Effect of ultrasound exposure duration on the state of microcirculation and hemostasis system in rats

In modern society, people are often subjected to chronic unavoidable stress provoking depressive-like states that play a significant role in the formation mechanisms of psycho-emotional stress. In view of this, the need to create an experimental model of psycho-emotional stress as a cause of cardiovascular disease development arose. By means of ultrasound waves which cause the depressive-like state in animals, it is possible to create such model as long as ultrasound has material properties and certain energy. We used the parameters of microcirculation and hemostasis as criteria for psycho-emotional stress development. Stress stimulation results in vasoconstriction and can be associated with the development of microcirculatory disorders due to a significant increase in blood levels of catecholamines. The development of acute tissue ischemia depends both on the state of neurohumoral regulation of vascular tone, and on the rheological blood properties. Hemostasis system is one of the most reactive body systems, and hemostasiological parameters play an important role in the process of adaptation to the effect of stress factors. Currently, studies aimed at finding possible predictors of cardiovascular diseases and their complications are relevant. In this regard, it seems promising to study the role of microcirculatory and hemostasis parameters as criteria for psycho-emotional stress development. The aim of this research was to assess the effect of ultrasound exposure duration on the state of microcirculation and hemostasis system in rats. The study was performed on 42 Wistar male rats divided into three groups: 1 control group and 2 experimental groups subjected to a 24-hour (Group 1) and a 7-day (Group 2) ultrasound exposure using a repellent-generator “Filin” (SPE “DonKont” Ltd., Russia) at a frequency of 25 kHz. Emitters were installed in a vertical position at a distance of 10 cm on both sides of the side cell walls made of coarse-meshed wire. The microphone of the ultrasonic vibration meter was located inside the cell and oriented towards the generator “Filin”. The sound pressure level was 89.0 dB and the power flow density was 7.73±0.03 W/cm². After exposure termination, microcirculation parameters were studied by laser Doppler flowmetry (LDF) method with analysis of the amplitude-frequency spectrum of blood flow oscillations by LAKK-02 apparatus (SPE “LAZMA” Ltd., Russia). The optical probe was fixed at the base of the animal's tail. The recording duration of LDF-gram was 5 minutes. The main microcirculation parameters were recorded, and the analysis of the amplitude-frequency spectrum of blood flow oscillations in the frequency range of 0.005 to 3 Hz was conducted. Four non-overlapping frequency ranges were formed in this range that allowed to estimate the state of “active” and “passive” links of micro-blood flow regulation. Blood levels of ACTH and cortisol were determined by enzyme immunoassay (EIA). The hemostasis system was assessed by an integral method, thromboelastometry. Thromboelastometry was performed by the “Rotem” device (“Pentapharm GmbH”, Germany) using the “Natem” reagent which includes calcium chloride. The statistical significance was assessed using the non-parametric Mann-Whitney U-test. The use of rats in experiments was carried out in accordance with the requirements of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986). In this research we revealed that in experimental rats, the 24-hour ultrasound exposure, primarily, caused significant disorders in microcirculation area in the form of vasoconstriction and dilation reserve reduction, and, secondly, it led to significant adverse changes in the hemostasis system that is a sign of stress. Evidence of the development of stress reaction was significantly increased concentration of ACTH by 227% (p=0.001) and cortisol by 37% (p=0.01) in the blood of these animals and the test results of the animals according to the “Open Field” method. A statistically significant decrease in the studied active factors of blood flow modulation, microcirculation and flax rates (by 66% and 68%), that characterize the role of the myogenic component as a reason of increased value of the wall shear stress was observed. The reduction of passive factors, pulse and respiratory waves, was also obtained. In experimental rats after the 24-hour ultrasound exposure a drop by 75% and 69% in the parameters was recorded compared to the control animals, and in the 7-day exposure group it was by 79% and 71% (See Table 1). In summation, these changes prove a formed spasm of microcirculation vessels. Reduction of blood flow into microcirculation in experimental animals of both groups was registered on the basis of reducing the amplitude of endothelial waves (by 75 and 63%) and vasomotor waves (by 78 and 74%) which inevitably leads to the development of stasis and disruption of tissue metabolism due to a blood flow bypass. Unidirectional changes in microcirculation in rats of the two experimental groups were accompanied by secondary changes in the hemostasis system. Based on the analysis of deviations of hemostasiological parameters from the control values, stress reaction development was recorded in rats exposed to the 24-hour ultrasound and the tendency of smoothing the deviations of hemostasis parameters after the 7-day exposure (See Table 2) was observed. We found that after the 24-hour and the 7-day exposures there was a decrease in the maximum lysis (ML) by 100 and 75% compared with the control which indicates the inhibition of fibrinolytic activity and represents a risk factor for venous thromboembolism and arterial thrombosis. Lower evidence of the decline in ML after the7-day exposure to stress factors together with the absence of signs of infringement of fibrin polymerization process and the growth of the clot density amplitude in the 10th minute after the beginning of its formation shows a tendency towards the gradual normalization of the hemostatic system. Thus, ultrasound exposure simulates the state of chronic unavoidable stress in experimental animals. The state of psycho-emotional stress is confirmed by the data on the increased concentration of hormones (ACTH and cortisol) in blood as well as by the results of testing on animals using the “Open Field” method. The study results indicate that the diagnosis of the microcirculation and hemostasis parameters is a sensitive way to assess the development of psycho-emotional stress and organism adaptedness. The return of some parameters of the hemostasis system in response to the 7-day stress exposure compared to the 24-hour exposure to the indicators specific for control animals can be regarded as a manifestation of the initial stages of adaptation to the stress factor. The paper contains 2 Tables and 30 References.

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