 days. The criterion for cessation of exposure was the achievement of a rectal temperature below 20°C by experimental animals, which corresponded to an ultra-high degree of hypothermia. Exposure time was individual and constituted 55±5 minutes. The control group of the animals was placed in water at a temperature of 30°C. Blood sampling was carried out on the 1st, 2nd, 5th, 10th, 14th, 20th, 30th days of daily cooling. Results: A consistent change in the response of the hemostatic system to daily cooling was observed. Thus, on the 1st experimental day, hypocoagulation was recorded. However, starting from the 2nd day, a hypercoagulation shift was recorded. The most pronounced hy-percoagulation was recorded on the 14th day. By the end of the experiment (on the 30th day), hypercoagulation was recorded against the background of an increase in the rate of formation of the fibrin network and an indicator of the maximum clot density. Conclusions. Daily cooling to an extremely high degree of hypothermia was accompanied by phased changes in the hemostasis system. On the 1st experimental day, hypocoagulation was recorded. Starting from the 2nd experimental day, the development and increase of the hypercoagulation was registered. The maximum hypercoagulation was observed on the 14th experimental day and was accompanied by the shortest clotting time recorded over the entire experimental period. On the 30th day, hypercoagulation and a reduction in the time of blood clot formation were observed, with the formation of the most dense clot. Key words: hypothermia, hemostasis, rats.
Adaptation to cold is an integral process which involves all organs and systems of the body in one way or another, but the main changes are aimed at preserving and increasing heat production. The preservation of heat in the body during adaptation to cold is primarily due to the reduction of heat transfer from the body surface, which can be achieved in various ways [1]. Also, temperature thresholds are increased for the occurrence of shivering and the start of a number of metabolic reactions that increase the body's cold tolerance [2]. Numerous studies are devoted to the adaptation of the respiratory, cardiovascular and endocrine systems to the action of cold [3, 4]. At the same time, among the works devoted to the study of prolonged exposure of the body to cold, there are only a few publications reflecting the state of the hemostasis system, which does not allow to evaluate the influence of the hypothermic effect on the development of adaptive changes in coagulation and fibrinolysis systems. Basically, these works are devoted to a comparative analysis of the hemostat-ic status of indigenous peoples of the North and newcomers [5, 6, 7]. All of the above marked the purpose of our study. The aim of the work was to study the effect of daily cold influences on the possibility of formation and the nature of adaptive changes in the hemostasis system.
Materials and methods
The studies were performed on 140 male Wis-tar rats weighing 300 ± 15 g. Immersion hypothermia was modeled by placing animals in individual cages in the water at 5 °C and air at 7 °C. The criterion for the termination of exposure was the achievement by experimental animals of rectal temperature <20 ° C, which corresponded to the ultra-high degree of hypothermia. The exposure time was individual and constituted 55 ± 5 minutes. The control group of animals was placed in water at a temperature of 30 °C. Cooling was reproduced daily. The state of the hemostatic system was assessed on the 1st, 2nd, 5th, 10th, 14th, 20th and 30th days. Thromboelastometry was performed on the Rotem instrument (Pentapharm GmbH, Germany) using the Natem reagent containing calcium chloride. Blood for research in a volume of 5 ml was obtained by taking a hepatic sinus into a polystyrene syringe containing a 0.11 M (3.8%) solution of sodium citrate (9: 1 ratio of blood and citrate). Prior to the experiment during the weekly adaptation to the conditions of the vivarium, all rats were kept in standard conditions according to the requirements of GLP. 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 Directive 86/609 / EEC. Comparison of the obtained results was carried out by calculating the median (Me) and percentiles (25% and
75%). Statistical analysis was performed using the non-parametric Mann-Whitney test on a personal computer by means of statistical software package Statistica 6.0 (StatSoft, USA). The critical level of significance by testing statistical hypotheses in this study was assumed to be 0.05.
Results and discussions
The results of the study of indicators of the hemostasis system in animals of all experimental
According to Table 1, on the 1st day, immediately after stopping of cooling, the clotting time, which characterizes the duration of the clot activation and coagulation phases, increased compared to the control group of animals by 16% (p = 0.046). The MCD, which gives an idea of the maximum density of the fibrin clot, decreased by 2.7 times (p = 0.024).
On the 2nd day, immediately after stopping of cooling, a decrease in clotting time was observed by a factor of 1.6 (p = 0.031) in comparison with the indicator of the control group. The time of clot formation increased by 20% (p = 0.045) in comparison with the value recorded in the previous group of animals and did not differ from that of the control group. An increase in clot density by 3.8 times was recorded, compared with this indicator on day 1 (p = 0.0004), and by 1.5 times compared with the control group (p = 0.034).
On the 5th day, the coagulation time increased by 1.5 times compared with the same indicator on the 2nd day (p = 0.0045) and did not differ from
groups are presented in Table. 1. Due to the lack of statistically significant differences in hemostasis in rats of the control groups throughout the experiment, the table shows the averaged values of the studied parameters. The comparison was carried out between the control and experimental groups at the corresponding time points.
the control group. At the same time, the clot formation time decreased by 1.5 times (p = 0.0075) as compared with the previous day and did not differ from the indicator of the control group. The maximum density of the fibrin clot exceeded the control values by 1.3 times (p = 0.028) and did not differ from the value recorded in the previous experimental group.
On the 10th day immediately after exposure to the hypothermic factor, a 1,5 factor decrease in clotting time was recorded, both in comparison with the indicator of the control group and in comparison with this indicator on the 5th day (p = 0.0024). The maximum clot density decreased by 60% (p = 0.036) in comparison with the analogous parameter of the previous experimental group and did not differ from the control values.
On the 14th day, the clotting time decreased to the minimum value recorded throughout the experiment, and, compared to the control group, decreased by 2.0 times (p = 0.013). The clotting time and angle a were not statistically changed. The
Table 1
Hemostatic system parameters in rats at different periods of hypothermia
CT, sec CFT, sec a ° MCD
Cjntrol (n=70) 245,0 [232,0-259,0] 107,0 [77,5-93,0] 69,5 [61,0-74,5] 35,0 [28,5-39,0]
Day 1 (n=10) 286,0 [233,0-343,0]* 84,0 [70,0-95,0] 73,0 [71,0-77,0] 13,0 [9,5-18,5]*
Day 2 (n=10) 148,0 [124,0-180,5]*# 103,5 [99,0-116,5]# 71,0 [71,0-72,0] 50,5 [43,0-61,0]*#
Day 5 (n=10) 234,0 [221,5 -243,0]# 69,0 [65,0-92,5]# 76,0 [65,5-81,5] 67,0 [64,5-71,0]*
Day 10 (n=10) 147,0 [126,0-181,0]*# 96,5 [76,0-104,5] 71,5 [69,0-75,0] 25,0 [17,0-37,0]#
Day 14 (n=10) 108,0 [102,0-124,0]* 92,0 [78,0-104,0] 75,0 [73,0-79,0] 69,5 [52,5-71,0]*#
Day 20 (n=10) 185,0 [164,0-211,5]*# 86,5 [73,5-101,0] 73,5 [64,0-79,5] 42,0 [31,5-58,0]
Day 30 (n=10) 196,5 [176,0-216,5]* 51,0 [45,0-57,0]*# 80,5 [79,0-82,0]* 77,0 [76,5-77,5]*#
Note: the data are presented in the form Me - sample median, [25 - 75] - sample percentile; * - statistically significant difference between the values of the control and experimental groups (p <0.05, p <0.01); # - statistically significant difference between the studied and previous experimental groups (p <0.05, p <0.01); CT - clotting time; CFT - clot formation time; a - angle a, reflects the intensity of thrombosis; MCD - maximum clot density.
maximum clot density value was close to the value recorded on the 5th day, and increased by 2.7 times (p = 0.0023) as compared to the value of the index on the 10th day. The value of this indicator also exceeded its level in the control group by 1.9 times (p = 0.038).
On the 20th day, when comparing with the control group parameter, a decrease in clotting time by 25% was recorded (p = 0.0025).
On the 30th day, a 20% decrease in coagulation time was observed compared with the control group (p = 0.028). The time of clot formation also decreased both in comparison with the control group and in comparison with the previous group (20th day) by 2 and 1.6 times, respectively (p = 0.0021). An increase was observed both in the angle alpha, reflecting the process of fibrin formation, by 7 ° (p = 0.045), and in the maximum clot density, by 1.8 times compared with the values recorded on day 20, and 2.2 times in comparison with the value of the indicator in the control group (p = 0.0011).
Thus, the study showed staged changes developing in the hemostasis system under the influence of daily cooling to an ultra-high degree of hypothermia. According to the literature, the primary reaction of the hemostatic system to stress is hy-percoagulation [8]. In the study, hypocoagulation recorded on the 1st experimental day is due to a decrease in the activity of enzymes under the influence of hypothermia, and demonstrates secondary disturbances in the hemostasis system [9]. Stress-reaction under the action of low temperatures is mediated by the activation of the sympa-tho-adrenal system and is accompanied by the release of catecholamines in the blood, as evidenced by the development of a hypercoagulable shift and an increase in the maximum clot density on the 2nd experimental day immediately after the stop of cooling. Since the adaptation of the body to cold is associated with an increase in the concentration of catecholamines, a number of authors described a sharp increase in the concentration of norepineph-rine in the peripheral blood: on the 5th cooling day - 1.5 times; on the 15th day - 1.7 times, and by the 30th day of the experiment - up to 2 times [10, 11, 12]. In addition, it has been shown that in developing adaptation to cold exposure in the body, there arise and strengthen mechanisms to prevent the processes of lipid peroxidation (LPO). At the same time, there is a separation of phosphorylation and free-radical oxidation in favor of the latter [13]. Significant increase in LPO products on the 7th, 14th, 21st and 28th days of the experiment [14, 15, 16] is considered a sign of failure of the response. At the same time, it has been established that LPO products stimulate the aggregation potential of blood cells and contribute to the development of hemor-heological disorders [17]. In our study, hypercoag-ulation increases gradually, starting from the 2nd day, and reaches a maximum on the 14th day. After
2 weeks of daily cooling, the most powerful hyper-coagulative shift for the entire experimental period is recorded, characterized by a minimum clotting time and a large clot density. By the end of the experiment (on the 30th day), hypercoagulation is aggravated by an increase in the rate of formation of the fibrin network (in terms of angle a) and an increase in clot density to the maximum values recorded throughout the experiment. The described changes correlate with the literature data and indicate the development of tension in functional systems formed to maintain an adequate functioning of the hemostasis system [6].
Conclusions:
1. Daily cooling to an ultra-high degree of hypothermia is accompanied by staged changes in the hemostasis system.
2. On the 1st experimental day, a hypocoagulation shift is recorded. Starting from the 2nd experimental day, the development and increase of hypercoagulative changes are observed.
3. The maximum hypercoagulative shift is observed on the 14th experimental day, which is accompanied by the smallest clotting time recorded for the entire experimental period.
4. On the 30th day, there is registered hyperco-agulation, an increase in the rate of formation of the fibrin network and an increase in the maximum density of the clot.
"The study was performed with the financial support of the Russian Foundation for Basic Research in the framework of the scientific project No. 16-34-60054 mol_a_dk."
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Contacts
Corresponding author: Lycheva Natalia Alek-sandrovna, Doctor of Biological Sciences, Senior Researcher of the Biomedical Laboratory of the Center for Biomedical Research, Altai State Medical University, Barnaul. 656038, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566928. Email: natalia.lycheva@yandex.ru
Author information
Makushkina Daria Aleksandrovna, 5th year student of the Medical Department, Altai State Medical University, Barnaul. 656038, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566928. Email: science@agmu.ru
Sedov Anton Vyacheslavovich, 5th year student of the Medical Department, Altai State Medical University, Barnaul.
656038, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566928. Email: science@agmu.ru
Shakhmatov Igor Ilyich, Doctor of Medical Sciences, Head of the Department of Normal Physiology, 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, Altai State Medical University, Barnaul.
656038, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 566928. Email: vik@mail.ru
