Scholarly article on topic 'Comparison of Electroacupuncture Frequency-related Effects on Heart Rate Variability in Healthy Volunteers: A Randomized Clinical Trial'

Comparison of Electroacupuncture Frequency-related Effects on Heart Rate Variability in Healthy Volunteers: A Randomized Clinical Trial Academic research paper on "Medical engineering"

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Abstract of research paper on Medical engineering, author of scientific article — Jong-Ho Lee, Kyu-Hyeong Kim, Jin-Woo Hong, Won-Chul Lee, Sungtae Koo

Abstract This study aimed to compare the effects of high frequency electroacupuncture (EA) and low-frequency EA on the autonomic nervous system by using a heart rate variability measuring device in normal individuals. Fourteen participants were recruited and each participated in the high-frequency and low-frequency sessions (crossover design). The order of sessions was randomized and the interval between the two sessions was over 2 weeks. Participants received needle insertion with 120-Hz stimulation during the high-frequency session (high-frequency EA group), and with 2-Hz stimulation during the low-frequency session (low-frequency EA group). Acupuncture needles were directly inserted perpendicularly to LI 4 and LI 11 acupoints followed by delivery of electric pulses to these points for 15 minutes. Heart rate variability was measured 5 minutes before and after EA stimulation by a heart rate variability measuring system. We found a significant increase in the standard deviation of the normal-to-normal interval in the high-frequency EA group, with no change in the low-frequency EA group. Both the high-frequency and low-frequency EA groups showed no significant differences in other parameters including high-frequency power, low-frequency power, and the ratio of low-frequency power to high-frequency power. Based on these findings, we concluded that high-frequency EA stimulation is more effective than low-frequency EA stimulation in increasing autonomic nervous activity and there is no difference between the two EA frequencies in enhancing sympathovagal balance.

Academic research paper on topic "Comparison of Electroacupuncture Frequency-related Effects on Heart Rate Variability in Healthy Volunteers: A Randomized Clinical Trial"

J Acupunct Meridian Stud 2011;4(2):107-115

I RESEARCH ARTICLE I

Comparison of Electroacupuncture Frequency-related Effects on Heart Rate Variability in Healthy Volunteers: A Randomized Clinical Trial

Jong-Ho Lee1t, Kyu-Hyeong Kim1t, Jin-Woo Hong2, Won-Chul Lee2, Sungtae Koo1*

1Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Yangsan, Korea

2Division of Clinical Medicine, School of Korean Medicine, Pusan National University, Yangsan, Korea

Abstract

This study aimed to compare the effects of high frequency electroacupuncture (EA) and low-frequency EA on the autonomic nervous system by using a heart rate variability measuring device in normal individuals. Fourteen participants were recruited and each participated in the high-frequency and low-frequency sessions (crossover design). The order of sessions was randomized and the interval between the two sessions was over 2 weeks. Participants received needle insertion with 120-Hz stimulation during the high-frequency session (high-frequency EA group), and with 2-Hz stimulation during the low-frequency session (low-frequency EA group). Acupuncture needles were directly inserted perpendicularly to LI 4 and LI 11 acupoints followed by delivery of electric pulses to these points for 15 minutes. Heart rate variability was measured 5 minutes before and after EA stimulation by a heart rate variability measuring system. We found a significant increase in the standard deviation of the normal-to-normal interval in the high-frequency EA group, with no change in the low-frequency EA group. Both the high-frequency and low-frequency EA groups showed no significant differences in other parameters including high-frequency power, low-frequency power, and the ratio of low-frequency power to high-frequency power. Based on these findings, we concluded that high-frequency EA stimulation is more effective than low-frequency EA stimulation in increasing autonomic nervous activity and there is no difference between the two EA frequencies in enhancing sympathovagal balance.

ELSEVIER

Received: Nov 5, 2010 Accepted: Feb 22, 2011

KEY WORDS:

autonomic nervous system;

electroacupuncture; frequency;

heart rate variability; sympathovagal balance

1. Introduction

Electroacupuncture (EA) is a type of stimulation method applied to traditional manual acupuncture (MA) using modern electronics. EA originated in the

18th century in Japan and in the 19th century in France, and it has been widely practiced in both the East and the West. EA is known to have more rapid and longer lasting results than MA, and it has specific effects on pain, relaxation, circulation and muscle

Corresponding author. Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Beomeo-ri Mulgeum-eup, Yangsan, Gyeongnam 626-870, Republic of Korea. E-mail: stkoo@pusan.ac.kr "•These authors contributed equally to this work.

©2011 Korean Pharmacopuncture Institute

that are different from those of MA [1]. In EA, needles are inserted into acupoints, which are anatomical locations that were described by ancient physicians to be effective in curing diseases when stimulated. Needles are then connected in pairs to an EA device that produces electric pulses. Needles are usually positioned either along a meridian or in a particular area. Intensity and frequency of electric pulses and selection of acupoints are the main parameters known to influence the effects of EA.

There are many clinical trials and laboratory studies that support the therapeutic value of EA and that have attempted to discover the mechanisms of EA. In these trials and studies, the EA parameters were optimally manipulated to obtain positive outcomes. Some neurochemical studies showed that specific opioid receptors are involved in the EA frequency-induced effects [2-5]. One study reported that different frequencies of EA exert different effects in humans [6]. It suggested that the different effects are also mediated via different mechanisms. There are also some studies that explained the effects of EA frequency on suppression of inflammation, and on modulation of ovarian blood flow in relation to the autonomic nervous system (ANS) in animal experiments [7-9]. In addition, some studies found that EA stimulation of different frequencies commonly activate the sympathetic nervous system by inducing different mechanisms in rats [10-12]. Among human studies, one study demonstrated that the different frequencies of EA show a variation of analgesic effects after surgery [13] and another study found that EA produces hypoalgesia by eliciting an increase in muscle sympathetic activity [14]. However, studies of autonomic responses to different frequencies of EA stimulation are limited, and no study has tested this in humans under normal healthy conditions.

Some methods have been used to study autonomic responses following acupuncture. Sympathetic nervous system activity has been evaluated indirectly by implementation of measures for skin sympathetic nervous activity [15,16], microneurography [14,17], skin temperature [18], and thermography [19]. Taken together, these methods provide an overall representation of the changes in the ANS in the body, as each method focuses on different specific parts of the body. In one study, pharmacological drugs such as atropine and propranolol were given to humans to study the sympathetic and parasympathetic contributions, but the disadvantage of this method was the requirement of regular injections to the study participants [20].

In contrast to the injection method mentioned above, heart rate variability (HRV) has been developed as a quantitative noninvasive marker calculated from the electrocardiogram, and it is usually

used to assess the role of ANS fluctuations in normal healthy individuals and in patients with various cardiovascular and noncardiovascular disorders. All the HRV parameters are calculated on the "normalto-normal" (NN) interbeat intervals (or NN intervals) caused by normal heart contractions induced by sinus node depolarization. The variations in heart rate are analyzed by two methods: the time domain method and the frequency domain method. The time domain method analyzes the statistical values of the mean and the variance of all NN intervals that occur during measurement, where a NN interval is the interval time between the successive R peaks of the QRS complex of an electrocardiogram wave. The variance is mathematically equivalent to the total power of spectral analysis, and reflects all the cyclic components of the variability in a recorded series of NN intervals. Standard deviation of the NN intervals (SDNN) is equivalent to the square root of the variance of NN intervals, and it is measured in milliseconds. It is one of the main indices that reflect the state of ANS activity, and represents the fine beat-to-beat timing of heart contractions. In power spectrum analysis of HRV (the frequency domain analysis), total power is the variance of all NN intervals, and is an estimate of the total power of power spectral density in the range of frequencies between 0 and 0.4 Hz. Total power also indicates the sum of powers in all spectrum bands that can be divided into power in the ultra-low-frequency range (ULF), power in the very low-frequency range (VLF), power in the low-frequency range (LF), and power in the high-frequency range (HF). Therefore, total power can be used as an indication of overall autonomic activity of the body. These frequency domain indices are calculated in milliseconds squared (ms2). The low-frequency component of HRV (LF) corresponds to a band of power spectrum in the low-frequency range (0.04-0.15 Hz), and it is known as a strong marker of modulation of the sympathetic nervous system that reflects activities of both sympathetic and parasympathetic (vagal) systems. For example, the parasympathetic predominance is represented by the LF value when a person's respiration rate is lower than seven breaths per minute or during a person taking a deep breath. Therefore, when a person is in a state of relaxation with slow and even breathing, the LF value can be very high, indicating increased parasympathetic activity rather than increased sympathetic regulation. The high-frequency component of HRV (HF) corresponds to a band of power spectrum in the high-frequency range (0.15-0.4 Hz) and it mainly reflects parasym-pathetic activity. HF is also known as a "respiratory" band because it corresponds to the NN variations caused by respiration. Heart rate is increased during inhalation and decreased during exhalation.

Rest HRV EA HRV Rest HRV EA HRV

10min 5 min 15min 5 min 10min 5 min 15min 5 min

4-1st session -► 4-2nd session-►

Figure 1 Experimental procedure. In this crossover design, all the participants made two visits to the clinic where the experiment was held. The interval between the sessions was over 2 weeks. In each session, the participants first had a 10-minute rest and their heart rate variability (HRV) parameters were recorded for 5 minutes. Subsequently, electroacupuncture (EA) was administered for 15 minutes, followed by another heart rate variability recording session. The order of sessions was randomized.

The LF/HF ratio is the ratio between the powers of low-frequency and high-frequency bands. It is considered to indicate the sympathovagal balance. Higher values reflect domination of the sympathetic system, while lower values reflect that of the parasympathetic system. The guidelines for the standards and the interpretations of measurements, and the clinical uses of HRV have been previously proposed [21].

The present study aimed to investigate whether ANS activity could change in accordance with EA frequency using a HRV measuring system in normal healthy individuals.

2. Methods 2.1. Participants

The experimental procedures were approved by the Institutional Review Board of the School of Korean Medicine in Pusan National University, and were performed in accordance with the ethical standard laid down in the 1964 Helsinki Declaration. Written informed consent was obtained from each subject before they participated in the study. Considering that HRV indices are dependent on age and sex [22,23], we limited age to a narrow range and adjusted the sex ratio. Participants aged 28.86 ± 3.28 years were recruited from students within the medical campus of Pusan National University and they were composed of seven males and seven females. The participants were healthy as confirmed by their medical histories and did not suffer from cardiovascular or autonomic disease and were not taking any medications. They also never experienced any adverse effects related to acupuncture nor did they suffer from skin lesions from previous acupuncture. They abstained from caffeine on the day of the study, as well as from eating 2 hours before the commencement of the study.

2.2. Experimental design

Since this trial was crossover-designed, each subject participated in two sessions. During the high-frequency session, the participants received needle insertion with 120 Hz stimulation (high-frequency EA group) and at the low-frequency session, they received insertion with 2 Hz stimulation (low-frequency EA group). The order of sessions was given by a computer-generated randomization sequence that was unknown to the participants. The latter session was separated from the former session by a period of more than 2 weeks. To avoid the influence of circadian fluctuation on HRV, every session was scheduled to be at the same time (between 4:00 pm and 6:00 pm) and the room temperature was maintained at 24-25°C. Participants were instructed to keep their respiration uncontrolled to minimize any possible influences on the HRV indices. Figure 1 shows the experimental procedure. For each session, the participants first had a 10-minute rest in the supine position. This was followed by the measurement of HRV for 5 minutes. Acupuncture needles were then inserted into two acupoints. EA was administered for 15 minutes and then needles were removed. Finally, the participants rested for 5 minutes in the same supine position during which HRV was measured again. The participants remained in supine positions throughout the periods of resting, measurement of HRV and EA stimulation.

2.3. Acupuncture

All the acupuncture insertions were performed by the same acupuncturist, who had 2 years of training. The skin was cleaned with alcohol before each insertion. Acupuncture needles (stainless steel, 0.25 x 30 mm length; Dongbang Acupuncture, Inc., Korea) were inserted perpendicularly into LI 4 and LI 11 of

the right arm. Their locations were decided based on the WHO standards that LI 4 is located on the lateral side of the second metacarpal bone in the middle of the dorsal thenar muscle, and that LI 11 is located on the proximal part of the brachiora-dial muscle, near the end of the lateral transverse elbow crease when the forearm is flexed 90° [24]. We selected LI 4 and LI 11 for the two electric poles of EA. These acupoints are two of the most frequently used acupoints in clinical practice, and the distance between them is close enough for EA to work well. In addition, there are several studies suggesting that LI 4 and LI 11 are related to activity of the ANS. LI 4 is anatomically related to autonomic nervous responses [31] and it is related to muscle and skin sympathetic nervous activity as well as regulation of blood pressure [10-12,14,15]. LI 11 is known to have thermoregulatory and analgesic effects related to ANS [25].

2.4. Electroacupuncture

Electric pulses of either 2 Hz (low) or 120 Hz (high) EA were delivered to the two needles for 15 minutes with an electrical stimulator (Cellmac, STN-100; Stratek Co., Ltd., Korea) that has been clinically used in Korea. Intensity (voltage) of the electric pulses given in the continuous mode was fixed to a maximally tolerated (i.e., without discomfort to the participants) value, which was determined from a pre-experimental test. The electric pulses measured by oscilloscope consisted of alternating symmetrical biphasic waves composed of two impulses in a period. The peak value of the intensity was approximately 15 V and the impulse duration was 50 |is. In the case of 2 Hz EA, one period was 500 ms in duration, and there were +15 V and -15 V relatively short impulses of 50 |is duration. The duration of these impulses was too short for the measurement of current to take place. The electrical stimulator was placed in a location where the participants could not see the frequency being administered, and the acupuncture physician was instructed only to insert needles and was blinded to the administration of the EA frequencies.

2.5. Measurement of heart rate variability

Participants were connected to a HRV measuring system (SA-3000P; Medicore Co., Ltd., Korea) for 5 minutes before and after EA. This system collects signals of an electrocardiogram via three electrodes positioned on both wrists and the left ankle. To calculate the HRV indices, the system analyzes the changes in heart rate using the methods of time domain and frequency domain.

2.6. Statistics

The differences between the HRV indices measured before EA stimulation and those measured after EA stimulation were evaluated by using the paired t test (SPSS version 17.0; SPSS Inc., Chicago, IL, USA). Similarly, the differences between the high-frequency EA group and the low-frequency EA group were also analyzed by the paired t test. A value of p < 0.05 was considered to be the level of statistical significance.

3. Results

3.1. General characteristics

Participants consisted of seven male and seven female individuals (Table 1). The physiological parameters of the volunteers were within the normal range (Table 1). There were no differences in general characteristics between the high-frequency EA group and the low-frequency EA group.

3.2. Comparison of HRV parameters before and after EA

When we compared the HRV indices measured before EA stimulation with those measured after EA stimulation irrespective of the EA frequency, we found that EA stimulation significantly decreased the HR index (baseline vs. after EA: 61.8 ± 8.3 vs. 60.1 ± 8.4, p < 0.05), but it had no significant effects on the other indices including SDNN, total power, LF/HF ratio, LF and HF (Table 2).

We then analyzed the differences between the HRV indices measured before EA stimulation and those measured after EA stimulation for the two groups. We found that high-frequency EA stimulation significantly decreased HR (baseline vs. after EA: 59.9 ± 7.9 vs. 58.0 ± 7.7, p<0.05) and increased SDNN (baseline vs. after EA: 57.2 ± 16.1 vs. 62.2 ± 17.0, p < 0.05), but there were no significant variations of

Table 1 General characteristics

Participants

Age (yr) 28.9±3.3

Male:female 7:7

Body temperature (°C) 36.9±0.2

BP (mmHg)

Systolic 107.9 ± 6.4

Diastolic 69.2±5.6

Values are mean ± standard deviation. BP=blood pressure.

Table 2 Comparison between heart rate, standard deviation of the normal-to-normal interval (SDNN), total power,

low-frequency power/high-frequency power ratio (LF/HF), LF, and HF before and after electroacupuncture

stimulation*

HRV parameter Before EA After EA p

Heart rate (cycle/min) 61.8+8.3 60.1+ 8.4 0.005T

SDNN (ms) 55.0 + 15.5 58.2+15.9 0.081

Total power (ms2) 2360.4 + 1286.4 2799.2+1815.8 0.171

LF/HF 1.14 + 0.89 1.34 + 0.81 0.380

LF (ms2) 587.6 +403.3 731.9+499.6 0.251

HF (ms2) 683.3 +485.0 657.3+472.3 0.637

'Groups were not divided as LF and HF EA groups; values are mean ± standard deviation. The p value was evaluated using the paired

t test to determine differences between HRV parameters before and after EA. 1p<0.05. EA=electroacupuncture; HRV=heart

rate variability.

Table 3 Comparison between heart rate, standard deviation of the normal-to-normal interval (SDNN), total power, low-frequency power/high-frequency power ratio (LF/HF), LF, and HF before and after electroacupuncture stimulation*

HRV parameter

High-frequency EA group

Before EA

After EA

Low-frequency EA group

Before EA

After EA

Heart rate (cycle/min)

SDNN (ms)

Total power (ms)

LF (ms2)

HF (ms2)

59.9+7.9 57.2 + 16.1 2437.0 + 1214.5 1.1 + 1.1 590.6 + 404.9 784.8 +463.9

58.0+7.7 62.2 + 17.0 2993.2 + 1781.7 1.2+0.8 751.7 +436.1 808.2 + 245.3

0.017T

0.036T

63.7+8.5 52.8 + 15.2 2283.8+ 1396.2 1.2+0.7 584.5 + 416.9 581.9 + 44.6

62.1±8.8 54.3+14.3 2605.3 + 1895.4 1.4 + 0.8 712.1 ± 572.2 506.4+54.8

0.104 0.605 0.562 0.397 0.535 0.254

'Groups were not divided as LF and HF EA groups; values are mean ± standard deviation; the p value was evaluated using the paired t test to determine differences between HRV parameters before and after EA; Tp < 0.05. EA=electroacupuncture; HRV=heart rate variability.

other HRV indices in both the high-frequency and low-frequency EA groups (Table 3).

Since we had found a significant decrease in HR independently of the EA frequency, but no significant change in SDNN (Table 2), subsequent analysis of the data for the two groups showed a statistically significant increase in SDNN in the high-frequency EA group, but not in the low-frequency EA group.

3.3. Comparison of HRV parameters between the high-frequency EA (120 Hz) group and the low-frequency EA (2 Hz) group

At baseline, there was a significant difference in HR between the high-frequency and low-frequency EA groups (59.9 ± 7.9 vs. 63.7 ± 8.5, p<0.05). After EA stimulation, significant differences between the two groups were observed in HR, SDNN, and HF (HR: 58.0 ± 7.7 vs. 62.1 ± 8.8; SDNN: 62.2 ± 17.0 vs. 54.3 ± 14.3; HF: 808.2 ± 245.3 vs. 506.4 ± 54.8, p<0.05) (Table 4). Therefore, taking into consideration the

baseline values, the indices that were still significantly different between the two groups after EA stimulation were SDNN and HF (Figures 2 and 3).

4. Discussion

The present study showed that at different EA frequencies, EA stimulations exert different effects on HRV. High-frequency EA caused a significant increase in SDNN, while low-frequency EA showed no significant changes in SDNN. SDNN can be interpreted as the ability to adapt and accommodate to circumstance variations [21]. SDNN is an indicator of ANS. Our study results suggest that high-frequency EA is more effective than low-frequency EA in facilitating ANS. SDNN is an important index of activity in heart function and a decrease in SDNN is related to an increase in mortality after myocardial infarction

[26]. SDNN is also an exclusive predictor of overall mortality, and has a close relation to sudden death

[27]. In addition, it has been reported that SDNN is

Table 4 Comparison of heart rate variability (HRV) parameters between high-frequency electroacupuncture (EA) and low-frequency EA

Before EA

After EA

HRV parameter

High EA

Low EA

High EA

Low EA

Heart rate (cycle/min)

SDNN (ms)

Total power (ms)

LF (ms2)

HF (ms2)

59.9±7.9 57.2 ± 16.1 2437.0 ± 1214.5 1.1± 1.1 590.6 ± 404.9 784.8 ± 463.9

63.7±8.5 52.8 ± 15.2 2283.8 ± 1396.2 1.2±0.7 584.5 ± 416.9 581.9 ± 44.6

0.016*

58.0±7.7 62.2 ± 17.0 2993.2 ± 1781.7 1.2±0.8 751.7 ±436.1 808.2 ± 245.3

62.1±8.8 54.3±14.3 2605.3 ± 1895.4 1.4 ± 0.8 712.1 ± 572.2 506.4± 54.8

0.006*

0.031*

0.025*

Values are mean ± standard deviation. The p value was evaluated using the paired t test to determine differences between high-frequency and low-frequency EA. *p < 0.05. F=frequency; SDNN=the standard deviation of all normal-to-normal intervals; LF= power in the low frequency; HF=power in the high frequency; LF/HF=LF/HF ratio.

I I High frequency EA I I Low frequency EA

Before EA

After EA

Figure 2 Comparison of the standard deviation of the normal-to-normal interval variations (SDNN) between the high-frequency and low-frequency electroacupuncture (EA) groups. High-frequency (120 Hz) EA stimulation to the LI 4 and LI 11 acupoints significantly increased the SDNN, while low-frequency (2 Hz) EA stimulation to the same acupoints had no significant effects on the SDNN. In addition, while there was no significant difference in the SDNN at baseline between the high-frequency and low-frequency EA groups, this was not the case after EA stimulation, with a significant increase in the SDNN in the high-frequency EA group compared with that in the low-frequency EA group. Values are expressed as mean ± standard error of the mean. The p value was evaluated using the paired t test between the high-frequency and low-frequency EA groups, and between the HRV parameters before and after EA. *p < 0.05.

significantly lower with hypertension [22]. Considering these findings, we postulated that high-frequency EA is more efficient than low-frequency EA in activating heart function associated with ANS.

The present study demonstrated that a variation in EA frequencies resulted in changes in HF, because there was a significant difference in HF between the high-frequency group and the low-frequency

1200 -,

1000 -

I I High frequency EA I I Low frequency EA

Before EA

After EA

Figure 3 Comparison of the power in the high-frequency range (0.15-0.4 Hz) between high-frequency electroacupuncture (EA; 2 Hz) and low-frequency EA (120 Hz). While there was no significant difference in the power in the high-frequency range at baseline between the high-frequency and low-frequency EA groups, this was not the case after EA stimulation, with a significant increase in power in the high-frequency range in the high-frequency EA group compared with that in the low-frequency EA group. Values are expressed as mean ± standard error of the mean. The p value was evaluated using the paired t test between high-frequency and low-frequency EA and also between heart rate variability parameters before and after EA. *p < 0.05.

group after EA stimulation, but not at baseline. Since HF is a major contributor of efferent vagal activity, as well as an index that reflects parasympathetic activation [21], we speculated that EA stimulation has different effects on parasympathetic nerve activity at different EA frequencies. However, it is still unknown what the difference of the effects is on parasympathetic nerves between the high-frequency and low-frequency EA groups.

Ogata et al [6] reported that the sweating rate can be reduced by low-frequency EA, the mechanisms

of which occur in both the brain and the areas situated below the supraspinal structure (probably the spinal cord). The sweating rate can also be reduced by high-frequency EA through the mechanisms of which occur in the areas situated below supraspinal structure only. These results suggest that EA stimulations of different frequencies act on the ANS via different mechanisms. It has been shown that the suppressive effect on inflammation induced by both low- and high-frequency EA is mediated through different systems [7]. Low-frequency EA mediates the suppressive effect on inflammation through sympathetic post-ganglionic neurons, whereas high-frequency EA acts on the sympathoadrenal medullary axis for suppression of inflammation. Stener-Victorin et al [8] found that low-frequency EA stimulation increases ovarian blood flow via the sympathetic nervous system, while high-frequency EA stimulation decreases ovarian blood flow via systemic changes in the circulation. This result suggests that low frequency EA has an effect on the ANS by modulating sympathetic nerves [9]. Based on these findings, we speculate that EA of different frequencies may have distinctive effects on the ANS via different neural mechanisms, and that the differences of SDNN and HF among the HRV parameters between the high-frequency and low-frequency EA groups in our study might have been caused by such differences in neural mechanisms.

It has been reported that high-frequency EA stimulation induces a pressor effect by eliciting a chronotropic effect via p-adrenergic receptors triggering an excitation of the sympathetic cardiac nerve, whereas low-frequency EA stimulation induces a pressor effect via a-adrenergic receptors through an excitation of the sympathetic vasoconstrictive nerve [28]. From this report, we speculate that the increase in SDNN in only the high-frequency EA group might have been due to a cardiac chro-notropic effect induced by high-frequency EA stimulation. On the other hand, it was reported that high-frequency EA stimulation induces the release of dynorphin, which interacts with the K-opioid receptor in the spinal cord, while low-frequency EA stimulation induces the release of enkephalin, pendorphin and endomorphin, which all interact with the and 8-opioid receptors in the central nervous system [2-5]. Since the presynaptic K-opioid receptor, but not the ^-opioid or 8-opioid receptor, inhibits the release of noradrenaline from the sympathetic neurons innervating the sinus node [29,30], we consider that high-frequency EA stimulation acts on the K-opioid receptor to modulate the sympathetic nerves in the heart. This hypothesis could account for the increase in SDNN in the high-frequency EA group of our study. However, more studies are needed to elucidate the exact mechanisms.

In the present study, we did not observe a difference in effects on the LF/HF ratio between the high-frequency and low-frequency EA groups. It is generally accepted that the LF/HF ratio reflects sympathovagal balance [21,31]. Therefore, an increase in the LF/HF ratio indicates a relative increase in sympathetic nervous activity, whereas a decrease in the LF/HF ratio indicates a relative increase in parasympathetic nervous activity. Based on these findings, we concluded that the different EA frequencies did not produce different effects on sympathovagal balance in healthy participants. It has been found that both high- and low-frequency EA stimulations into LI 4 similarly activate the sympathetic nervous system, despite the fact that each frequency acts via a different mechanism: high-frequency EA stimulation induces a phasic response, while low-frequency EA stimulation elicits a tonic response [10-12]. In this respect, we presumed that even though there was no difference in effects on sympathovagal balance between the high- and low-frequency EA groups, the two groups had their own distinctive mechanisms.

Our present study showed that there was an increase in the LF/HF ratio in both the high-frequency and low-frequency EA groups, although this was not statistically significant. We postulate that the increase in the LF/HF ratio might have been caused by a reciprocal coordination of the following two factors. One factor refers to the potential increase in sympathetic nervous activity after EA stimulation applied at the LI 4 and LI 11 acupoints. Knardahl et al [14] reported that EA stimulation into the LI 4 and LI 11 acupuncture points produces moderate hy-poalgesia by eliciting a significant increase in muscle sympathetic nervous activity. Moreover, manual acupuncture stimulation into the LI 4 point induces a transient increase in skin sympathetic nervous activity [15] and EA stimulation to the corresponding point facilitates sympathetic nervous activity [10-12]. The explanation for these results may lie in the fact that the LI 4 acupuncture point is innervated by sympathetic nerve fibers [32]. The second factor is the fact that after EA stimulation, the LF/HF ratio has a tendency to converge towards a value that reflects a healthy state of an individual. This factor can be explained by the results of two previous studies, which showed significantly different effects: one study showed that in response to manual acupuncture stimulation, basal sympathetic nervous activity increased in participants [15], while another study showed that acupuncture was able to normalize autonomic dysfunction in stressed participants [33]. In addition, this concept of the LF/HF ratio tending to converge towards a value of a healthy individual after EA stimulation is suggested by a report that acupuncture can only normalize a

leukocyte pattern that has deviated from the norm and it does not change a normal leukocyte pattern

[34]. This report is relevant to the present study in that the immune system is modulated by the ANS

[35]. In the present study, the baseline value of the LF/HF ratio was slightly below the healthy value range that was set by studies on HRV standards [21,22]. We speculate that the insignificant increase in the LF/HF ratio might have been due to the above mentioned second factor. Therefore, since the basal value of the LF/HF ratio was initially near the normal range, despite the capability of EA stimulation to normalize a deviated LF/HF ratio, this potential was not fully manifested. We speculate that if the basal value of the LF/HF ratio had deviated far from the normal range, EA may have induced a significant variation in the value.

It has been reported that in the normal state, acupuncture produces conflicting effects on the ANS depending on various factors, whereas in stressful states, acupuncture restores autonomic dysfunction [33]. In this respect, we assumed that the participants under normal conditions in the present study may have been limited in showing more potent effects of EA. In addition, we became aware of some other limitations of our study design. Since there was no control group, we could not confirm the effects of EA. The crossover design of our study might have triggered a carryover effect. Furthermore, it is conceivable that we might have failed to use the optimal time of measurement because we only measured the HRV indices 5 minutes before and after EA stimulation.

Although the intensity of EA was fixed in our experiment, it is possible that EA stimulations at low intensities might have occurred, and thus only partial effects on the ANS were shown in a number of participants [8,11,14].

In summary, our study analyzed the HRV parameters in relation to ANS activity and sympathovagal balance. Based on our result that only high-frequency EA significantly increased SDNN and there were no significant differences in other indices, we conclude that clinically, high-frequency EA is better than low-frequency EA in increasing ANS activity, and that there is no difference between the two EA frequencies in enhancing sympathovagal balance.

Acknowledgments

This research was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0003928). We express our gratitude to Mr. John Shim for his excellent assistance in editing the manuscript.

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