Investigation of Specificity of Auricular Acupuncture Points In

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Investigation of specificity of auricular acupuncture points in

regulation of autonomic function in anesthetized rats

Xin-Yan Gao a,b, Shi-Ping Zhang a,, Bing Zhu b, Hong-Qi Zhang a

a School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Kowloon, Hong Kong, Chinab Institute of Acupuncture & Moxibustion, China Academy of Chinese Medical Sciences, Beijing, China, 100700

Received 2 August 2007; received in revised form 29 September 2007; accepted 22 October 2007

Abstract

Auricular acupuncture has been used for various autonomic disorders in clinical practice. It has been theorized that different auricular

areas have distinct influence on autonomic functions. The present study aims to examine the effects of acupuncture stimulation at different

auricular areas on cardiovascular and gastric responses. In male SpragueDawley rats anesthetized with pentobarbital sodium, five auricular

areas, which were located at the apex of the helix (A 1), the middle of the helix (A2), the tail of the helix (A3), the inferior concha (A4) and the

middle of the antihelix (A5), had been selected for stimulation with manual acupuncture (MA) and different parameters of electroacupuncture

(EA). A mild depressor response (6%12% decrease from baseline) was evoked from A1, A3 and A4 by MA and from all five areas by EA

(100 Hz1 mA). The biggest depressor response (18.4 3.1 mmHg, pb0.001) was evoked from A4. A small bradycardia was evoked by

MA from A4 and by EA at A3, A4 and A5. Increase in intragastric pressure (814 mmH2O) was evoked by MA from A1, A3 and A4 and by

EA at A2. These results show that similar patterns of cardiovascular and gastric responses could be evoked by stimulation of different areas of

the auricle. The present results do not support the theory of a highly specific functional map in the ear. Rather, there is a similar pattern of

autonomic changes in response to auricular acupuncture, with variable intensity depending on the area of stimulation.

2007 Elsevier B.V. All rights reserved.

Keywords: Acupuncture; Blood pressure; Heart rate; Gastric motility; Vagus nerve; Ear map

1. Introduction

Auricular acupuncture (AA) is the application of needle

stimulation to the external ear, which is a form of acupuncture

practice that has been described in ancient China as well as

Egypt, Greece and Rome (Oleson, 2003a). In 1950s, Frenchphysician Nogier reawakened the interest in AA by proposing

a theory that there is a somatotopic and viscerotopic rep-

resentation on the auricle, such that different body regions

and organs appear on the external ear to form a humunculus

of inverted fetus (Nogier, 1987). The selection of AA point

for treatment, therefore, has been based on this theory. Hence,

a disorder from a particular part of the body is treated by the

corresponding point in the ear (Oleson et al., 1980; Nogier,

1987). Since then, AA has been used for pain relief

(Usichenko et al., 2005a,b; Goertz et al., 2006), anxiety and

sleep disorders (Chen et al., 2007, Pilkington et al., 2007), as

well as various autonomic disorders including hypertension(Huang and Liang, 1992), gastrointestinal disorders (Taka-

hashi, 2006), lower urinary tract symptoms (Capodice et al.,

2007) and postoperative vomiting (Kim et al., 2003). How-

ever there was no scientific evidence supporting Nogier's

theory, and the specificity of AA points is still a matter of

conjecture. This can be problematic when selecting treatment

and control points in clinic trials (e.g., Margolin et al., 1996).

A better understanding of the specificity of auricular points

will help to improve clinical practice and facilitate AA

research.

Autonomic Neuroscience: Basic and Clinical 138 (2008) 5056

www.elsevier.com/locate/autneu

Corresponding author. Tel.: +852 34112466; fax: +852 34112461.

E-mail address: [email protected] (S.-P. Zhang).

1566-0702/$ - see front matter 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.autneu.2007.10.003
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It has been documented that the human auricle receives

innervations from cervical and cranial nerves including the

auricular branch of the vagus nerve, the great auricular nerve

and the auriculotemporal nerve (Peuker and Filler, 2002).

Study of the origin and peripheral distribution of the primary

afferent fibers in the auricle of cats and rats showed that

innervations of the central region of the auricle mainly came

from the trigeminal ganglion, the geniculate ganglion, the

superior ganglion of the vagus nerve, and to a lesser degree,

the spinal ganglia. In contrast, the peripheral region of the

auricle was mostly innervated by spinal nerves (Satomi and

Takahashi, 1991; Folan-Curran et al., 1994). The central

projections of the auricular branch of the vagus nerve, socalled the Arnold's nerve, had been examined by the trans-

ganglionic horseradish peroxidase (HRP) tracing method in

cats. HRP-labelled neuronal somata were seen in the superior

ganglion of the vagus nerve and terminal labelling was seen

mainly in the ipsolateral solitary nucleus (Nomura and

Mizuno, 1984). Thus, in the theorized ear map, the areas

representing somatic parts such as the helix and the scapha

are mainly innervated by somatic afferents, and those rep-

resenting visceral organs such as the superior and inferior

concha are primary innervated by cranial nerves especially

the vagus nerve (Fig. 1). In human, it has been suggested that

the sensory vagus nerve is responsible for the ear-coughreflex seen clinically (Tekdemir et al., 1998). As well,

stimulation of the inferior concha could induce a significant

increase in parasympathetic activity as demonstrated by

heart rate variability (Haker et al., 2000).

In the study of point specificity, Young and McCarthy

(1998) reported that stimulation of the sympathetic point at

the lower limb of the antihelix (AH6 in Fig. 1) significantly

decreased the stimulus-evoked electrodermal response when

compared with stimulation of a controlled point in the mid-

dle of the helix (HX9 in Fig. 1). It had been found that AA at

specific points such as Lung (CO14 in Fig. 1) and Shenmen

(TF4 in Fig. 1) produced better analgesic effect than at non-

acupuncture points on the helix (Usichenko et al., 2005a,b).

Taken together, evidence from anatomical studies and

clinical observations suggest that there might be location

specificity for auricular points. In this study, we set out to

examine the specificity of auricular acupuncture points in

regulation of autonomic function using gastric and cardio-

vascular changes as indicators.

2. Methods and materials

2.1. Animal preparation

The experimental protocol was approved by the animalethics committee of the Hong Kong Baptist University.

Male SpragueDawley rats (n =18), weighing 300380 g,

were fasted overnight with free access to water. They were

Fig. 1. Drawings showing auricular zones and nerve distribution of the human auricle. A, auricular zones based on recommendations of the 1990 WHO auricular

nomenclature committee (Oleson, 2003b). Large dots indicate areas stimulated in the present study or in previous studies as mentioned in the text. B, C and D

show nerve distribution (indicated by small dots) of the human auricle according to Peuker and Filler (2002).

Fig. 2. Photograph of the right ear of the rat showing areas being stimulated

in the present experiments. A1, A2, A3, A4, and A5 correspond to HX6, HX9,

HX12, CO15, and AH9 in the human auricular map as described in Fig. 1,

respectively.

51X.-Y. Gao et al. / Autonomic Neuroscience: Basic and Clinical 138 (2008) 5056


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anesthetized with an initial dose of pentobarbital sodium

(i.p., 50 mg/kg, Alfasan), with i.v. supplement (1520 mg/

kg/h) via the jugular vein using a syringe pump to maintain

the level of anesthesia at a depth in which the withdrawal

reflex to a noxious toe pinch was absent. After tracheal

cannulation, animals breathed spontaneously and the core

temperature was maintained at 37.00.5 C by a feedback-

controlled electric blanket. Animals were sacrificed huma-

nely with an overdose of pentobarbital sodium at the end of

the experiments.

For recording of mean arterial pressure (MAP), the left

common carotid artery was cannulated with a polyethylene

tubing filled with normal saline containing heparin (500 IU/

ml, Sigma), and connected to a blood pressure transducer.

Needle electrodes were placed at the forelimbs and the left

hind limb for ECG recording (Bio Amp, ADInstruments),

from which heart rate (HR) was derived. For recording of

intragastric pressure (IGP), a midline laparotomy was made

with a small latitudinal incision in the duodenum wall about

23 cm from the pylorus. A balloon (1 cm in diameter) made

of flexible condom rubber was inserted into the pyloric

antrum and kept in position by tying the connecting poly-

ethylene tubing (ID 0.58 mm, OD 0.96 mm, PORTEX) to the

duodenum (Kametani et al., 1979). The balloon was filled

Fig. 3. Computer chart records of a typical experiment. Changes in blood pressure (BP), heart rate (HR) and intragastric pressure (IGP) during manual acupuncture

(MA) at five areas of the rat auricle are shown in the boxes. Schematic drawing of the right ear of the rat shows the location of the auricular areas being stimulated.

52 X.-Y. Gao et al. / Autonomic Neuroscience: Basic and Clinical 138 (2008) 5056


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with 0.30.8 ml water pre-warmed to 37.00.5 C and kept

at a baseline pressure of 40250 mmH2O. IGP was measured

from the intragastric balloon with a transducer connecting to

an amplifier (ADInstruments). Background gastric motility

was recorded for at least 30 min before AA stimulation.

2.2. Auricular acupuncture stimulation

When the depth of anesthesia was stable, showing that the

fluctuation of MAP and HR were less than 5% within a minuteand the gastric peristaltic wavewas stable, responses to manual

(MA) or electroacupuncture (EA) were examined. Five

auricular areas, which were located at the apex of the helix

(A1), the middle of the helix (A2), the tail of the helix (A3), the

inferior concha (A4) and the middle of the antihelix (A5),

corresponding to HX6, HX9, HX12, CO15, and AH9 in

human auricle respectively, were selected for MA and EA

(Figs. 1 and 2). Acupuncture needle (0.2 mm13 mm Hwato,

China) was inserted to a depth of 12 mm from the skin

surface before each stimulation. For MA, the needle was

twisted right and left once every second for 30 s and then

removed. For EA, a pair of needles was inserted into the same

auricular area separated by approximately 1.5 mm with the aid

of a piece of cotton wool. The EA stimulation consisted of

biphasic rectangular pulses (0.45 ms) at four different in-

tensities and frequencies: low frequencylow intensity (LL:

4 Hz0.4 mA), low frequencyhigh intensity (LH: 4 Hz

1.0 mA),high frequencylow intensity (HL: 100 Hz0.4 mA),

high frequencyhigh intensity (HH: 100 Hz1.0 mA). Eachepisode of EA stimulation lasted for 30 s. All four parameters

of EA induced twitch of the ear, indicating effective

stimulation of auricular muscles. When tested on human

subjects, LL and HL of EA were just above the detection

threshold and were reported non-painful, whereas LH and HH

of EA were sometimes reported to be painful but tolerable

(personal observation). In a given experiment, MA and EA

were examined for different auricular areas and the stimula-

tions were arranged randomly and repeated 34 times.

2.3. Statistical analysis

Baseline values of MAP and HR were measured by taking

the average of 1 second record after needle insertion and just

before acupuncture stimulation, when the signals were stable.

Baseline IGP was determined by the last peak value of the

peristaltic wave just before the stimulation. Changes in MAP

and HR were measured by taking the average of a 1 second

record with the biggest change during acupuncture stimulation,

and change of IGP was measured by selecting the highest peak

of peristaltic wave during the acupuncture stimulation period.

Comparison between means was made by Student's-ttest and

one-way ANOVAwith LSD post-hoc test. Datawere presented

as mean S.E.M., and pb0.05 was considered significant.

3. Results

3.1. Cardiovascular and gastric responses evoked by

manual acupuncture (MA)

Data had been obtained from 12 successful experiments.

Before the start of any stimulation, the MAP was 116.4

8.9 mmHg, and the HR was 386.0 8.4 beats per minute (bpm).

As seen in Fig. 3, MA at auricular areas of A1, A3, and A4produced significant depressor responses (8.01.6 mmHg,

7.5 2.6 mmHg and 18.4 3.1 mmHg respectively,

Fig. 4. Histograms showing mean values ( S.E.M.) of mean arterial pressure

(MAP), heart rate (HR) andintragastric pressure (IGP) beforeand after manual

acupuncture (MA) in five different areas of the auricle (A1-5). *" s inside the

bars indicates statistical significant difference between pre-MA and post-MA

(: pb0.01; : pb0.01;: pb0.001; paired t-test). #s indicates statistical

significant differencecompared with A4 (one wayANOVAwith LSDpost-hoc

test; #: pb0.05; ##: pb0.01; ###: pb0.001).

Fig. 5. Histograms showing the effect of needle insertion on mean arterial

pressure (MAP). indicates statistical significant difference between

pre- and post-insertion in MAP (pb0.001, paired t-test).



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pb0.05, Fig. 4), which outlasted the duration of the stimulation.No significant change in MAP was observed during stimulation

of A2 and A5. Changes in HR were rather variable and statistical

difference could only be found during stimulation of A4 (4.5

1.6 bpm,pb0.05). MA also led to an increase in IGP at A1, A3,

A4 (14.7 4.3 mmH2O, 11.9 5.5 mmH2O and 12.8

3.8 mmH2O, pb0.05, respectively). Thus, MA at A1, A3 and

A4 evoked simultaneous changes in MAP and IGP, but

simultaneous changes in MAP, and IGP as well as HR were

evoked only at A4. A4 showed the biggest depressor response

compared to other areas (pb0.01).

Interestingly, it was observed that insertion of the needle

evoked significant depressor responses at all auricular areas

(pb0.001, Fig. 5). Compared to MA, the duration of thedepressor responses evoked by insertion of the needle was

less than that evoked by MA, lasting for a few seconds only,

but the magnitude of the response was similar to that of

manual acupuncture. However, no significant change in HR

or IGP was observed during needle insertion.

3.2. Autonomic changes evoked by electroacupuncture (EA)

As seen in Fig. 6, of the four types of EA applied to the

five AA areas, HH evoked significant decrease in MAP

at all areas (8.8 3.7 mmHg at A1, pb0.05; 8.6

2.6 mmHg at A2, pb0.01; 9.63.5 mmHg at A3, pb0.05;

Fig. 6. Histograms showing changes in mean arterial pressure (MAP), heart rate (HR) and intragastric pressure (IGP) for five auricular areas (A 15) under

different parameters of electroacupuncture. LL, low frequency (4 Hz), low intensity (0.4 mA) stimulation; LH, low frequency (4 Hz), high intensity (1 mA)

stimulation; HL, high frequency (100 Hz), low intensity (0.4 mA) stimulation; and HH, high frequency (100 Hz), high intensity (1 mA) stimulation. s inside

the bars indicate statistical significant change compared with baseline (pb0.05; : pb0.01; paired t-test); #s outside the bars indicate statistical significance

compared with HH (one way ANOVA with LSD post-hoc test; #: pb0.05; ##: pb0.01; ###: pb0.001).



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7.61.9 mmHg at A4, pb0.01 and 12.92.5 mmHg at

A5,pb0.01). HH at A3, A4 and A5 induced small bradycardia

(7.01.4 bpm, 5.7 1.7 bpm and 8.31.8 bpm respec-

tively, pb0.05), and at A2 produced significant increase in

IGP (7.6 2.4 mmH2O, pb0.05).

EA of HL evoked significant depressor effect at A3 (5.1

1.7 mmHg, pb0.05). Low frequency EA at either low (LL) orhigh intensity (LH) did not evoke any significant change in

MAP, HR or IGP (Fig. 6).

Comparing the responses evoked by MA and EA of HH

for a given area, the overall patterns of response appeared to

be similar, except the depressor effect produced by MA was

significantly bigger than that evoked by EA of HH at A4(pb0.01), and the bradycardia evoked by EA of HH was

bigger than that evoked by MA at A3 (pb0.05).

4. Discussion

This is the first study that examines systematically thelocation specificity and response characteristics of auto-

nomic changes using different methods of stimulation in

auricular acupuncture. The major finding from this study is

that stimulation of the auricle with either MA or strong EA

(100 Hz1 mA) can evoke a characteristic pattern of res-

ponse including decrease in blood pressure, bradycardia and

gastric contraction, whereas mild EA is ineffective. Decrease

in blood pressure is the most consistent response evoked by

MA and EA. On the other hand, changes in HR and gastric

contraction are more variable, depending on the area and

method of stimulation.

Cardiac dysrhythmias and bradycardia have been re-

ported during insertion of an ear speculum and initial ma-nipulation of the ear (Moorthy et al., 1985). Activation of the

parasympathetic system by auricular acupuncture in man, as

measured by heart rate variability, has also been reported

before, although the same study fails to elicit any change in

blood pressure and heart rate (Haker et al., 2000). The

observation in the current study that needle insertion, manual

needle manipulation and strong EA are effective in evoking

autonomic responses, whereas weaker forms of EA are not,

suggest that strong or even painful stimulation may be

required to produce these responses.

In a separate series of experiments aimed to explore the

mechanism of the auricular-autonomic responses (Gao et al.,unpublished data), we had observed that the responses evoked

from A4 were abolished by blockade of vagal transmission

with intravenous injection of the muscarinic receptor blocker

atropine sulphate (0.5 mg/kg). That is, before atropine block-

ade the changes in MA, HR and IGP were16.54%,2.3

0.5% and 49.513.2%, respectively (n = 5, pb0.05, paired

t-test, compared with pre-acupuncture baseline), and after

the blockade the changes in MA, HR and IGP were 2.7

1.7%, 0.70.6% and 4.52.9%, respectively (n = 5, pN0.05,

paired t-test, compared with post-atropine, pre-acupuncture

baseline). These results suggest that the depressor response,

bradycardia and gastric contraction induced by AA may be

due to an increase in vagal output, mediated by auricular-

vagal reflexes. In contrast, a different efferent pathway has

been demonstrated in depressor response evoked by body

acupuncture. For example, Ohsawa et al. (1995) showed in

the rat that acupuncture-like stimulation at the hind limb

reduced renal sympathetic nerve activity coupled with the

depressor response. It is interesting to note that MA at onlyA4 of the inferior concha area evoked consistent bradycardia,

whereas MA at other areas evoked increase or decrease in

heart rate. Sato et al. (1976) reported that cutaneous noxious

stimulation in anesthetized rats produced an increase in heart

rate in about 70% of their tests, while in the other 30%

induced either biphasic responses or no change. Activation of

hind limb muscle afferents in the dog was also associated with

either increase or decrease in heart rate (Tallarida et al., 1985).

Taken together, the inferior concha represents a distinct region

in which decrease in heart rate could be evoked consistently.

It has been observed that for a given area, the patterns of

response evoked by MA and EA can be different. This maybe due to the fact that EA stimulates local receptors as well as

nerve fibers that innervate other area passing through the

area of stimulation, and that the effective stimulation area of

EA may well exceed the area between the two inserted

needles as a result of spreading of the electrical current. In

contrast, manual manipulation stimulates receptors primary

at the site of needle insertion. The differences in the response

evoked by different methods of AA should be taken into

consideration in clinical practice and research.

As for the analysis of functionalanatomical relationships on

the auricle, the results obtained from MA stimulation may be

more precise, for reasons discussed above. The inferior concha

produced the biggest depressor effect during MA in the presentexperiments, andit was the only site where bradycardia could be

elicited. This coincides with previous findings that the conchae

receives major innervations from the auricular branch of the

vagus nerve (Fig. 1). However, apart from the inferior concha,

the areas in which depressor response and gastric contraction

have been elicited by MA in the current experiments include the

apex of the helix and tail of the helix, which are innervated

predominately by the auriculotemporal nerve and the great

auricular nerve, respectively. This indicates that the hypotensive

and gastric responses are not elicited solely from areas in-

nervated by the vagus nerve. This is in agreement with previous

findings that denervation of the auricular branch of the vagusnerve did not abolish gastric response evoked by auricular

acupuncture in the rabbit, but complete denervation of all the

nerves tothe ear did (Liu et al., 1990). Taken together, it appears

that the AA-evoked autonomic responses are mediated by the

auricular branch of the vagus nerve, as well as other sensory

nerves innervating the ear.

Direct vagal nerve stimulation has been used in clinical

practice for treatment of refractory epilepsy and major depres-

sion (Shafique and Dalsing, 2006). Recently, Kraus et al (2007)

showed that non-invasive electrical stimulation of the outer

auditory canal, aiming to activate vagal afferences, produced

enhancement of well-being score coupled with deactivation of



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limbic and temporal brain structures, whereas stimulation of the

ear lobe did not. It is interesting to note that AA point Heart

(CO15 in Fig. 1 or A4 in Fig. 2) in the inferior concha has also

been used for mood disorders in clinical practice (Pilkington

et al., 2007; Wang et al., 2001). Taken together, convergent

evidence supports that the concept that vagal afferents on the

auricle provide a unique situation whereby autonomic and othernervous disorders may be corrected with the use of appropriate

stimulation, such as acupuncture.

5. Conclusions

Previous schemas of auricular acupuncture suggest that

auricular points have high specificity. On the other hand,

anatomical studies of auricular innervations have described

an overlapping network of distribution of somatic and cranial

nerves, although some areas do receive preferential innerva-

tions from one or two nerves. Our experiments show that the

same pattern of reflex responses can be elicited from dif-ferent areas with supposedly distinct nerve innervations.

Taken together, evidence from anatomical and physiological

studies does not support the concept of a highly specific

functional map in the ear. Rather, there is a general pattern

of autonomic changes in response to auricular acupuncture,

with variable intensity depending on the area of stimulation.

Our study suggests that the inferior concha is the most

powerful site for regulation of autonomic functions. How-

ever, in clinical study, it should be cautious in using auricular

points outside the conchae as control points, as they also

have the potential to influence autonomic functions.

Acknowledgements

Dr. Xin-Yan Gao is supported by the Wofoo Postdoctoral

Fellowship Scheme of Chinese Medicine from the Hong

Kong Baptist University. The technical assistance of Miss

Nickie Chan is appreciated.

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Investigation of Specificity of Auricular Acupuncture Points In