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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 62  |  Issue : 6  |  Page : 256-260

Changes in the microvasculature and hemostatic system in rats after insonation


Department of Normal Physiology, Federal State Budgetary Educational Institution of Higher Education “Altai State Medical University” of the Ministry of Health of the Russian Federation, Barnaul, Altai Territory; Laboratory of Physiology and Pathology of Hemostasis and Hemodynamics, Federal State Budgetary Scientific Institution “Scientific-Research Institute of Physiology and Basic Medicine”, Novosibirsk, Russia

Date of Submission28-Jan-2019
Date of Acceptance16-Oct-2019
Date of Web Publication29-Nov-2019

Correspondence Address:
Dr. Yuliya Bondarchuk
Altai State Medical University, Lenin Prospekt, 40, Barnaul 656038
Russia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_9_19

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  Abstract 

Ultrasound, one of the most physically impactful factors of the modern human living environment, can cause hemodynamic changes in the microvasculature and the hemostatic system. Such shifts can be considered as possible predictors of cardiovascular diseases and their complications. This study aimed to examine the effect of 7-day in-air insonation on the microvasculature and hemostatic system of rats. The study included 28 male Wistar rats. A group of study animals was insonated over 7 days at a frequency of 25 kHz. The emitters were installed in a vertical position at a distance of 10 cm from both the sidewalls of a coarse wire cage. The sound pressure was 89.0 dB and power flux density was 7.73 ± 0.03 W/cm2. The microvasculature values of the study rats obtained by laser Doppler flowmetry were compared to those of control animals. To evaluate the hemostatic system, an integral research method, thromboelastography, was used. In the study rats, in response to 7-day insonation, statistically significant decreases in the active and passive factors of blood circulation modulation were observed compared to the control animals: microcirculation, flux, amplitude of endothelial and vasomotor vibrations, and amplitude of respiratory and pulse waves. According to the thromboelastography data, prolonged coagulation time at the initial stage and inhibited fibrinolytic activity were recorded. Thus, the study animals showed signs of a stress reaction based on changes in their microcirculatory parameters confirmed by increased serum concentrations of adrenocorticotropic hormone and cortisol and analysis of behavioral reactions in the open-field test.

Keywords: Hemostatic system, insonation, microvasculature, stress


How to cite this article:
Bondarchuk Y, Nosova M, Shakhmatov I. Changes in the microvasculature and hemostatic system in rats after insonation. Chin J Physiol 2019;62:256-60

How to cite this URL:
Bondarchuk Y, Nosova M, Shakhmatov I. Changes in the microvasculature and hemostatic system in rats after insonation. Chin J Physiol [serial online] 2019 [cited 2019 Dec 8];62:256-60. Available from: http://www.cjphysiology.org/text.asp?2019/62/6/256/272030


  Introduction Top


Microvasculature disorders may be the underlying cause of many pathological processes. Local changes in the microcirculation are visible at an early stage of a number of abnormalities (myocardial infarction, angina pectoris, and arterial hypertension), and as pathological processes spread, they become systemic; therefore, data obtained from one microcirculation area can represent the microvasculature as a whole.[1]

Ultrasound, one of the most physically impactful factors of the modern human living environment, can cause hemodynamic changes in the microcirculation system, particularly in the capillaries. Ultrasound is a high-frequency (over 20 kHz) mechanical vibration propagating in elastic media. Contact insonation can cause tissue circulatory failure due to spasms of the blood vessels, particularly the capillaries. Long-term systematic insonation in the air causes changes in the function of the nervous, cardiovascular, and endocrine systems as well as auditory and vestibular analyzers. The influences of physical factors on rat behavior including ionizing and nonionizing radiation and vibration have been described.[2],[3] Inactivity combined with low-dose radiation can shift the balance of nervous processes toward excitation.[4] However, there are insufficient data on the effects of in-air ultrasonic waves on the body. In animals that are capable of perceiving ultrasonic waves, they cause psychoemotional stress. Thus, emitters with a frequency of 35, 38, 40, and 50 kHz irritated and demobilized rats.[5] We previously demonstrated that hemostatic parameters, sensitive markers of the body's adaptive potential, quickly respond to stress.[6]

This study aimed to analyze the effect of 7-day in-air insonation on the microvasculature and the hemostatic system of rats.


  Materials and Methods Top


The study included 28 male Wistar rats (14 each in the study and control groups) weighing 250 ± 20 g each.

The study group was insonated over 7 days using a Filin repellent generator (DonCont Research and Production Enterprize, Russia). During the study, the external meteorological parameters of the room were as follows: air temperature, +22.5°С ± 0.2°С; atmospheric pressure, −101.3 ± 9.3 kPa, and air density, −1.19 ± 0.03 kg/m3. The emitters were installed in a vertical position at a distance of 10 cm from both the sidewalls of the coarse wire cell (57 cm × 37 cm × 20 cm). Insonation parameters were studied using an Ecofizika-110 A sound-and-vibration meter spectrum analyzer (Russia) with an ultrasonic microphone at a frequency of 25 kHz. The ultrasonic microphone was located inside the cell and turned toward the generator. The sound pressure was 89.0 dB, while the power flux density or strength of the ultrasonic vibrations was 7.73 ± 0.03 W/cm2.[7]

After the exposure, microvasculature values were studied using laser Doppler flowmetry with the analysis of the amplitude-frequency spectrum of blood flow oscillations using LAKK-02 device (Lazma Research and Production Enterprize, Russia). The head of the optical probe was fixed at each animal's tail base. The duration of the laser Doppler flowmetry recording was 5 min. The main microcirculation parameters were recorded, and the amplitude–frequency spectrum of blood flow fluctuations from 0.005 to 3 Hz was analyzed. Four nonoverlapping frequency ranges were formed in the frequency interval, which made it possible to assess “active” and “passive” parts of the microcirculation regulation.

Adrenocorticotropic hormone (ACTH) and cortisol blood levels were estimated by enzyme-linked immunosorbent assay with an Immulite Analyzer (Siemens, USA).

Before the trial, during a week-long adaptation to the vivarium conditions, all rats were kept in standard conditions according to the Good Laboratory Practice requirements. The rats were used in accordance with the Directive 2010/63/EU of the European Parliament, the Council of September 22, 2010, on the protection of animals used for scientific purposes.[8] The protocol of experiment is approved by the Local Ethical Committee of Federal State Budgetary Educational Institution of Higher Education “Altai State Medical University” of the Ministry of health of the Russian Federation (the Protocol No. 8 of 22.10.2018).

Anesthesia and animal mortifications were performed in accordance with the rules of working with experimental animals.

In the present work, an integral research method, thromboelastography, was used to evaluate the hemostatic system. The thromboelastogram was recorded on a Rotem device (Pentapharm GmbH, Germany) using Natem reagent, which included calcium chloride.

During the thromboelastogram examination, the following values were considered:

Coagulation time (CT) is the time from reagent administration until the thromboelastogram findings reach an amplitude of 2 mm. This interval represents coagulation initiation.[9]

Clot formation time (CFT), the thromboelastogram amplitude changes from 2 to 20 mm, represents thrombus formation amplification.[9]

Alpha angle (ALP), the angle formed by the longitudinal axis and a straight line drawn tangentially to the thromboelastogram from a point corresponding to a clot amplitude of 2 mm, reflects clot formation kinetics and represents the propagation phase.[10]

Maximum clot firmness (MCF) represents the maximum clot amplitude and reflects platelet and fibrinogen functions.

Maximum lysis (ML), the level of maximum fibrinolysis recorded during the analysis, is defined as the lowest amplitude after reaching MCF.

A10, the clot density amplitude at the 10th min from the beginning of its formation (from the end of the CT), is determined by the thromboelastogram amplitude after 10 min.[11]

The statistical analysis was performed on a personal computer using the MedCalc Version 17.9.7 statistical software package (license BU556-P12YT-BBS55-YAH5M-UBE51). Statistical significance was evaluated using the nonparametric Mann–Whitney U-test since the signs did not follow the normal distribution. The differences were considered significant at values of P < 0.05.


  Results Top


Microcirculation values were studied immediately after the 7-day insonation. Insonation significantly decreased the active blood flow modulation factors. Thus, microcirculation and flux decreased by 32% and 63%, respectively, while the amplitude of the endothelial and vasomotor vibrations decreased by 63% and 74%, respectively. A decrease in passive blood flow modulation factors has been reported as well; compared to control animals, the amplitude of respiratory waves decreased by 71%, while the amplitude of pulse waves was decreased by 79% [Figure 1].
Figure 1: Changes in microcirculation parameters (Δ, %) in control and study rats after 7-day insonation. Parameters of control rats are considered 100%. PM, microcirculation index; SKO (σ), lax, standard deviation of blood flow oscillation amplitudes; VLF, endothelial waves; LF, vasomotor waves; HF1, respiratory waves; CF, pulse waves. *Significant difference between the control and study rats (P < 0.05).

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According to the thromboelastogram findings [Figure 2], the CT from the initial stage to a clot density amplitude of 2 mm was prolonged by 37%, while the CT amplitude growth at the 10th min from the beginning of clot formation was 62%. There was no statistically significant change in CFT recorded from the start of clot formation to a density amplitude of 20 mm or in serum ALP in the control animals. There were no significant changes in MCF. The inhibition of fibrinolytic activity was evidenced by a 75% decrease in the ML time.
Figure 2: Changes in hemostasis parameters (Δ, %) in control and study rats after 7-day insonation. Parameters of control rats are considered 100%. CT: Coagulation time; CFT: Clot formation time; ALP: Alpha angle; MCF: Maximum clot firmness; ML: Maximum lysis; A10: Clot density after 10 minutes. *Significant differences between control and study rats (P < 0.05).

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  Discussion Top


In our study, insonation with a frequency of 25 kHz was used since vibrations with a wave frequency of 22–25 kHz are emitted by animals in danger after a defeat in a fight or during pain exposure;[12] therefore, they cause stress in rats. Under prolonged exposure, animals may develop a depressive-like state that plays a significant role in the formation of psychoemotional stress.[13] In our trial, the development of a stress reaction was confirmed by the statistically significant increase in the animals' serum ACTH and cortisol levels. The mean ACTH level increased by 227.5%, while the mean cortisol level increased by 36.7% [Table 1]. In addition, as shown in previously published results (open-field test), insonation caused stress and affected individual behavior elements in 90% of the tested rats.[14]
Table 1: Parameters of the concentration of hormones in the blood of control and experimental rats

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A significant role in the regulation of blood flow in the vessels of the microvasculature is assigned to tone-forming factors that act through vascular wall muscles.[15] A decrease in the modulation of blood flow by active factors is indicated by a decrease in their amplitude in response to the 7-day insonation. Against the background of increased sympathetic impulse as a result of stress reactions, there was an increase in the tension of smooth muscle cells of the vascular wall, resulting in vasoconstriction and a decrease in dilatation reserve. Data on decreased active vasomotor factors of blood flow modulation, microcirculation, and flux after insonation confirm the role of the myogenic component as a cause of an increase in wall shear stress; a consequence of the latter is the development of endothelial dysfunction associated with a decrease of endothelial nitric oxide.[16] The observed decrease in the amplitude of endothelial waves also speaks in favor of a blood flow modulation reduction and a lower blood flow into the microvasculature. The stasis signs appearing therein cause blood flow shunting, which violates tissue metabolism.

An important question is the role of passive factors in modulating blood flow, which are external factors outside the microcirculation zone, namely pulse waves originating at the terminal arteries at the entrance to the microvasculature and respiratory waves from the veins at the exit. The observed decrease in amplitude and the smoothing of the pulse wave peak after insonation occurred due to an increase in the tone of the resistive vessels (small arteries and arterioles). Respiratory waves are directly related to modulation of blood flow in venous regions, and respiratory oscillations reflect the distribution of perfusion and pressure in venules. In the study animals, arteriovenous pressure increased since there was a decrease in the respiratory wave amplitude.[17]

Hemodynamic parameters formed for transcapillary exchange are normally optimal; due to the alternation of contraction and relaxation of vascular wall smooth muscles (active factors), the periodically changing blood volume (passive factors) is modulated.[15] On the contrary, the resulting spasm depletes the nutritional blood flow, causing ischemia and decreasing perfusion.

Based on our thromboelastography data, we ascertain the secondary nature of disorders in the hemostatic system in the study animals with respect to the primary disturbance of the microcirculation, which contributes to increasing blood flow resistance due to vasospasm. Thromboelastography makes it possible to qualitatively and quantitatively characterize thrombus formation, analyze clot density, clot stability, and the interaction of platelets and fibrinolysis, and indirectly evaluate the platelet content of the blood.[11] Whole blood serves as the material for this study. At high parietal shear stress rates, the maximum of which occurs on arterial vessels with a diameter of about 20 μm,[18] blood viscosity increases according to the plasma viscosity, hematocrit, and microrheological properties of red blood cells.[19] On the one hand, the occurrence of acute tissue ischemia largely depends on the state of neurohumoral regulation of vascular tone in the microvasculature and the hemostatic system, which determines the rheological properties of the blood. On the other hand, ischemic events stimulate the release of pro-inflammatory cytokines into the bloodstream, which have a powerful procoagulant effect.[20] In addition, the emerging turbulent blood flow due to the inflow of arteriovenous anastomoses into the venules at a significant angle also contributes to the release of proaggregants and procoagulants due to the collision of blood corpuscles with each other and the vessel walls.[1]

The comparison of coagulation shifts due to stress-related factors of any nature revealed the initial appearance of hypercoagulation, followed by hypocoagulation changes.[6] After the 7-day insonation period, we revealed an increase in the CT at the initial stage, which is usually associated with a deficiency in coagulation factors or a shift in the balance of the activator–inhibitor system in favor of the latter.

The absence of statistically significant changes in CFT and ALP values favors the assumption that the insonation did not disrupt the fibrin polymerization or the deficiency of platelets and/or fibrinogen, which is also confirmed by an increase in the clot density amplitude at the 10th min from the beginning of its formation. The absence of a significant change in MCF indicates platelet count stability and platelet aggregation ability. The decrease in the ML time indicates the inhibition of fibrinolytic activity. Our data are consistent with those of a study of a 7-day hyperthermic effect on rats, in which similar reactions were noted: hypocoagulation and inhibited the fibrinolytic activity of blood plasma with an unchanged fibrinogen concentration.[21]

Thus, we can conclude that significant changes in the microvasculature occur in response to 7-day insonation. The latter are not accompanied by as significant shifts in hemostatic parameters as shown in studies of the effects of other physical factors during a single exposure, causing a stress response;[21] on the contrary, they show a tendency to develop an adaptive reaction as in the case of taking an adaptogen.[22]


  Conclusions Top


  1. Seven-day insonation in rats causes significant changes in the microcirculation, including inhibiting active and passive mechanisms of blood flow modulation, spasms of the small arteries and arterioles, microcirculation system congestion, and endothelial dysfunction
  2. Changes in the microvasculature are accompanied by an increase in CT at the initial stage and the inhibition of fibrinolytic activity
  3. In the microvasculature, signs of a stress reaction are observed in response to 7-day insonation.


Financial support and sponsorship

This study was financially supported by the Federal State Budgetary Educational Institution of Higher Education “Altai State Medical University” of the Ministry of Health of the Russian Federation.

Conflicts of interest

There are no conflicts of interest.



 
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