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Pharmacological Research 51 (2005) 183–188

Antinociceptive mechanisms associated with diluted bee venomacupuncture (apipuncture) in the rat formalin test: involvement

of descending adrenergic and serotonergic pathways

Hyun Woo Kima,1, Young Bae Kwona,1, Ho Jae Hanb, Il Suk Yanga,Alvin J. Beitzc, Jang Hern Leea,∗

a Department of Veterinary Physiology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, San 56-1,

Shilim-dong, Kwanak-gu, Seoul 151-742, South Koreab Hormone Research Center, College of Veterinary Medicine, Chonna m National University, Kwang-ju, South Korea

c Department of Veterinary and Biomedical Science, University of M innesota, St. Paul, MN, USA

Accepted 21 July 2004

Abstract

In a previous report, subcutaneous injection of diluted bee venom (dBV) into a specific acupuncture point (Zusanli, ST36), a procedure

termed apipuncture, was shown to produce an antinociceptive effect in the rat formalin pain model. However, the central antinociceptive

mechanisms responsible for this effect have not been established. Traditional acupuncture-inducedantinociception is considered to be mediated

by activation of the descending pain inhibitory system (DPIS) including initiation of its opioidergic, adrenergic and serotonergic components.

The purpose of the present study was to investigate whether the antinociceptive effect of apipuncture is also mediated by the DPIS. Behavioral

experiments verified that apipuncture significantly reduces licking behavior in the late phase of formalin test in rats. This antinociceptive

effect of apipuncture was not modified by intrathecal pretreatment with naltrexone (a non-selective opioid receptor antagonist), prazosin (a

1 adrenoceptor antagonist) or propranolol (an adrenoceptor antagonist). In contrast, intrathecally injected idazoxan (an 2 adrenoceptorantagonist) or intrathecal methysergide (a serotonin receptor antagonist) significantly reversed apipuncture-induced antinociception. These

results suggest that apipuncture-induced antinociception is produced by activation of 2 adrenergic and serotonergic components of the DPIS.

© 2004 Elsevier Ltd. All rights reserved.

Keywords: Bee venom; Acupuncture; Antinociception; 2 Adrenergic; Serotonergic

1. Introduction

Although the precise antinociceptive mechanisms under-

lying acupuncture remain to be elucidated, this procedure hasbeen widely used to relieve various types of acute and chronic

pain [1]. Electrical or mechanical stimulation of an acupunc-

ture point (acupoint) is the most popular form of acupunture

and this type of stimulation produces significant antinocicep-

tive effects [2]. Recently, we have demonstrated that chem-

∗ Corresponding author. Tel.: +82 2 880 1272; fax: +82 2 885 2732.

E-mail address: [email protected] (J.H. Lee).1 These authors contributed equally to this study.

ical stimulation of an acupoint with subcutaneous injection

of diluted bee venom (dBV), a procedure termed apipunc-

ture, produces a very potent and long-lasting antinociceptive

effect in both acute andchronic rodentpain models [4,5]. Col-lectively, these data imply that apipuncture may be a more

effective acupoint stimulant than other forms of acupunc-

ture. While apipuncture appears to be a more effective form

of acupuncture, there is very little known regarding the neu-

ronal mechanisms underlying apipuncture-induced antinoci-

ception as compared to other types of acupuncture.

Both spinal and supraspinal mechanisms contribute to

acupuncture-induced analgesia [3]. We have recently demon-

strated that dBV injection into the Zusanli acupoint (ST36)

1043-6618/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.phrs.2004.07.011



184 H.W. Kim et al. / Pharmacological Research 51 (2005) 183–188

dose dependently decreased paw licking time and spinal Fos

expression in the formalin inflammatory pain model. While

the antinociception produced by apipuncture was clearly

demonstrated in this study, the central mechanisms respon-

sible for this analgesic effect are currently unknown. In an

earlier study, apipuncture was shown to produce visceral

antinociception by activation of the DPIS involving spinaladrenoceptors. In the present study, we further investigated

the involvement of opioidergic, adrenergic and serotonergic

components of the DPIS in the apipuncture-induced antinoci-

ceptive effect observed in the rat formalin test.

2. Materials and methods

2.1. Animals

Experiments were performed on male Sprague–Dawley

rats weighing 200–250 g. All experimental animals were ob-tained from the Laboratory Animal Center of Seoul National

University. They were housed in colony cages with free ac-

cess to food and water and were maintained in temperature

and light controlled rooms (23 ± 0.5 ◦C, 12/12 h light/dark

cycle with lights on at 07:00 h) for at least 1 week prior to

the study. All of the methods used in the present study were

approved by the Animal Care and Use Committee at SNU

and conform to NIH guidelines (NIH Publication No. 86-23,

revised 1985). In addition, the ethical guidelines for investi-

gating experimental pain in conscious animals recommended

by theInternational Association forthe Study of Pain [6] were

followed.

2.2. Surgery for intrathecal catheterization

Rats were anaesthetized by intraperitoneal injection of

chloral hydrate (400 mg kg−1, Merck, Germany). A piece of

PE10 tubing (0.28 mm i.d., 0.61 mm o.d., Becton and Dick-

inson, MD, USA) wasinserted via the atlanto-occipital mem-

brane using a stereotaxic instrument as previously described

[7]. In order for the catheter to reach the lumbar enlargement,

an intraspinal length of 8 cm was used. The dead space of the

catheter was about 5l. To verify proper catheter placement

in the lumbar enlargement, an intrathecal injection of lido-caine was performed. When the intraspinal location of the

catheter was correct, then motor paralysisof theanimal’s hind

limb lasting for a period of 20–30 min was observed immedi-

ately after lidocaine injection. Catheterized animals were not

used for intrathecal studies until 7 days post-implantation.

To determine if catheterized animals showed normal motor

function, a spontaneous activity chamber (MED Associates

Inc., USA, model # SG-506) was used to measure freely trav-

eled distance as previously described [8]. Any animals that

displayed a dysfunction of the limbs while walking were not

used in this study.

2.3. Drugs

Diluted bee venom of Apis mellifera, idazoxan, prazosin,

propranolol were purchased from Sigma (St. Louis, MO,

USA). Naltrexone was purchased from Research Biochem-

icals (Natick, MA, USA) and methysergide was purchased

from Tocris (UK). All drugs were dissolved in physiologicsaline (0.9% (w/v) NaCl) just before use.

2.4. Experimental protocol

Anesthesia was induced in catheterized rats by inhala-

tion of 5% isoflurane (Baxter, USA) in a mixed N2O/O2 gas

for 30 s and then maintained with 3% isoflurane. Naltrexone

(NTX, 10g per rat), idazoxan (IDZ, 50g per rat), pra-

zosin (PRA, 50g per rat), propranolol (PRO, 50g per rat),

or methysergide (MET, 30 g per rat) in a volume of 10 l

saline (Sal) was intrathecally injected. The dose of each re-

ceptor antagonist was determined from the literature and was

based on previous studies in which intrathecal administra-tion of the antagonist effectively blocked its receptor and a

spinal cord associated antinociceptive effect [9–15]. Control

animals received an equivalent volume of physiologic saline

through the same route of administration. Animal groups

names are abbreviated throughout the text and in the fig-

ures based on their specific treatment. Thus, the animal group

that received intrathecal injections of saline (Sal) followed by

subcutaneous injection of bee venom (BV) and then forma-

lin injection is designated “Sal-BV-F”, while the group that

received intrathecal saline, subcutaneous saline and forma-

lin is designated “Sal-Sal-F”. Similarly animals receiving in-

trathecal injection of the drugs, naltrexone (NTX), idazoxan(IDZ), prazosin (PRA), propranolol (PRO), or methysergide

(MET) plus subcutaneous BV and then formalin were desig-

nated “NTX-BV-F”, “IDZ-BV-F”, “PRA-BV-F”, “PRO-BV-

F”, and “MET-BV-F”, respectively.

Ten minutes after intrathecal drug treatment, dBV

(0.08mgkg−1 in a volume of 20l saline) was subcu-

taneously administered into the Zusanli (ST36) acupoint

located 5 mm below and lateral to the anterior tubercle of the

tibia. Thirty minutes post-dBV administration, 1% formalin

(20l) was subcutaneouly injected under the plantar surface

of the right hindpaw. The formalin test is a commonly used

model of persistent pain and formalin-induced behavior is

characterized by two phases: an early phase and a late phase.

Pain behaviors during the early phase are thought to be due

to direct chemical stimulation of nociceptors, while pain

behaviors associated with the late phase are attributed to

inflammatory pain induced by formalin-released inflamma-

tory mediators including histamine and prostaglandin [16].

Following intraplantar injection of formalin, the animals

were immediately placed on an acrylic observation chamber

(40 cm high, 20 cm diameter), and behaviors were recorded

using a video camera for 30 min. Following the video-taping,

paw licking time (in seconds per each 5 min increment) was

calculated by two experienced investigators, blinded to the



H.W. Kim et al. / Pharmacological Research 51 (2005) 18 3–188 185

experimental conditions, during both the early phase (phase

1; 0–10 minpost-formalin injection) andthe late phase (phase

2; 10–30 min post-formalin injection) of the formalin test.

Subsequently, the mean licking time was calculated from the

data obtained by these two experimenters and the mean lick-

ing time values were subsequently analyzed statistically to

determine significant differences among the various groups.

2.4.1. Data and statistical analysis

Thepaw licking time at early and late phase wasexpressed

as the mean ± S.E.M. Data was analyzed by either an inde-

pendent t -test or an analysis of variance (ANOVA) followed

by post-hoc comparisons using Fisher’s least significant dif-

ference test. Statistical significance was determined at p <

0.05.

3. Results

3.1. Antinociceptive effect of dBV on formalin-induced

pain behavior

Pretreatment with dBV (0.08 mg kg−1, s.c.) strongly sup-

pressed the formalin-induced paw licking time in the late

phase (Fig. 1). Total paw licking time in the late phase was

92.83 ± 8.35 s in the Sal-Sal-F group and 23.17 ± 9.04 s in

the Sal-dBV-F group (∗∗∗ p < 0.001). In addition, spontaneous

activity of i.t. catheterized and naı̈ve animals was not statisti-

cally different during 10 min (freely traveled distance: naı̈ve

= 346.10 ± 79.92 cm; catheterized = 325.28 ± 55.52 cm).

Fig. 1. This graph shows the antinociceptive effect of dBV on formalin-

induced painbehavior 10 and30 minpost-formalin injection. Treatment with

dBV remarkably reduced formalin-induced pain behavior in the late phase

(∗∗∗ p < 0.001, two-tailed t -test). Administration sites and routes were: i.t.

injection–Zusanli (s.c.)–paw (s.c.). Abbreviations: dBV, diluted bee venom;

Sal, saline; F, formalin.

Fig. 2. The effect of the non-selective opioidergic antagonist naltrexone on

dBV-induced antinociception is illustrated in this graph. Pretreatment (i.t.)

with NTX (10g 10l−1) did not alter the dBV-induced antinociception

observed during the late phase of the formalin test as compared with the

NTX–saline treated (NTX-Sal-F) group (∗∗∗ p < 0.001, two-tailed t -test).

Administration sites and routes were: i.t. injection–Zusanli (s.c.)–paw (s.c.).

Abbreviations: dBV, diluted bee venom; Sal, saline; F, formalin; NTX, nal-

trexone.

3.2. Opioidergic involvement in dBV-induced

antinociception

Pretreatment (i.t.) with the non-selective opioid receptor

antagonist, naltrexone (NTX, 10g 10l−1) did not reverse

the dBV-induced antinociception in the late phase of the for-malin test (Fig. 2). Paw licking time in the NTX-Sal-F group

was 87.20 ± 6.69 s and in the NTX-dBV-F group was 25.00

± 6.87 s (∗∗∗ p < 0.001).

3.3. 1 and adrenergic involvement in dBV-induced

antinociception

Both i.t. pretreatment with the 1 adrenoceptor antago-

nist prazosin (PRA, 50g 10l−1) and the adrenoceptor

antagonist propranolol (PRO, 50g 10l−1) did not inhibit

the dBV-induced antinociceptive effect on formalin-evoked

pain behavior in the late phase (Fig. 3A and B). In the late

phase, paw licking times were 90.60 ± 20.58 s (PRA-Sal-F),

8.11 ± 2.46 s (PRA-dBV-F), 90.40 ± 11.95 s (PRO-Sal-F)

and 27.33 ± 6.90 s (PRO-dBV-F).

3.4. 2 adrenergic and serotonergic involvement in

dBV-induced antinociception

The 2 adrenoceptor antagonist, idazoxan (IDZ,

50g 10l−1) significantly blocked the antinociceptive

effect of dBV on the formalin-induced pain behavior in the

late phase (Fig. 4A). During this phase, the total sum of

the paw licking time was increased from 81.80 ± 5.00 s in




Fig. 3. A graph summarizing the effect of intrathecal administration of an 1 and a adrenergic antagonist on dBV-induced antinociception. In the late phase

of the formalin test, dBV treatment still had a significant antinociceptive effect on formalin-induced pain behavior in animals pretreated intrathecally with PRA(50g 10l−1; A) or PRO (50g 10l−1; B) ( ∗∗∗ p < 0.001, two-tailed t -test). Administration sites and routes were: i.t. injection–Zusanli (s.c.)–paw (s.c.).

Abbreviations: dBV, diluted bee venom; Sal, saline; F, formalin; PRA, prazosin; PRO, propranolol.

the IDZ-dBV-F group to 88.80 ± 9.63s in the IDZ-Sal-F

group. The antinociceptive effect of dBV was also blocked

by i.t. pretreatment with the serotonin receptor antagonist

methysergide (MET, 30g 10l−1) (Fig. 4B). In the late

phase, the total sums of paw licking time were 98.58 ±

8.66 and 92.75 ± 12.01s in the MET-Sal-F group and the

MET-dBV-F groups, respectively.

4. Discussion

A variety of stimulation techniques including elec-

troacupuncture (EA),moxibustion and acupressure have been

used to stimulate acupoints in order to produce antinocicep-

tive effects that are selectively mediated by the activation of

descending modulatory systems [17–21]. Two major compo-

nents of this endogenous descending antinociceptive system

have been implicated in inhibition of nociceptive input at the

level of thespinal cord. Oneis theintrinsicopioidergicsystem

and the other is a descending monoaminergic (i.e., serotonin

and adrenaline) system in the brainstem [2]. It has been pro-

posed that acupuncture’s antinociceptive effect is mediated

by different neuronal mechanisms depending on the type of

stimulation that is applied to an acupoint [22,23]. For in-

stance, low frequency electroacupuncture-induced analgesia

appears to be mediated by the endogenous opioidergic sys-

tem, while the analgesic effect of high frequency EA is me-

diated by a non-opioidergic system [24]. In the present study,

we observed that the antinociceptive effect of dBV induced

by acupoint stimulation (apipuncture) was totally reversed by

intrathecal pretreatment with the 2 adrenoceptor antagonist

idazoxan or the non-selective serotonin receptor antagonist

methysergide. In contrast, apipuncture-induced antinocicep-

tion was not affected by intrathecal injection of antagonists

of other adrenoceptor subtypes or by i.t. injection of a non-

selective opioid receptor antagonist. These results imply that

the antinociceptive effect of apipuncture is mediated by spe-

cific descending monoaminergic pathways rather than by the

intrinsic opioidergic system. A similar phenomenon has also

been observed in an acetic acid-induced visceral pain model

[5]. Based on these findings, it is suggested that apipuncture-

induced antinociception is mediated by the spinal release of

norepinephrine and/or serotonin and the subsequent activa-

tion of specific adrenoceptors and/or 5-HT receptors in thespinal cord. It is well documented that the adrenergic and

serotonergic components of the descending pain modula-

tion system arise principally from the nucleus raphe mag-

nus (RMg) and the locus coeruleus (LC), respectively [19].

Therefore, it is likely that the activation of these nuclei by

acupoint stimulation with dBV produces an antinociceptive

effect via activation of spinal 2 and/or serotonergic recep-

tors. Support for this hypothesis comes from recent work

in our laboratories showing that BV acupoint stimulation

increases neuronal activity in brainstem catecholaminergic

neurons [25]. While this hypothesis seems likely for nora-

drenergic neurons, it remains to be determined if the 5-HT

specific antinociceptive mechanism of dBV is mediated via

higher brain centers including the RMg.

We observed that the2 adrenoceptor antagonist idazoxan

antagonized apipuncture-induced antinociception. It is well

known that intrathecal administration of the non-selective

adrenergic antagonist phentolamine or the selective2 adren-

ergic antagonist yohimbine attenuates descending inhibition

of nociceptive reflexes produced by electrical and/or chem-

ical stimulation in the PAG, nucleus raphe magnus, nucleus

reticularis paragigantocellularis, and locus coeruleus [26]. In

addition, intrathecal administration of noradrenergic recep-

tor agonists including clonidine is antinociceptive/analgesic



H.W. Kim et al. / Pharmacological Research 51 (2005) 183–188 187

Fig. 4. This graph illustrates the effect of i.t. pretreatment with the2 and serotonergic receptor antagonist, IDZ (50g 10l−1) and MET (30g 10l−1) on

dBV-induced antinociception. Both IDZ (A) and MET (B) pretreatment significantly reversed the antinociceptive effect of dBV on the formalin-evoked pain

behaviors during the late phase of the formalin test. Administration sites and routes were: i.t. injection–Zusanli (s.c.)–paw (s.c.). Abbreviations: dBV, diluted

bee venom; Sal, saline; F, formalin; IDZ, idazoxan; MET, methysergide.

[26]. Currently, it is presumed that norepinephrine inhibits A

and C fiber-mediated nociceptive transmission to spinal cord

through the activation of the 2 adrenoceptors (probably the

2A subtype) that are present on primary afferent terminals

[27].

While serotonin was initially proposed as a purely

antinociceptive transmitter, the influence of 5-HT upon the

activity of nocisponsive neurones in the dorsal horn is het-

erogeneous, with observations of both inhibition and less fre-

quently excitation. In this regard, a variety of supraspinalstructures can inhibit and/or facilitate spinal nociceptive re-

flexes via activation of serotonergic receptors [26]. In the

present study, we have shown that intrathecal administration

of the non-selective serotonin receptor blocker methysergide

antagonized apipuncture-induced antinociception suggesting

that a serotoninergic spinal cord component is involved in

apipuncture analgesia.

It is interesting that the effects of BV were principally

on the late phase of the formalin response. As mentioned

earlier, pain behaviors during the early phase of the for-

malin test are thought to be due to direct chemical stimu-

lation of nociceptors, while pain behaviors associated with

the late phase are attributed to inflammatory pain induced

by formalin-released inflammatory mediators including his-

tamine and prostaglandin [16]. This concept is supported

by a recent study showing that a novel non-peptide antag-

onist of the bradykinin (BK) B1 receptor also inhibits only

the late phase of the formalin response suggesting that this

phase is related to formalin-released inflammatory mediators

[28]. These results taken together with our own data sug-

gest that apipuncture is affective against the inflammatory

state induced by formalin injection that occurs 10–55 min

post-formalin injection, while it is ineffective in blocking the

initial direct formalin stimulation of nociceptors.

In summary, we conclude that the antinociceptive effect of

apipuncture is mediated by the selective activation of spinal

2 adrenergic and serotonergic receptors. Conversely, neither

spinal opioidergic nor 1 or adrenergic components of the

descending pain inhibitory system are involved with dBV-

induced antinociception in the rat formalin test.

Acknowledgements

This research was supported by a grant

(M103KV01000903K220100940) from the Brain Re-

search Center of the 21st Century Frontier Research

Program funded by the Ministry of Science and Technology

of the Republic of Korea. The publication of this manuscript

was also supported by a Research Fund from the Research

Institute for Veterinary Science (RIVS) in the College of

Veterinary Medicine, Seoul National University, as well as

the Brain Korea 21 project.

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apipuntura

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