The Effect of Electroacupuncture on Plasma Fibrinogen Levels in Restraint Stress Wistar Rats

Abstract

Objective: Blood is a suspended cellular element that dissolves in plasma. Blood transports oxygen and nutrients to all body cells and carries metabolic waste from the whole body. The nature of blood flowing through vessels is a factor that plays an important role in oxygen delivery and tissue perfusion. In acute stress conditions, various reactions can occur and affect the blood flow in blood vessels. Electroacupuncture (EA) is an additional therapy with minimal side-effects that can improve the quality of blood flow. This study investigated the effect of EA bilaterally at ST 36 (Zusanli) on plasma fibrinogen.

Materials and Methods: Eighteen male Wistar rats were divided randomly into 3 groups: (1) a control group (n = 6); (2) a restraint-stress group (n = 6); and (3) a restraint-stress-and-EA group (n = 6). EA was performed in group 3 after the rats were subjected to 3 hours of restraint stress.

Results: There were significant mean differences in plasma fibrinogen levels (P = 0.048; 95% confidence interval: 0.5–109.5) between the restraint stress groups and the control group.

Conclusion: This study showed that the EA on ST 36 lowered levels of plasma fibrinogen of Wistar rats in restraint stress model.

Introduction

Blood is a two-element suspension (with erythrocytes, leukocytes, platelets) dissolved in a solution of organic molecules, proteins, and salts called plasma.¹ Blood transports oxygen and nutrients to every cell of the body and carries waste products out of the body. To accomplish this, blood must pass through a complex vascular network consisting of blood vessels with diameters varying from 5 μm to 3 cm at appropriate circulation, and do this at flow rates that can meet organ's and tissue's metabolic needs. The purpose of blood flow is to maintain O₂ delivery and tissue perfusion. Blood flow is dependent on the pressure generated from the heart, vascular resistance (such as diameter and length), and rheologic properties of the blood itself.²

The main factors that contribute to blood flow are blood pressure (BP) and blood hydrodynamic characteristics. The relationship between control of vascular resistance and the hydrodynamic characteristics of blood is very important but is still unclear.³ One of the hemorheologic factors that plays a role in circulation is blood viscosity. When blood viscosity increases, it can increase flow resistance; therefore, this increases the work of the heart and interferes with organ perfusion.²

Epidemiologic studies show that rheologic variables, such as hematocrit and blood viscosity, are related to mortality rates in acute cardiovascular events—such as myocardial infarction and stroke—and can increase the risk of cardiac thrombosis and ischemia. Among patients with coronary artery disease, the hemoconcentration effect of acute stress influences parameters related to myocardial supply (e.g., coronary thrombosis, decreased microcirculation flow) and needs (e.g., increased workload for the heart to pump blood that has high viscosity). Furthermore, hemoconcentration due to stress will increase shear stress on the walls of blood vessels, which will trigger the formation of atherosclerotic plaques that can lead to plaque rupture and the formation of blood clots with the potential to cause myocardial ischemia.⁴ Clinical studies have also confirmed the relationship between blood fluidity and the severity of circulatory disorders, although the causal relationship might not be very visible.⁵

According to Poiseuille's law, a decrease in blood fluidity can increase vascular resistance and increase blood coagulation.⁶ Decreasing blood fluidity can cause circulatory-system disorders—such as arterial sclerosis or embolization—and cause vascular endothelial-cell damage due to hypertension; degradation of glucose tolerance; chronic inflammation; degradation of blood-vessel flexibility due to hyperlipidemia and aging; weakness of blood cells; and plasma plasticity degradation.⁷ Currently, blood viscosity is known as cardiovascular disease (CVD) indicators for peripheral arterial disease, arterial hypertension, diabetes mellitus, high cholesterol and triglyceride levels, and crescent anemia.⁸

Anticoagulant or antithrombotic therapy is often used in patients with a risk of venous thromboembolism (VTE) who are either hospitalized or who will undergo an examination or therapy procedure. Approximately 40% of patients have 3 or more risk factors for thromboembolism, so most inpatients receive thromboprophylaxis therapy. However, to lower the risk of bleeding after antithrombotic administration, it is necessary to follow the guidelines for administration of antithrombotic therapy, including appropriate pharmacologic agents, the level of anticoagulation needed, and the duration of continued therapy. In general, it is very difficult to determine the correct balance between the risk factors and benefits of antithrombotic therapy in patients who will undergo surgery.⁹

In 2016, the American Heart Association (AHA)¹⁰ through a Heart Disease and Stroke Statistics Update reported that 15.5 million people ages ≥20 years in the United States have coronary heart disease (CHD). CHD is the leading cause of adult deaths in the United States, accounting for one-third of all deaths of people over age 35. Researchers in 2014 used data from the World Health Organization (WHO) originating from 49 countries in Europe and northern Asia, reporting that more than 4 million deaths per year are caused by CVD disease,¹¹ and ∼17.9 million people died each year from CVD worldwide, constituting 31% of all global deaths. This report also stated that 85% of all deaths are caused by heart attacks and strokes.¹²

Based on Riskesdas' data in 2013,¹³ the highest prevalence of CVD in Indonesia is due to CHD, which is at 1.5% of the population. From this prevalence, the highest number is in the NTT province (4.4%) and the lowest is in Riau province (0.3%). CHD is most prevalent in the people ages 65–74 (3.6%), followed by people ages 75 and older (3.2%), those who are ages 55–64 (2.1%), and those who are ages 35–44 (1.3%). Whereas, according to economic status, CHD occurs mostly in people at a lower economic level (2.1%) or a lower middle-class level (1.6%). Of all deaths from CVD, 7.4 million (42.3%) were caused by CHD and 6.7 million (38.8%) were caused by stroke.¹³

Stress means different things to different people under different conditions. Stress is a condition wherein an individual becomes anxious due to a challenge that is out of his or her control.¹⁴ Stress is a major risk factor in several multifactorial diseases, such as CVD, psychiatric disorders such as anxiety and depression, and neurodegenerative diseases such as Alzheimer's and Parkinson's diseases.¹⁵ Physical and mental stresses cause sympathetic excitation and results in a rapid increase of heart rate (HR) and BP, which will exacerbate the development of acute coronary syndrome disease.¹⁶ Scientific evidence shows that acute mental stress can reduce blood plasma volume and increase the concentration of red blood cells (RBCs). Mental stress also influences other factors related to hemoconcentration including colloid osmotic pressure and plasma and blood density as well as plasma and blood viscosity.⁴ To understand the effect of stress on blood viscosity, experimental animals such as rats are used, so the types of stressors received can be equated. In experimental animals, stress responsiveness can be measured noninvasively by stressing animals through restraints, which is a psychophysical stressor that is not physically harmful and is the most-frequently used stressor.¹⁵

Acupuncture is believed to have the ability to regulate various biologic functions. Research shows that acupuncture affects blood cytokine levels, hormonal levels, and leukocyte counts. Smooth blood flow affects the body's supply of oxygen and nutrients and prevents the formation of substances that produce pain and the emergence of reactive oxygen species (ROS). Physiologically, the main driver of BP is heart pressure. However, blood flow is also influenced by the hemodynamic characteristics of the blood. Changes in the composition of blood cells and plasma components can affect blood fluidity in the long term and affect blood-cell activities such as red blood–cell agglutination, leukocyte adherence, and platelet aggregation in the short term.⁷

Theoretical Overview

Complete blood is a concentrated suspension of cellular elements, including RBCs or erythrocytes, white blood cells or leukocytes, and platelets. These cellular elements are suspended in plasma, a liquid solution of ionic protein with a normal viscosity of 1.5–1.8 times that of water. The average person has between 4.5 L and 6 L of blood or about 6%–8% of body weight in healthy individuals. During pregnancy, blood volume increases by 50%.¹⁷

Blood viscosity is controlled by psychochemical characteristics and the quantity of various cell components as well as the constitution and levels of plasma components, coaxial components of cells and plasma components, and the psychochemical environment of the blood. This condition varies in the same blood supply, but the viscosity coefficient changes.⁷ Impaired blood viscosity can affect tissue perfusion significantly and result in declining normal body functions, especially if vascular properties are also disrupted by the disease.¹ A decrease in blood fluidity increases the risk of thrombosis, high BP, and the formation of ROS. Various causes of psychologic and physical stress can cause disturbances through humoral and neuronal mechanisms.⁷ The two most important parameters of blood viscosity are hematocrit and plasma proteins (e.g., fibrinogen). Lipid profiles such as low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides also affect low-shear blood viscosity more than high-shear blood viscosity.¹⁸

Research shows that endothelium plays an important role in regulation of blood viscosity. Endothelium acts as a sensor and effector for blood viscosity that is not only regulated by blood viscosity, but also plays an important role in the regulation of feedback mechanisms to control this viscosity. The effect of blood viscosity on organisms depends on endothelial-cell function. Under normal conditions, increased blood viscosity increases the shear stress of the vascular wall and increases the production of factors that relax the endothelium such as nitric oxide (NO) and prostacyclin. These vasoactive substances not only cause vasodilation to decrease mean arterial pressure (MAP) but also decrease blood viscosity by reducing erythrocyte deformability, thereby suppressing erythrocyte aggregation and platelet aggregation and adhesion. In pathologic conditions, many dangerous factors—such as mental stress, inflammation, and high sugar levels—cause increased production of ROS, fibrinogen, immunoglobulins, and hematocrit, which results in increased abnormal blood viscosity. Furthermore, this can lead to endothelial dysfunction—directly and indirectly—and disruption of regulatory feedback mechanisms. Therefore, abnormal changes in blood viscosity and endothelial dysfunction trigger each other and form a vicious cycle.¹⁹

Plasma is mainly composed of water (∼ 90%–92%) with dissolved organic and inorganic substances (∼ 1%–2%), various proteins (especially albumin, globulin, and fibrinogen), and other components. Plasma's main physiologic function is to transmit dissolved substances, nutrients, and waste products, and cellular elements forming throughout the circulatory system. Plasma also plays an important role in maintaining homeostasis (constant osmotic-colloidal pressure), buffer function, and coagulation.¹⁷ Fibrinogen is one of the plasma proteins that plays a role in blood viscosity. According to a 17-year cohort study by Peters et al., which examined the effect of blood and plasma viscosity on cardiovascular disease and mortality, it was found that plasma viscosity plays a role independently in the pathogenesis of CVD.²⁰

Increased concentration of cellular components and blood constituents caused by a decrease in plasma volume results in changes in blood-flow properties. Hemoconcentration due to stress results in thickening blood. Blood viscosity contributes to total peripheral resistance and BP.²¹ Plasma viscosity is influenced by proteins contained in plasma. The amount of a group of plasma proteins such as fibrinogen, known as acute-phase reactants, increases during the inflammatory reaction. This concentration of fibrinogen in inflammatory conditions can increase five times, therefore increasing plasma viscosity significantly. Plasma fibrinogen concentration is also an important factor in the emergence of red blood–cell aggregation and is a major determinant of plasma viscosity and overall blood viscosity, wherein the increase will result in an increase in total peripheral resistance and greater systemic arterial pressure.²²

In several epidemiologic studies, an increase in plasma fibrinogen concentrations was identified as a strong predictor of vascular problems (such as myocardial infarction and stroke) that increase with age.⁵ Erythrocytes carry a negative charge on the surface of the membrane. Fibrinogen is not only the most acute-phase protein but is also a large positively charged protein molecule. If there is a lot of fibrinogen in plasma, it will make erythrocytes tend to aggregate with each other and become difficult to separate. In a laboratory examination, an increased rate of erythrocyte sedimentation can be seen.²³ Fibrinogen is also sensitive to acute mental stress, wherein an increase is noted from blood samples taken as soon as possible after challenging tasks are faced, and partial recovery occurs after 45 minutes.²²

In conditions of acute mental stress, there is a cascade of changes in the nervous, cardiovascular, endocrine, and immune systems. These changes indicate a stress response and are usually adaptive, at least in the short-term. Stress hormones are produced by the sympathetic nervous system (SNS) and the adrenocortical–hypothalamic–pituitary axis. The SNS stimulates the adrenal medulla to produce catecholamines (e.g., epinephrine). At the same time, the paraventricular nucleus at the hypothalamus produces corticotropin-releasing factor and then stimulates the pituitary to produce adrenocorticotropin. Adrenocorticotropin then stimulates the adrenal cortex to secrete cortisol. Catecholamines and cortisol increase available energy sources by triggering lipolysis and conversion of glucogen to glucose (e.g., glucose).²⁴

Stress in animals triggers changes in perception, behavior, thermoregulation, social interaction, sleep, cognition, endocrine secretion, neurotransmission, reproductive ability, immune defense, cardiovascular and gastrointestinal functions, metabolism, and susceptibility to harmful substances that have shown a pattern among mammal species. From the perspective of a stress model, three important consequences that must be considered include: (1) duration of the stress-indicator assessment: (2) duration of the stress challenge, and (3) the habituation phenomenon.²⁵ Most laboratory studies regarding the effects of social stress use rodents, especially laboratory rats and sometimes hamsters. Various parameters can be used to monitor responses in animal stress models, including behavioral, physiologic, metabolic, neurochemical, endocrine, and immunologic parameters.²⁶

Medical acupuncture is one of the minimally invasive therapeutic modalities with fine matchmaking—an adaptation of Chinese acupuncture based on anatomy, physiology, and pathology using the principles of scientific evidence-based medicine. The application of clinical acupuncture in curative, rehabilitative, preventive, and promotive efforts uses the basis of scientific evidence.²⁷ Acupuncture works primarily by stimulating the nervous system, and acupuncture's mechanism of action includes local axon reflexes, segmental and extrasegmental neuromodulations, and other central nervous system effects. The method that can be used is involves inserting a needle while rotating it or tapping into a specific part of the skin known as an acupuncture point.²⁸

Increased sympathetic nerve activity elevates BP through constricting arterioles and increasing the strength and rate of contraction of the heart to increase cardiac output. The acupuncture mechanism can be centrally activated. The acupuncture mechanism can centrally activate n. arcuatus in the hypothalamus then stimulates the ventrolateral periaqueductal gray to inhibit sympathoexcitatory premotor neurons in the ventrolateral rostral medulla, resulting in a decrease in sympathetic activity. A decrease in sympathetic activity makes blood vessels tend to vasodilate, which indirectly will facilitate blood flow.²⁹

The mechanism underlying the effect of acupuncture to improve blood viscosity cannot be explained with certainty. There have been several animal studies^{3,6,7,8,30,33} conducted by the same group of researchers, examining the effect of acupuncture on blood fluidity. From these several studies, it can be concluded that acupuncture affects blood flow from parasympathetic autonomic stimulation, decreases catecholamine levels, and stimulates of endothelial NO expenditure.

The mechanism underlying the activation of NO by acupuncture stimuli might explain how acupuncture can increase mediators and neurotransmitters—such as acetylcholine, bradykinin, and histamine—and these mediators can activate endothelial NO synthase (eNOS) through the dependent calcium pathway. In addition, acupuncture affects viscosity by reducing erythrocyte aggregation that can affect hemodynamic shear stress, which can activate eNOS through independent calcium pathways. Increased NO levels function as potent vasodilators and inhibit platelet adhesion by inhibiting soluble platelet selectin (sP-selectin)–activation. The inhibition of platelet aggregation reduces the risk of thrombus as a major cause of cardiovascular events.³⁰ In studies of women with polycystic ovary syndrome (PCOS), low-frequency EA caused a decrease in plasminogen activator inhibitor–1 activity, circulating fibrinogen levels, and tissue plasminogen activator.³¹

EA is defined as the use of a part of an electric current that pulses through the body's tissues through one (or more) pair(s) of acupuncture needles for therapeutic purposes. The electricity flow used in EA has various characteristics, including polarity, frequency, amplitude/intensity, mode, pulse duration, and waveform. The advantages of EA include being more effective in some situations; requiring less time than repeated manipulations with needles; if manual acupuncture does not produce an effect, EA can be used to produce a good effect, enabling stronger and more-continuous stimulation with less tissue damage and more control that can be standardized and measured objectively.³²

EA at a frequency of 2 Hz results in a significant increase in enkephalin immunoreactivity, not in dinorphine immunoreactivity, while 100 Hz increases dinorphine immunoreactivity but not enkephalin immunoreactivity. In the brain, enkephalin and endorphins associated with opioid receptors μ and δ have been shown to be more important for modulating cardiovascular action, compared to dynorphine (κ opioids).²⁹

Materials and Methods

The current study was a randomized, controlled, double-blinded, laboratory experimental post-test conducted in December of 2018. The study was conducted at the Experimental Animal Laboratory, Biomedical Research Center and Basic Health Technology, Research and Development Agency, in the Ministry of Health of the Republic of Indonesia.

The population tested was an experimental species of adult male Wistar rat who met the inclusion criteria. Rats were obtained from the Try Animal Laboratory, Biomedical Research Center and Basic Health Technology, Research and Development Agency, of the Ministry of Health of the Republic of Indonesia. Inclusion criteria included Wistar male rats,^30,33 ages 8–10 weeks,³⁴ weighing 250–350 g,³⁵ having good appetites and drinking habits,^34,36 and fur that did not fall out.³⁶ Exclusion criteria were inflammation and infection in the acupuncture-point area.³⁰ Fall criteria included dead rats during the study⁸ and lysis blood samples.⁸

The samples were estimated by using a sample-size calculation formula for experimental research by Federer,³⁷ based on the principle of reducing the use of animals in research to use as few as possible to obtain optimum results; the number of samples for the current study was 18 rats. These study subjects were randomly divided into 3 groups, namely, (1) a control group, (2) a restraint-stress group, and (3) another restraint-stress group who received EA.

In the control group, the rats did not get any treatment. In the restraint stress group, the rat were subjected to restraint stress inside a tube for 3 hours.³⁴ The restraint stress–and-EA group received restraint stress for 3 hours and then were punctured on ST 36 (Zusanli) and stimulated with continuous-wave EA, at a frequency 2 Hz and an intensity of 1 mA for 15 minutes (Fig. 1).³⁰ The 3 groups were given the same anesthetic agents—injections of ketamine (0.075 mg/g body weight) and xylazine (0.02 mg/g body weight) intramuscularly.

FIG. 1.

In the control group, rats did not get any treatment. In the restraint-stress group, the rats were subjected to restraint stress inside a tube for 3 hours.³⁴ The restraint stress–and-electroacupuncture (EA) groups received restraint stress for 3 hours, were then needled on ST 36 (Zusanli) and stimulated with continuous-wave EA, at a frequency of 2 Hz and an intensity of 1 mA for 15 minutes.³⁰ The 3 groups were given the same anesthetic agents—injections of ketamine (0.075 mg/g of body weight) and xylazine (0.02 mg/g of body weight) intramuscularly.

The collected research data were entered into a master table and processed by using SPSS 20.0.

This study was approved by the Health Research Ethics Committee of the Faculty of Medicine RSCM Jakarta (No: 1315/UN2.F1/ETHICS/2018) on December 17, 2018. In conducting the study, the researchers applied the principles of the “3Rs” in the study protocol: (1) replacement; (2) reduction; and (3) refinement.

Results

The study was conducted on 18 male Wistar rats who met the inclusion criteria. The rats were divided into 3 groups: (1) a normal control group (no treatment); (2) a negative control group (only restraint stress); and a restraint stress–and-EA group (received restraint stress and EA stimulation). Each group consisted of 6 rats. No rats were declared dead; thus all 18 rats completed the study.

There were no statistically significant differences (P > 0.05) in the basic characteristics of the rats (Table 1), so it could be concluded that the 3 groups of rats were equivalent and worthy of comparison.

Table 1.

Characteristics of the Study Subjects

	Groups
	Control	Negative control	Restraint stress-and-EA
Characteristics	(n = 6)	(n = 6)	(n = 6)	P-value*
Ages	8.5 ± 0.84	8.17 ± 0.41	8.83 ± 0.75	0.531*
(weeks)
(Mean ± SB)
Body weights (g)	244.83 ± 26.83	248.67 ± 17.20	259.17 ± 22.03	0.278*
(Mean ± SB)
Body temperatures (°C)	35.7 ± 0.27	35.52 ± 0.39	35.53 ± 0.57	0.717*
(Mean ± SB)
HRs	358.33 ± 23.17	362.02 ± 23.66	360.00 ± 23.67	0.990*
(beats/min)
(Mean ± SB)

^*ANOVA statistical test.

There was no statistically significant difference (P > 0.05) in the basic characteristics of the research subjects (rats), so it could be concluded that the 3 groups were equivalent and worthy of comparison.

EA, electroacupuncture; SD, standard deviation; HRs, heart rates; min, minute.

Table 2 shows that the results of an analysis of variance test on average plasma fibrinogen levels showed a statistically significant difference (P = 0.001), so a post hoc test to see the comparison of average plasma fibrinogen levels in these 3 groups was performed (Table 3).

Table 2.

Average Description of Plasma Fibrinogen Levels in the 3 Groups

	Groups
	Control	Negative control	Restraint stress-and-EA
Characteristics	(n = 6)	(n = 6)	(n = 6)	P
Fibrinogen (mg/dL)	154.50 ± 38.91	247.50 ± 31.14	192.50 ± 34.65	0.001*

^*Statistical one-way ANOVA.

The results of the ANOVA statistical test on average plasma fibrinogen levels showed a statistically significant difference (P = 0.001), so a post-hoc test was performed to compare the average plasma fibrinogen levels in these 3 groups.

EA, electroacupuncture; ANOVA, analysis of variance.

Table 3.

Analysis of Post-Hoc Comparison of Mean Fibrinogen Levels Among the Groups

Group	Mean difference (mg/dL)	Trust interval 95%		Average value P*
		Min	Max
Control vs. negative control	93	38.5	147.5	0.001 *
Control vs. restraint stress-and-EA	38	16.5	92.5	0.240 *
Negative control vs. restraint stress-and-EA	55	0.5	109.5	0.048 *

^*Post-hoc test on Tamhane's. There were 6 rats in each group.

Note: The mean plasma fibrinogen level in the control group was 154.50 mg/dL, while it was higher in the negative control group at 247.50 mg/dL. The mean difference of 93 mg/dL was statistically significant (P = 0.001). When the control group was compared to the restraint-and-EA group, the mean difference was 38 mg/dL, wherein the restraint stress-and-EA group's mean fibrinogen level was lower than that of the control group. The post-hoc test result indicated that there was no difference in the levels of fibrinogen plasma between the control group and restraint-and-EA group (P = 0.240). The plasma fibrinogen mean level for the restraint stress-and-EA group was 192.50 ± 34.65 mg/dL, which was lower than that of the negative control group, which had a mean level of 247.50 ± 31.14 mg/dL. The mean difference of 55 mg/dL was a statistically significant difference (P = 0.048).

EA, electroacupuncture; min, minimum; max, maximum.

The plasma-fibrinogen-levels examination in the control group showed that mean plasma fibrinogen levels were 154.50 mg/dL in this group; the mean levels were higher in the negative control group, at 247.50 mg/dL. This mean difference of 93 mg/dL was statistically significant (P = 0.001). In the comparison between the control group and the restraint stress–and-EA group, the mean difference between the 2 groups was 38 mg/dL, wherein the latter group's mean levels were lower than the those of the control group. The result of the post hoc test indicated that there was no significant difference in the levels of fibrinogen plasma between the control group and restraint stress–and-EA group (P = 0.240). The mean levels of plasma fibrinogen for the latter group was 192.50 ± 34.65 mg/dL, which were lower than those of the negative control group, which were 247.50 ± 31.14 mg/dL. The mean difference of 55 mg/dL was a statistically significant difference (P = 0.048).

Discussion

This was a randomized, double-blinded, laboratory experimental study with a post testing conducted on 18 subjects who met the inclusion criteria. The rats were divided randomly into 3 groups—(1) a control group, (2) a negative control group (restraint stress), and (3) a restraint stress–and–EA group—using a computer-based random allocation program. All of the rats finished the study.

Rats were chosen as the study subjects to minimize other factors that could cause acute stress, so the similarity of acute stressors were found in all rats except for those in the control group.²⁶ Male rats were chosen to reduce the risk of bias caused by hormonal influences that might have occurred if female rats had been used.^25,26

Before treatment, all of the rats' weights, body temperatures, and HRs were calculated, then the rats were divided randomly into the 3 groups. Each group consisted of 6 rats. Groups 2 and 3 received restraint stress for 3 hours and then group 3 was punctured on ST 36 and stimulated with continuous-wave EA, at a frequency 2 Hz and an intensity of 1 mA for 15 minutes.³⁰

The acupuncture point used in this study was ST 36. In research conducted by Hisamitsu and Ishikawa, in 2014, ST 36 provided the most-significant results with respect to determining blood fluidity; this was followed by LI 4 (Hegu) and SP 6 (Sanyinjiao), while PC 6 (Neiguan) and BL 23 (Shenshu) points did not give significant results on blood-flow time, which is an indicator of blood viscosity, wherein higher blood viscosity takes more time to flow in the blood vessels.³⁰

In the current study, only 1 acupuncture point (ST 36) was chosen in order to learn more about the effect of acupuncturing at this point on changes in plasma fibrinogen, as previous studies have shown that ST 36 can increase blood fluidity.^8,30,33 In addition, acupuncture at ST 36 is also known to reduce sympathetic activity with a decrease in noradrenaline^8,29; increase blood flow by increasing eNOS protein and mRNA, and decreasing platelet aggregation (shown by decreases in hydroperoxide and sP-selectin)³; reduce proinflammatory cytokine interleukin (IL)–6 and erythrocyte sedimentation rate; increase plasma superoxide dismutase, glutathione reductase catalase activity, glutathione-stimulating hormone, and plasma adenosine triphosphate concentrations; and reduce blood viscosity, plasma viscosity, and d-dimer levels.^38,39

This study used EA because EA parameters can be controlled precisely so the that results can be repeated. EA has been used in many related studies on CVD. Ishikawa et al., in 2012, compared the effect of stimulation with 1-Hz and 100-Hz frequencies on blood fluidity.³³ The researchers found that blood fluidity increased at both frequencies and blood fluidity was not affected by frequency of stimulation.³³ The frequency of the selected EA in the current study was 2 Hz because this frequency results in a significant increase in enkephalin and endorphin immunoreactivity, but not in dynorphine immunoreactivity, while 100-Hz EA increases dynorphine immunoreactivity but not enkephalin immunoreactivity. In the brain, enkephalin and endorphins, associated with opioid receptors μ and δ, have been shown to be more important in modulating cardiovascular action, compared to dynorphine (κ opioids).²⁹ In a deep vein thrombosis study, by Hou et al., low-frequency EA stimulation prophylactically increased lower limb blood flow and decreased blood viscosity.³⁹

EA stimulation in the current study was performed for 15 minutes, based on research conducted by Ishikawa et al., in 2012,³³ on the effect of EA stimulation time on ST 36 on blood fluidity. A comparison between 15 minutes and 60 minutes of stimulation time showed that both stimulation times had the same effect. This showed that, even with a short stimulation time, there was an immediate effect of acupuncture stimulation on blood fluidity.³³

The restraint-stress tool is used to cause acute mental stress in rats. Psychologic stress that occurs is marked by measuring increases in the temperature of the tube after a new rat is inserted into the tube; and this continues every hour until the restraint-stress test is completed. Given that rodents were used, psychologic stressors included placement of the animals in new environments, and limitations of animal activities, noise, and handling in various ways to trigger hyperthermia. In addition to changes in temperature, stress responses in animals are also indicated by changes in the pulses of animals undergoing stress restraint, as shown in the measurements of the pulses before and after the study took place, wherein an acute stress reaction activated the autonomic nervous system, which regulates the release of catecholamines, such as epinephrine and norepinephrine.⁴⁰

The restraint stress used in the current study lasted 3 hours because, according to research conducted by Melia et al. in 1994, acute exposure to restraint stress for 2–3 hours activated the pituitary–adrenal axis significantly, which resulted in an increase in plasma adrenocorticotropic hormone and corticosterone levels—the markers of acute stress.⁴¹

Acute stress triggers activation of the sympathoadrenal medullary system, which, in turn, activates the coagulation and fibrinolysis system and results in hypercoagulability. Stress-hypercoagulability protects healthy organisms from bleeding excessively in fight-or-flight situations. Acute mental stress, negative emotions, and psychologic traumata are also trigger factors for atherothrombotic events and venous thromboembolisms.²² It is now suggested that the inflammatory response to acute mental stress results in unstable atherosclerotic plaques that tend to rupture. It is well-known that acute psychologic stress increases arterial BP and changes in blood rheology, such as decreased plasma volume and increased blood viscosity. The consequences of increasing shear stress can result in plaque disruption. Hemostatic reactions to mental stress indicate activation of the coagulation cascade. Therefore, if plaque ruptures when mental stress occurs and exposes the contents of thrombogenic factors to procoagulant, clot formation will occur quickly.⁴²

Fibrinogen plasma is an acute-phase protein that is the main determinant of plasma viscosity and overall blood viscosity, wherein increased blood viscosity will increase total peripheral resistance and systemic arterial pressure. In this current study, fibrinogen responses to acute stress were seen in the mean differences between the control group and the group undergoing restraint stress alone, wherein plasma fibrinogen levels increased significantly (P = 0.001) after rats underwent acute stress procedures in the tube. This is in line with the research conducted by Steptoe et al., in 2016, wherein state plasma fibrinogen was sensitive to acute mental stress characterized by increased recorded fibrinogen from samples taken immediately after testing behavioral challenges.²²

The mean comparison between the negative control group and the restraint stress–and-EA group showed a statistically significant difference (P = 0.048), so it can be concluded that EA plays a role in decreasing the raised plasma fibrinogen levels caused by exposure to acute mental stress. This was consistent with studies of women with PCOS conducted by Stener-Victorin et al., in 2012, wherein the fibrinogen levels of the study subjects decreased after 2-Hz frequency EA was performed.³¹

Acupuncture at ST 36 can stimulate the release of eNOS protein and mRNA; these vasoactive substances cause vasodilatation, and, therefore, lower MAP; they also reduce blood viscosity by improving RBC deformability. This suppresses RBC aggregation, and platelet aggregation and adhesion.¹⁹ EA at ST 36 can reduce fibrinogen levels because this EA has anti-inflammatory effects; this was indicated by decreasing IL-6 levels after acupuncture at ST 36. This reduction of proinflammatory cytokines will reduce liver activity in removing acute-phase proteins, especially fibrinogen, which have been known to play very important roles in blood viscosity. In addition, by decreasing proinflammatory cytokines, EA at ST 36 can also reduce the potential for erythrocyte deformability and endothelial dysfunction. Therefore, efforts to improve blood viscosity and endothelial function can be more optimal.²³

There were no side-effects in this study, so it can be concluded that it was safe. It is hoped that this research can be used as a basis for further research in both animal and human experiments. The limitation of this study was the study time was too short, so longer observations could not be performed.

Conclusions

EA in restraint-stress, male Wistar rats affected plasma fibrinogen levels, inducing lower levels, compared with similar rats who did not receive EA; the difference was statistically significant (P = 0.001).

Further research is needed in experimental animals to see the effect of EA on other hemorheologic factors that play roles in blood viscosity, such as hematocrit, erythrocyte deformability, plasma viscosity, platelet aggregation, and inflammatory factors (IL-6), with a longer period of time and a comparison between EA with manual acupuncture or laserpuncture.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

The funding of this research was wholly provided by the first author.