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Research Report

Extrasegmental analgesia of heterotopic electroacupuncture stimulation on visceral pain rats

Jianhua Liu, Wenbin Fu, Wei Yi, Zhenhua Xu, Yuan Liao, Xinran Li, Jiesi Chen, Xuefang Liu, Nenggui Xu
Guangzhou University of Traditional Chinese Medicine, 12 Jichang Road, Guangzhou 510006, PR China

A R T I C LE I N FO Article history: Accepted 5 December 2010 Available online 14 December 2010 Keywords: Finance Asset pricing Information

AB S T R A C T Acupuncture has been applied in the clinic to treat visceral pain for a long time. However, the underlying mechanism still remains unknown. In the present study, extrasegmental analgesia of electroacupuncture (EA) at orofacial acupoints on visceral pain rats was investigated. The results revealed that nociceptive EA stimulation applied at heterotopic acupoints or nonacupoints to activate A and/or C fibers induced c-fos expression in the paratrigeminal nucleus (PTN) and significantly inhibited acetic acid-induced abdominal contractions and c-fos expression in the nucleus of the solitary tract (NTS). However, nonnociceptive EA or non-EA stimulation applied at heterotopic acupoints was totally ineffective. After infraorbital nerves transaction or pretreated by capsaicin, the EA analgesia was dramatically inhibited. Snake venom pretreatment had no influence on this analgesia. Consequently, heterotopic EA stimulation trigger the pain-inhibiting effect of diffuse noxious inhibitory controls (DNIC), in which PTNNTS secondary neural pathway may be involved and small-diameter (A and/or C) fibers are crucial. 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Visceral pain is a common form of pain in the clinic, which is mainly originated from viscera and induced by various stimuli including mechanical stretch, muscle spasm, inflammation and ischemia, etc. Much of what we know about pain comes from somatic and not visceral pain, therefore, visceral pain has been considered as a variant of somatic pain for a long time. Visceral shares some features in common with somatic pain; however, they also have important differences. Compared with somatic pain, it has some characteristics as follows: (1) it is diffuse and location of pain is inaccurate; (2) it mainly shows chronic pain and progressive enhancement; (3) hollow organs are sensitive to distension and traction stimulation but insensitive to noxious stimuli such as cutting
Corresponding author. Fax: +86 20 39358505. E-mail address: ngxu8018@gzhtcm.edu.cn (N. Xu).

and cauterization; (4) it is always accompanied with automatic reflexes, such as nausea, vomiting, and cardiovascular and respiratory changes, which often produced unhappy emotional reactions; (5) it always induces pain or hyperalgesia on the body surface far away from location of lesion (referred pain) (Cervero, 1999). At present, the underlying mechanism of visceral pain still remains unknown and the treatment in the clinic is not satisfactory. Therefore, it is necessary to develop new procedures for its management. Acupuncture has been widely applied in treating various kinds of acute and chronic pain for thousands of years. Recent studies have showed that acupuncture is effective for visceral pain (Iwa et al., 2005; Takahashi, 2006). Up to now, we know not much about visceral pain, and the precise mechanism of acupuncture in treating visceral pain remains unclear.

0006-8993/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.12.013

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control VP EA ST2 EA GB14 + VP EA ST2 + VP EA nonacu + VP EA ST6 + VP

Researches have shown that innocuous stimulation applied in the pain area produce segmental analgesia via large-diameter (A) fibers (Defrin et al., 2005; Johnson et al., 1991; Melzack and Wall, 1965), and noxious stimulation remote to pain area produce extrasegmental analgesia via small-diameter (A and/ or C) fibers (Le Bars et al., 1979a,b). The NTS is a major target of termination for visceral primary sensory afferents and involved in the somatovisceral processing. Various kinds of noxious visceral signal direct to the NTS via the vagus nerve, and somatic afferent stimuli evoke response of neurons in the NTS (Emch et al., 2000; Toney and Mifflin, 2000). In our previous works, electroacupuncture (EA) at orofacial acupoints or gastric distension induced c-fos expression in the nucleus of the solitary tract (NTS) (Liu et al., 2004). Meanwhile, extracellular recording showed that there were somatovisceral neurons in the NTS simultaneously reactive to somatic stimulation from acupuncture at orofacial acupoints and visceral stimulation from gastric distension (He et al., 2006). These data suggest that EA at orofacial acupoints have a regulation on visceral functions and the NTS may be involved in the process. Therefore, the purpose of this study is to investigate extrasegmental analgesic effects of EA at orofacial acupoints on visceral pain and its afferent pathway.

The number of abdominal contractions

100

80

60
**#

*#

*#

40

20
***### ***###

Fig. 1 Effects of EA at different orofacial acupoints on acetic acid-induced abdominal contractions. *P < 0.05, **P < 0.01, *** P < 0.001 vs. visceral pain group (VP), respectively; #P < 0.05, ### P < 0.001 vs. EA at GB14 and visceral pain group (EA GB14 + VP), respectively. and the EA analgesia was not influenced (46.0 10.39). Vehicle (44.3 11.1) or saline pretreatment (37.8 6.49) caused the same analgesic effects as the snake venom (Fig. 4).

2.

Results

2.4. Effects of EA at different orofacial acupoints on acetic acid-induced c-fos expression in the NTS
Acetic acid induced numerous c-fos expressions in the NTS (60.4 5.47), which were distributed along the rostrocaudal extent of the NTS and mainly located in the medial (mNTS), dorsomedial (dmNTS), and intermediate (imNTS) subnucleus of the NTS. EA at ST2 (receiving EA treatment alone) caused dramatic c-fos expression in the NTS (27.3 4.38, P < 0.01), caudal spinal trigeminal nucleus (cSTN), and paratrigeminal nucleus (PTN). Scattered c-fos positive neurons were also observed in the area postrema, dorsal motor nucleus of the vagus, and reticular formation. Pretreatment of EA at ST2 inhibited markedly acetic acid-induced c-fos expression in the NTS (45.2 5.85, P < 0.05). A similar phenomenon was also observed in the pretreatment of EA at ST6 or nonacupoints (33.8 7.72, 38.6 4.49, P < 0.01 for both groups). However, EA at

2.1. Effects of EA at different orofacial acupoints on acetic acid-induced abdominal contractions

I.p. injection of acetic acid produced characteristic writhing movement (88.0 16.66), which was composed of the contraction of the flank muscles together with inward movements of the hind limb or with whole body stretching and usually began 510 min following the injection and lasted about 1 h. Pretreatment of EA at Sibai (ST2) acupoints inhibited obviously acetic acid-induced abdominal contractions (43.2 11.19, P < 0.01). Pretreatment of EA at ST6 (36.2 13.50, P < 0.05) or nonacupoints (39.5 12.20, P < 0.05) caused similar inhibitory effects. However, pretreatment of EA at GB14 had no effects (82.5 16.24) (Fig. 1).

The number of abdominal contractions

2.2. Effects of different intensity of EA at ST2 on acetic acid-induced abdominal contractions

Non-EA or low intensity (1.0 mA) of EA had no effects on acetic acid-induced abdominal contractions (83.0 12.57, 80.5 11.05, respectively). Medium (2.0 mA) and high (4.5 mA) intensity of EA resulted in strong inhibitions (42.7 10.88, 37.5 12.23, P < 0.05, P < 0.05, respectively) (Fig. 2).

*#

100

*#

*#

VP Non EA + VP 1.0 mA + VP 2.0 mA + VP 4.5 mA + VP

80

60

40

2.3. Effects of EA at ST2 on acetic acid-induced abdominal contractions following ION pretreatment
Following infraorbital nerves (ION) transaction, inhibition of EA at ST2 on acetic acid-induced abdominal contractions was significantly attenuated (91.5 15.04 vs. 43.2 11.19, P < 0.01). Capsaicin pretreatment produced similar inhibitory effects (80.7 13.28 vs. 43.2 11.19, P < 0.05). After snake venom pretreatment, the ION showed typical demyelination (Fig. 3)

20

Fig. 2 Effects of different intensity of EA at ST2 on acetic acid-induced abdominal contractions. *P < 0.05 vs. medium intensity of EA group (2.0 mA + VP); #P < 0.05 vs. high intensity of EA group (4.5 mA + VP).

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(64.2 6.67, 57.8 4.35, respectively) and PTN (8.3 1.63, 9.2 1.80, respectively) (Figs. 9 and 10). Following medium (2.0 mA) or high (4.5 mA) intensity of EA at ST2, c-fos expression decreased in the NTS (41.2 4.29, 38.0 6.40, P < 0.05, P < 0.05, respectively) and increased in the PTN (21.3 4.93, 25.2 4.83, P < 0.01, P < 0.001, respectively) (Figs. 11 and 12).

2.7. Effects of EA at ST2 on acetic acid-induced c-fos expression in the NTS following ION pretreatment
The inhibitory effect of pretreatment of EA at ST2 on acetic acidinduced c-fos expression was completely abolished (71.3 8.52 vs. 45.2 5.85, P < 0.01) after ION transaction. Capsaicin pretreatment produced similar effects (68.5 10.26 vs. 45.2 5.85, P < 0.01). Snake venom (47.5 8.15) or vehicle (50.2 7.02) or saline pretreatment (41.2 6.12) had no obvious influence on the inhibition (Figs. 13 and 14).

Fig. 3 Photograph showing demyelination of infraorbital nerves following snake venom pretreatment. Scale bar represents 50 m.

2.8. Effects of EA at ST2 on c-fos expression in the PTN following ION pretreatment
GB14 (67.6 3.99) had no significant influence on acetic acidinduced c-fos expression in the NTS (Figs. 5 and 6). After ION pretreated by transaction or capsaicin, EA-induced c-fos expression was significantly decreased in the PTN (5.3 1.65, 11.7 2.44 vs. 22.3 4.72, P < 0.01, P < 0.05, respectively). Snake venom (20.8 6.04) or vehicle (26.3 4.50) or saline (19.5 2.47) pretreatment had no significant effects on EA-induced cfos expression in the PTN (Figs. 15 and 16).

2.5. Effects of EA at different orofacial acupoints on acetic acid-induced c-fos expression in the PTN
Acetic acid produced a few c-fos expressions in the PTN (6.0 1.32). However, EA at ST2 (receiving EA treatment alone) elicited numerous c-fos expression in the PTN (18.5 4.29). Pretreatment of EA at ST2 (22.3 4.72, P < 0.01) or ST6 (19.7 3.50, P < 0.01) or nonacupoints (15.2 3.11, P < 0.05) resulted in similar effects. Pretreatment of EA at GB14 had no influences on acetic acid-induced c-fos expression in the PTN (5.7 1.31) (Figs. 7 and 8).

3.

Discussion

3.1. PTNNTS secondary neural pathway involved in the analgesia of EA at orofacial acupoints on visceral pain rats
It is well known that i.p. injection of acetic acid in rats induced characteristic stretching movement in mice and rats, a visceral nociceptive response (Bonaz et al., 2000; Sinniger et al., 2004). The writhing test is regarded as a typical model of inflammatory visceral pain and has been widely applied for the screening of analgesic drugs for a long time (Koster, et al., 1959). The writhing movement consists of the contractions of the flank muscles together with inward movements of the hind limb or whole body stretching and usually begins 510 min after the injection and lasts about 1 h (Bonaz et al., 2000). In the present study, EA at orofacial acupoints (ST2 or ST6) or nonacupoints inhibited obviously acetic acid-induced abdominal contractions, suggesting that EA has analgesic effects on visceral pain rats. In addition, the results revealed that EA at ST2 or ST6 or nonacupoints inhibited acetic acid-induced c-fos expression in the NTS. NTS receives general and special visceral afferent fibers of VII, IX, and X nerves and is viewed as an important relay station of visceral primary sensory afferents. Both mechanical and chemical noxious sensory information from viscera direct mainly to the NTS via the vagus (Emch et al., 2000; Zhang et al., 1998) and the NTS neurons respond to somatic stimuli (Toney and Mifflin, 2000). Our previous study also indicated that gastric distention and EA at orofacial acupoints induced c-fos expression in the NTS (Liu et al., 2004), and NTS neurons simultaneously reacted to somatic

2.6. Effects of different intensity of EA at ST2 on acetic acid-induced c-fos expression in the NTS and PTN
Non-EA at ST2 or low intensity (1.0 mA) of EA at ST2 had no effects on acetic acid-induced c-fos expression in the NTS
control VP EA ST2 + VP ION transaction capsaicin vehicle snake venom saline

The number of abdominal contractions

##

## #

100

80

60

**

* **

40

20
***#

Fig. 4 Effects of EA at ST2 on acetic acid-induced abdominal contractions following ION pretreatment. *P < 0.05, **P < 0.01, *** P < 0.001 vs. visceral pain group (VP), respectively; #P < 0.05, ## P < 0.01 vs. EA at ST2 and visceral pain group (EA ST2 + VP), respectively.

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Fig. 5 Photographs of acetic acid-induced c-fos expression in the NTS at the level of area postrema following EA at orofacial acupoints. (A) Control group, (B) visceral pain group, (C) EA at ST2 group, (D) EA at GB14 and visceral pain group, (E) EA at ST2 and visceral pain group, (F) EA at nonacupoints and visceral pain group, (G) EA at ST6 and visceral pain group. Scale bar represents 100 m. stimulation from EA at ST2 acupoints and visceral stimulation from gastric distension (He et al., 2006). Consequently, NTS may be an important supraspinal structure in the somatovisceral processing and involved in the EA analgesia. ST2 is located in the infraorbital foramen and innervated by infraorbital nerve. ST6 is located in the mandibular angle on the cheek and its cutaneous and deep tissues are respectively innervated by auriculotemporal and masseter nerves. It is noteworthy that primary sensory afferents in orofacial areas mainly project to the spinal trigeminal nucleus (STN) and principal trigeminal nucleus. Branches of trigeminal nerves innervating orofacial areas (i.e., supraorbital, infraorbital, auriculotemporal, mylohyoid, mental nerves, etc.) have no direct projections to the NTS, except for the inferior alveolar, lingual, and buccal nerves (Marfurt, 1981; Panneton and Burton, 1981; Shigenaga et al., 1986). However, orofacial noxious stimuli, induced by orofacial cutaneous or deep tissue inflammation and temporomandibular joint injury, etc.,

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The number of c-fos immunoreactive neurons in the NTS

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70 60
*##

control VP EA ST2 EA GB14 + VP EA ST2 + VP EA Nonacu + VP EA ST6 + VP

50
**## **###

40 30 20 10 0
***### ***###

Fig. 6 Effects of EA at orofacial acupoints on acetic acid-induced c-fos expression in the NTS. *P < 0.05, **P < 0.01, ***P < 0.001 vs. visceral pain group (VP), respectively; ##P < 0.01, ###P < 0.001 vs. EA at GB14 and visceral pain group (EA GB14 + VP), respectively.

produced c-fos expression in the NTS (Bereiter and Bereiter, 2000; Zhou et al., 1999). Therefore, somatic sensory input in orofacial areas elicited by EA at ST2 or ST6 or nonacupoints should indirectly project to the NTS via secondary or multilevel neural afferent pathway. In the present study, EA at ST2 or ST6 or nonacupoints induced c-fos expressions in the PTN in visceral pain rats, which was contrary to the c-fos expression in the NTS. Following the ION transaction, EA at ST2 did not induce c-fos expression in the PTN and the analgesia was abolished. PTN is a small collection of medullary neurons, located in the dorsal lateral spinal trigeminal tract at the level of the rostral onethird of the caudal spinal trigeminal nucleus (cSTN) and caudal two-thirds of the interpolar spinal trigeminal nucleus (iSTN). PTN receives orofacial somatic sensory inputs via the trigeminal nerves, including infraorbital, auriculotemporal, and masseter nerves (Marfurt and Rajchert, 1991; Pfaller and Arvidsson, 1988; Shigenaga et al., 1988). Neuroanatomical and electrophysiological studies revealed that PTN and/or NTS neurons were activated by various kinds of noxious orofacial stimuli (Hayashi and Tabata, 1989; Shimizu et al., 2006; Tsujimura et al., 2009). Furthermore, the NTS receives afferent inputs from the PTN (Saxon and Hopkins, 1998; de Sousa Buck et al., 2001). It is reasonable to conclude that PTN may relay orofacial somatic sensory information to the NTS and orofacial areas; PTNNTS secondary neural pathway may be involved in the EA analgesia.

Fos, as the protein product of immediate early gene c-fos, has been classically viewed as a cellular marker of neuronal activation in response to somatic or visceral sensory stimuli to map neuronal pathway (Morgan et al., 1987). Many studies demonstrated that various forms of noxious mechanical, electrical and thermal, as well as chemical stimuli induced c-fos expression in the brain. However, non-nociceptive stimulation, such as water stimulation of the nasal mucosa or analgesia, induced c-fos expression in the PTN and NTS, respectively (Bonaz et al., 1994; Dutschmann and Herbert, 1997; Lanteri-Minet et al, 1993). Therefore, EA-induced c-fos expression is not enough to confirm whether EA stimulation is nociceptive. According to the previous study (Zhu et al., 2004), the EA stimulation intensity with 0.8 (non-nociceptive stimulation) and 1.0 times (slight nociceptive stimulation) of the threshold for the activation of A-fiber reflex and 1.0 times (strong nociceptive stimulation) of the threshold for the activation of C-fiber reflex was respectively 1.32 0.44 mA, 1.68 0.53 mA, and 4.78 0.45 mA. Therefore, low, medium, and high intensity of stimulation, including 1.0 mA (non-nociceptive stimulation) and 2.0 mA (slight nociceptive stimulation) as well as 4.5 mA (strong nociceptive stimulation), were used in this experiment. The results indicated that 2.0 mA or 4.5 mA significantly produced analgesic effects and 1.0 mA or nonEA did not. Meanwhile, capsaicin and snake venom, a selective neurotoxin for fine unmyelinated (C) fibers (Lynn, 1990) and large myelinated (A and A) fibers (Zhu et al., 2004), respectively, were applied to investigate the components of afferent fibers in EA analgesia. Capsaicin pretreatment of the ION attenuated dramatically the EA analgesia, but snake venom pretreatment did not. The results are in accordance with previous reports. When the stimulation intensity is strong enough to activate A fibers, noxious heterotopic EA stimulation displays a striking inhibition on the C-fiber reflex. However, non-noxious heterotopic EA stimulation below the threshold of A-fiber activation was totally ineffective (Zhu et al., 2004). Furthermore, heterotopic EA-induced analgesia was completely abolished in capsaicin-treated animals (Zhu et al., 1990b). Consequently, these results suggest that noxious EA stimulation produces analgesic effects on visceral pain via small-diameter (A and/or C) fibers.

3.3. Involvement of diffuse noxious inhibitory control in the extrasegmental analgesia elicited by heterotopic EA stimulation
When innocuous stimuli to activate large fibers (A) are applied in the pain focus, it inhibits nociceptive inputs of the spinal neurons and results in a segmental analgesia (Defrin et al., 2005; Johnson et al., 1991), which is known as Gate Control Theory (Melzack and Wall, 1965). However, when noxious stimuli are applied distant to pain focus, it displays a systemic or extrasegmental analgesia, which is termed as diffuse noxious inhibitory controls (DNIC) (Le Bars et al., 1979a,b). It is well known that, in animals, the DNIC is an electrophysiological phenomenon in which the activity of convergent neurons in the spinal dorsal horn and trigeminal nucleus is inhibited by noxious stimuli applied to various sites of the body, which are remote from the excitatory receptive fields of

3.2. A and/or C fibers involved in the analgesia of EA at orofacial acupoints on visceral pain
There has been controversy about components of the afferent fibers in acupuncture analgesia for a long time. Some investigations reveal that large-diameter (A) fibers are important (Lv, 1987; Toda, 2002), and others show that small-diameter (A and/or C) fibers are critical (Hou, 1989). It is well known that large-diameter fibers carry non-nociceptive information and small fibers carry nociceptive information.

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Fig. 7 Photographs of acetic acid-induced c-fos expression in the PTN following EA at orofacial acupoints. (A) Control group, (B) visceral pain group, (C) EA at ST2 group, (D) EA at GB14 and visceral pain group, (E) EA at ST2 and visceral pain group, (F) EA at nonacupoints and visceral pain group, (G) EA at ST6 and visceral pain group. Scale bar represents 100 m.

neurons (Le Bars et al., 1979a,b). Various conditioning stimuli, including electrical, pinch, heat, and chemical stimulations, trigger the DNIC effect (Dickenson et al., 1980; Le Bars et al., 1979a; Morton et al., 1987). However, the intensity of the heterotopic stimuli must be strong enough to activate small (A and/or C) fibers. Meanwhile, this phenomenon is also observed in humans. Heterotopic painful stimulation attenuates experimentally and clinical pain, such as tooth pain (Motohashi and Umino, 2001), muscle pain (Graven-Nielsen et al, 1998), nociceptive flexion (RIII) reflex (Roby-Brami et al, 1987), and blink reflex (Ellrich and Treede, 1998). The mechanism

underlying DNIC is viewed as the involvement of a spinal supraspinalspinal feedback loop producing a descending inhibition (Ellrich and Treede, 1998; Le Bars et al., 1979a,b; Roby-Brami et al, 1987). It has been shown that heterotopic acupuncture stimulation can trigger DNIC effect by inhibiting activities of trigeminal convergent neurons (Zhu et al., 1990a). The heterotopic acupuncture-induced DNIC effect is closely related to the stimulation intensity. Heterotopic EA stimulation to activate A and/or C fibers produced powerful inhibition of C-fiber reflex. However, heterotopic EA stimulation not to

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The number of c-fos immunoreactive neurons in the PTN

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**

25 20 15 10 5
**

** *

**

control VP EA ST2 EA GB14 + VP EA ST2 + VP EA nonacu + VP EA ST6 + VP

The number of c-fos immunoreactive neurons in the NTS

**##

70 60 50 40 30 20 10 0

*# *#

VP Non EA 1.0 mA EA 2.0 mA EA 4.5 mA EA

Fig. 8 Effects of EA at orofacial acupoints on acetic acid-induced c-fos expression in the PTN. *P < 0.05, **P < 0.01 vs. visceral pain group (VP) and EA at GB14 and visceral pain group (EA GB14 + VP), respectively. activate A and/or C fibers was completely ineffective (Zhu et al., 2004). Furthermore, regardless of the stimulation intensity, the pain-inhibiting effect of DNIC was unable to be observed in the spinalized or capsaicin-treated animals (Zhu et al 2004, 1990b). In the present study, EA at heterotopic ST2 acupoints or nonacupoints produced the same extrasegmental analgesic effects on visceral pain. Meanwhile, only noxious EA stimulation to activate A and/or C fibers can produce the analgesic effects, whereas non-noxious EA stimulation to activate A fibers is not effective. Consequently, all these findings suggest that DNIC is involved in the extrasegmental

Fig. 10 Effects of different intensity of EA at ST2 on acetic acid-induced c-fos expression in the NTS. *P < 0.05, **P < 0.01 vs. medium intensity of EA group (2.0 mA EA), respectively; # P < 0.05, ##P < 0.01 vs. high intensity of EA group (4.5 mA EA), respectively. analgesia elicited by noxious EA at orofacial acupoints on visceral pain rats.

4.
4.1.

Experimental procedures
Animals

Adult SpragueDawley rats of both sexes, weighing from 280 320 g, were used in this study. Each rat was housed in plastic

Fig. 9 Photographs of acetic acid-induced c-fos expression in the NTS following different intensity of EA at ST2. (A) Non-electroacupuncture group (non-EA), (B) low intensity of EA group (1.0 mA EA), (C) medium intensity of EA group (2.0 mA EA), (D) high intensity of EA group (4.5 mA EA). Scale bar represents 100 m.

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Fig. 11 Photographs of acetic acid-induced c-fos expression in the PTN following different intensity of EA at ST2. (A) Non-electroacupuncture group (non-EA), (B) low intensity of EA group (1.0 mA EA), (C) medium intensity of EA group (2.0 mA EA), (D) high intensity of EA group (4.5 mA EA). Scale bar represents 100 m.

compartments and controlled environmental conditions (25 1 C; relative humidity 40%60%; a 12 h/12 h lightdark cycle from 7:00 a.m. to 7:00 p.m.), with access to food and water ad libitum. The procedures in the present study were performed in accordance with the guidelines of Guangzhou University of Traditional Chinese Medicine Committee for Care and Use of Research Animals and the ethical guidelines for investigations of experimental pain in conscious animals (Zimmermann, 1983).

4.2. Visceral pain induced by intraperitoneal (i.p.) injection of acetic acid

Visceral pain was induced by i.p. injection of 0.6% acetic acid (10 ml/kg) in conscious rats, resulting in contraction of the abdominal muscle together with a stretching of the hind limbs. After injection, rats were placed into individual plastic compartments and visceral pain was assessed by counting abdominal contractions for 60 min (Sinniger et al., 2004). Animals in the control group received i.p. injection of normal saline (10 ml/kg). The assessment of the abdominal contractions was made by a single observer blinded to the experimental conditions.

The Number of Fos immunoreactive neurons in the PTN

30 25 20 15 10 5 0
*## **### *##

VP Non EA 1.0 mA EA 2.0 mA EA 4.5 mA EA

4.3.

Pretreatment of the ION nerve

Fig. 12 Effects of different intensity of EA at ST2 on acetic acid-induced c-fos expression in the PTN. *P < 0.05, **P < 0.01 vs. medium intensity of EA group (2.0 mA EA), respectively; ## P < 0.01, ###P < 0.001 vs. high intensity of EA group (4.5 mA EA), respectively.

ION pretreatment was performed five days before EA at ST2 and acetic acid injection. The animals were anesthetized by an i.p. injection of urethane given at a dose of 1 g/kg. A vertical incision was made in the skin overlying the infraorbital foramen to expose bilateral ION under a dissecting microscope. For the transaction of ION, the exposed ION was ligated at two separate points with silk suture, and the nerve bundle between two ligatures was transected. For the capsaicin pretreatment, the exposed ION was dissociated for 11.5 cm and wrapped by a piece of cotton silver soaked in a fresh solution of capsaicin (Sigma, St. Louis, MO, USA) dissolved in 20% Tween 80 in paraffin for 20 min. A plastic film was placed under the ION to prevent the capsaicin solution from soaking the peripheral tissues. Twenty minutes after capsaicin treatment, the cotton silver and the film were removed. For the snake venom pretreatment, the ION bundle received slowly intrathecal injection of 0.3 mg snake venom solution (Institute of Snake

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Fig. 13 Photographs of acetic acid-induced c-fos expression in the NTS at the level of area postrema following ION pretreatment and EA at ST2. (A) ION transaction group, (B) capsaicin group, (C) vehicle group, (D) snake venom group, (E) saline group. Scale bar represents 100 m.

The number of c-fos immunoreactive neurons in the NTS

80 70 60 50 40 30 20 10 0
***### *

##

##

* *

control VP EA ION transaction capsaicin vehicle snake venom saline

Venom of Guangzhou Medical College, Guangzhou, China), avoiding solution soaking the peripheral tissues. After finishing the experiment, the demyelination of ION induced by snake venom was identified by histology. As a control, capsaicin and snake venom were replaced by the vehicle (20% Tween in paraffin) and normal saline, respectively. After ION treatment, the wound was sutured and animals were survived for 5 days.

4.4.

EA treatment

Fig. 14 Effects of EA at ST2 on acetic acid-induced c-fos expression in the NTS following ION pretreatment. *P < 0.05, *** P < 0.001 vs. visceral pain group (VP), respectively; ##P < 0.01, ### P < 0.001 vs. EA at ST2 and visceral pain group (EA), respectively.

Prior to injection of acetic acid, rats were immobilized in a plastic box and were pretreated by EA stimulation for 20 min. Two stainless acupuncture needles (0.28 mm outer diameter) were subcutaneously inserted 5 mm into the acupoints on each side and were left for 20 min. The electrical stimulation was from a medical EA apparatus (model G6805-2; Shanghai, China). The stimulation parameters were a frequency of 2 and 20 Hz, alternatively, and an intensity of 15 mA. The stimulation intensity was mainly determined by the tolerance to the EA

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Fig. 15 Photographs of acetic acid-induced c-fos expression in the PTN following ION pretreatment and EA at ST2. (A) ION transaction group, (B) capsaicin group, (C) vehicle group, (D) snake venom group, (E) saline group. Scale bar represents 100 m.

The number of c-fos immunoreactive neurons in the PTN

***

30
**

** *

25 20 15 10
## #

control VP EA ION transaction capsaicin vehicle snake venom saline

5
###

and slight twitches of local tissues in acupoints without irritation and bray. In the non-electroacupuncture group, the needles were only inserted into ST2 acupoints with no electrical stimulation. Multiples of thresholds for the activation of A fibers or C fibers to elicit the nociceptive or non-nociceptive responses were applied in the present study and the intensities of EA stimulation were 1.0 mA (non-noxious stimulation) and 2.0 mA (slight nociceptive stimulation for the activation of A fibers reflex) as well as 4.5 mA (strong nociceptive stimulation for the activation of C fibers reflex), respectively. The locations of acupoints and nonacupoints were as follows: Sibai (ST2) is located in the infraorbital foramen; Jiache (ST6), 0.5 cm anterior and superior to the mandibular angle on the cheek; Yangbai (GB14), 2.5 cm directly above the pupil on the forehead; and nonacupoints, 1 cm lateral to the ST2.

Fig. 16 Effects of EA at ST2 on acetic acid-induced c-fos expression in the PTN following ION pretreatment. *P < 0.05, ** P < 0.01, ***P < 0.001 vs. visceral pain group (VP), respectively; # P < 0.05, ##P < 0.01, ###P < 0.001 vs. EA at ST2 and visceral pain group (EA), respectively; P < 0.05, P < 0.01, P < 0.001 vs. ION transaction group, respectively; P < 0.01 vs. capsaicin group.

4.5.

C-fos immunohistochemistry

Rats were sacrificed 2 h after i.p. injection of acetic acid (or normal saline). The c-fos immunohistochemistry was performed as previously described (Liu et al., 2004). Animals were deeply anesthetized by an intraperitoneal injection of urethane given

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(1 g/kg) and perfused through the ascending aorta, with 100 ml of 0.9% physiological saline followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 5060 min. The brainstem was immediately removed and post-fixed in the same fixative solution for 6 h, followed by cryoprotection from 20% sucrose solution in 0.1 M PB for overnight at 4 C. Frozen transverse sections (40 m) were obtained at 20 C by a freezing microtome (CM1850; Leica, Germany) and then were performed immunohistochemical staining for c-fos using biotinavidin horseradish peroxidase complex (ABC) methodology. Sections were rinsed twice in 0.01 M phosphate buffered saline (PBS; pH 7.4) and were then immersed in 0.3% H2O2carbinol for 30 min to block the intrinsic peroxidase activity, followed by permeation in 0.3% Triton X-100 for 30 min. After treatment with 10% normal goat serum (Santa Cruz, CA, USA) for 20 min, sections were incubated with a polyclonal rabbit anti-cfos antibody (Santa Cruz, CA, USA) at a dilution of 1:400 for 1 h at room temperature. Three washes for 5 min in 0.01 M PBS (pH 7.4) were performed between all two steps. The sections were incubated with biotinylated anti-rabbit antibody (Santa Cruz, CA, USA) at a dilution of 1:100 for 1 h. Following the washing step, the sections were incubated with the biotinavidin peroxidase complex (Santa Cruz, CA, USA) for 1 h. Finally, the sections were developed in 0.05% diaminobenzidine (DAB) 0.01% H2O2 0.05 M TrisHCl buffers (pH 7.6) for 35 min. The reaction was terminated by placing the sections in 0.01 M PBS (pH 7.4). Sections were mounted on polylysine-coated slides, dehydrated, cleared in xylene, and coverslipped with neutral gum. C-fos protein labeled mainly roundish and oval nuclei and showed brown or black in bright field microscopy. As a control, primary antibody was replaced by normal rabbit serum, and no c-fos positive neurons were observed.

Program of China (973 Program) (NO2010CB530500, NO2010CB530503), Program for New Century Excellent Talents in University (NCET-09-0083) and Gudong natural science fund committee (NO9351040701000001).

REFERENCES

4.6.

Qualitative and quantitative analysis

Anatomical location of the NTS and PTN (Fig. 5) was identified according to stereotaxic rat brain atlas (Paxinos and Watson, 1998). C-fos protein labeled mainly in the nucleus of neurons and c-fos positive neuron appeared round or oval, completely filled, and brown or black in color. C-fos count was performed as previously described (Liu et al., 2004). Every third section within the NTS region from 2.0 mm caudal to obex to 1.0 mm rostral to obex was selected from each brainstem. The number of c-fos positive cells in the NTS and PTN was counted under a light microscope by a single observer blinded to the experimental conditions.

4.7.

Statistical analysis

Data were presented as mean standard error of the mean (SEM). Differences between groups were considered to be statistically significant for P < 0.05. The data were analyzed using the one-way ANOVA followed by least significant difference (LSD) as post-hoc multiple comparisons test.

Acknowledgments
This work was supported by the National Natural Science Foundation of China (NO30873239), National Basic Research

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