Acupuncture Defecation in Rats

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Electro-acupuncture attenuates stress-induced defecation in rats with chronic visceral hypersensitivity via serotonergic pathway Xiao Yu Tian a , Zhao Xiang Bian a,⁎, Xu Guang Hu a,b , Xiao Jun Zhang a , Liang Liu a , Hongqi Zhang a a

School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR GuangDong Pharmaeutical College, P. R. China

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Acupuncture has long been used for patients with irritable bowel syndrome. However, it has

Accepted 3 March 2006

remained unclear. The aim of this study was to testify the effect of electro-acupuncture(EA)

Available online 2 May 2006

on (1) visceral hypersensitivity induced by the mechanical colorectal irritation during postnatal development of rats, and (2) stress-induced colonic motility changes on rats with

Keywords:

chronic visceral hypersensitivity. The abdominal withdrawal reflex (pain threshold and

Electro-acupuncture (EA)

score) for visceral hypersensitivity and fecal pellet output for motor dysfunction were

Chronic visceral hypersensitivity

selected as two indexes for measurement. In addition, the effect of EA on 5-HT4a receptor

(CVH)

and serotonin transporter (SERT) expression in the colon mucosa was analyzed semi-

Irritable bowel syndrome (IBS)

quantitatively through immunohistochemistry and 5-HT concentration in the colon tissue

Serotonin pathway

was observed through spectro-photo-fluorimeter detection, respectively. Our results showed that EA significantly elevated pain threshold, decreased the scores and also

Abbreviations:

decreased fecal pellet output during water avoid stress. Furthermore, EA decreased 5-HT

AWR, abdominal withdrawal reflex

concentration in colon in rats with CVH and CVH rats with water avoidance stress, and

CRD, colorectal distention

increased the 5-HT4a and SERT expression in rats with CVH. Thus, it can be concluded that

CVH, chronic visceral

EA attenuates behavioral hyperalgesia and stress-induced colonic motor dysfunction in

hypersensitivity

CVH rats via serotonergic pathway.

EA, electro-acupuncture

© 2006 Elsevier B.V. All rights reserved.

EC, enterochromaffin GABA, γ-aminobutyric acid IBS, irritable bowel syndrome 5-HT, serotonin SERT, serotonin transporter PTP, pain threshold pressure WAS, water-avoidance stress

1.

Introduction

Irritable bowel syndrome (IBS) is a common digestive disease characterized by recurrent abdominal discomfort or pain

associated with disturbance in bowel habit such as diarrhea or constipation and a lowered sensory threshold to rectal distention (Thompson et al., 1999; Bouin et al., 2002). Dysfunction of the bidirectional communication between the

⁎ Corresponding author. Fax: +86 852 34112929. E-mail address: [email protected] (Z.X. Bian). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.014

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gut (i.e., the enteric nervous system) and the brain (the braingut axis) has the most important impact on IBS, and such dysfunction modulated by various psychosocial and environmental factors (e.g. infection, inflammation) could cause serious symptoms, such as visceral pain and disturbance of bowel habit (Mach, 2004). Over the past 30 years, the main treatment of IBS has aimed to normalize gastrointestinal transit using laxatives or antidiarrheal agents, with or without the concurrent use of spasmolytics, but the results are unsatisfactory (Bueno, 2005). The recent pharmacological investigation has been focusing mainly on the serotoninrelated drugs, and alosetron, a 5-HT3 antagonist, is an approved drug for the treatment of diarrhea predominantIBS. However, the adverse effects of alosetron limit its usage (Andersen and Hollerbach, 2004). As the treatment for IBS has so far been suboptimal, many patients turn to alternative treatment modalities for IBS, including acupuncture that appears to be a promising treatment (Li et al., 1992; Xing et al., 2004). One trial reported that acupuncture has curative effect on IBS patients, including improvement of abdominal discomfort, defecation frequency, and overall well-being (Li et al., 1992). In addition, transcutaneous electrical acustimulation at acupoints can reduce rectal perception in diarrhea predominant-IBS patients (Xing et al., 2004). However, it has been debated whether the efficacy of acupuncture is a placebo response (Schneider et al., 2005). It is necessary to provide consolidate evidences to validate the efficacy of acupuncture on visceral hypersensitivity and related motility dysfunction in IBS. In the present study, we hypothesized that electro-acupuncture (EA) can attenuate the visceral hyperalgesia and inhibit colon motility in rats of chronic visceral hypersensitivity (CVH) model, and such effects are mediated by serotonin(5HT) signaling pathway in the colon. To test this hypothesis, the effect of EA on (1) visceral hypersensitivity induced by the mechanical colorectal irritation during postnatal development, and (2) stress-induced colonic motility changes on CVH rats were tested. Two standard methods were used to test the responses of the rats, involving testing abdominal withdrawal reflex (score and pain threshold) for visceral hypersensitivity and counting fecal pellet output for motor dysfunction. The effect of EA on 5-HT4a, and serotonin transporter (SERT) expression in the colon mucosa were analyzed semi-quantitatively through immunohistochemistry and 5-HT concentration in the colon tissue was measured through spectro-photo-fluorimeter detection, respectively.

2.

Results

2.1.

Effects of EA on visceral hypersensitivity of CVH rats

As shown in Fig. 1a, the AWR scores in response to graded CRD (20, 40, 60 and 80 mm Hg) were 0, 0, 2.3 ± 0.4, 3.2 ± 0.8 in the normal control group (n = 6), and 0.2 ± 0.4, 3 ± 0.65, 3.3 ± 0.5, 3.7 ± 0.4 in the CVH group (n = 6), respectively. PTP as showed in Fig. 1b in response to CRD was 53.2 ± 6.0 mm Hg in the normal control group (n = 6) and 37.4 ± 3.5 mm Hg in the CVH group (n = 6), respectively. Thus, a significant difference existed in the rectal sensitivity to CRD between the control

Fig. 1 – (a) Abdominal withdrawal reflex scores (AWR score) measured in response to graded colorectal distension show significant differences between the rats with chronic visceral hypersensitivity and the control groups at 40 mm Hg, and between the EA treatment group and the CVH group at 40, 60 and 80 mm Hg. Data are mean ± SE. *P < 0.05 vs. normal, #P < 0.05 vs. CVH. (b) Pain threshold pressure (PTP) measured in response to colorectal distension showed significant decrease in the rats with chronic visceral hypersensitivity comparing to the control group, and a significant increase in the EA treatment group comparing to the CVH group without EA treatment. Data are mean ± SE. *P < 0.05 vs. normal, #P < 0.05 vs. CVH.

and the CVH groups. These results suggest that CVH model exhibited visceral hypersensitivity. EA treatment significantly elevated PTP and reduced AWR scores of CVH rats. As shown in Fig. 1a, AWR scores in response to graded CRD (20, 40, 60 and 80 mm Hg) were 0,0.3 ± 0.8,1.5 ± 1.65,2.7 ± 0.4 after the EA treatment (n = 6), respectively. Fig. 1b showed that PTP in response to CRD was 62.7 ± 4.3 mm Hg after EA-treatment (n = 6), thus EA

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significantly decreased AWR scores and PTP. These results suggested that EA has analgesia effects on CVH rats.

2.2. rats

Effects of EA on WAS-induced colon transit in CVH

The effects of EA on colon transit were measured by fecal pellet output. There is no significant difference about fecal pellet output among normal rats (0.9 ± 1.4, n = 6) and CVH rats (0.8 ± 1.1, n = 6). WAS-induced fecal pellet output in CVH rats was significant higher (11.9 ± 2.0, n = 6) than that of CVH rats without WAS (0.8 ± 1.1, n = 6) or normal rat (0.9 ± 1.4, n = 6). Furthermore, EA significantly inhibited WAS-induced increase of fecal pellet output (4.0 ± 1.1, n = 6) (P < 0.05, Fig. 2). These results suggested that EA could inhibit WAS-induced increase of colon motility.

2.3. Effect of EA on 5-HT concentration in the colon tissue in CVH rats As shown in Fig. 3, CVH and WAS raised the 5-HT concentration in colon, while EA could significantly decrease it and increase the PTP. Significant differences of the 5-HT concentration in the colon segment among different groups existed, with 12.53 ± 6.45 ng in the normal rats (n = 6), 38.64 ± 8.01 ng in the CVH rats (n = 6) and 25.12 ± 8.82 ng in EA-treated CVH rats (n = 6). These results suggested that CVH have elevated 5-HT concentration in colon mucosa. Furthermore, WAS significantly raised 5-HT concentration in the colon of CVH rats (50.57 ± 7.88, n = 6), while EA significantly lowered the enhanced concentration induced by WAS (25.44 ± 6.38, n = 6). These results suggested that EA could inhibit WAS-induced defecation, with diminishing the 5-HT concentration.

Fig. 3 – 5-HT concentrations were detected among 5 groups involving normal group, CVH group, CVH rats receiving EA group, CVH group receiving WAS and EA treatment group plus WAS. CVH significantly elevated 5-HT concentration, while EA decreased it. WAS further increased 5-HT concentration, which decreased with EA treatment. Data are mean ± SE. *P < 0.05 vs. normal, #P < 0.05 vs. CVH, and P < 0.05 vs. CVH + WAS.

2.4. tissue

As shown in Figs. 4a–c, quantification of 5-HT4a and SERT expression by counting the gray level in the colon mucosa revealed that animals which received chronic colorectal distention have lower levels of 5-HT4a and SERT expression (137.66 ± 7.72, 135.23 ± 5.23, respectively) than that of normal rats (166.50 ± 18.42 and 166.52 ± 16.19, respectively). After EA, expression of 5-HT4a and SERT increased to 169.00 ± 5.10 and 162.88 ± 3.76, respectively. Moreover, WAS counteracted with EA treatment, by significantly suppressing the expressions of 5-HT4a (133.58 ± 6.69) and SERT (131.20 ± 3.20) that were elevated by EA treatment.

3.

Fig. 2 – Colon transit measured by fecal pellets per hour in normal rats, rats with CVH, rats with CVH plus WAS and rats with CVH receiving EA treatment, showed that there was a significant increase of fecal pellets after WAS, which was decreased by EA treatment. Data are mean ± SE. *P < 0.05 vs. normal, #P < 0.05 vs. CVH, and P < 0.05 vs. CVH + WAS.

5-HT4a and SERT immunohistochemistry of the colon

Discussion

Chronic visceral hyperalgesia is an important and characteristic feature of the irritable bowel syndrome (IBS), inflammatory bowel disease and other functional bowel disorders. Basic research into the mechanisms of chronic visceral pain has increased greatly in the last decade (Bueno et al., 1997). Visceral hyperalgesia is thought to be a pain state caused by central sensitization, that leads to abnormal perception of both painful stimuli (hyperalgesia) and non-painful stimuli (hyperanesthesia or, if perceived as pain, allodynia) (Mayer and Gebhart, 1994). Long-lasting pain states, chronic inflammation, genetic factors and many unknown factors contribute to the generation of visceral hyperalgesia/allodynia, and these have a fundamental role in chronic visceral pain states. It is believed that the development of sensitization in the nervous system induced by repetitive stimulation of peripheral

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Fig. 4 – (a) Gray level of SERT and 5-HT4a expression in the colon mucosa in 4 groups involving normal group, CVH groups, CVH receiving EA group and CVH plus WAS receiving EA group, showed that CVH group had lower expression of 5-HT4a than normal group, and EA increased the 5-HT4a and SERT expression in CVH rats. In addition, WAS decreased the expression level of SERT and 5-HT4a in EA-treated groups. Data are mean ± SE. *P < 0.05 vs. normal, #P < 0.05 vs. CVH + EA. (b) Photomicrograph findings of 5-HT4a expression in colon mucosa of rats. There was a significant decrease in 5-HT4a expression in the colon mucosa of animals with CVH, comparing with normal rats. EA significantly increased the expression of 5-HT4a in rats with CVH. In addition, WAS significantly elevated the expression of 5-HT4a in rats with CVH (×200). (c) Photomicrograph showed SERT expression in colon mucosa of rats. There was a significant decrease in SERT expression in the colon mucosa of animals with CVH, comparing with normal rats. EA significantly increased the expression of SERT in rats with CVH. In addition, water avoid stress significantly decreased the expression of SERT in rats with CVH (×200).

visceral input when it is vulnerable during the animal's postnatal development is one of important causes of chronic visceral hyperalgesia (De Deurwaerdere et al., 1998). Our study once again demonstrated that repetitive CRD stimulation on neonates subsequently developed visceral hypersensitivity in adulthood (Al-Chaer et al., 2000). Acupuncture has been used for pain management involving somatic pain and visceral pain for long time, such as in shoulder pain (Green et al., 2005) and labor pain (Lee and Ernst, 2004). Previous researches showed that the analgesic effect of acupuncture is mainly mediated through endogenous opioids and other neurotransmitters, including 5-HT, nitric oxide, γAminobutyric acid (GABA), etc., in the central nerve system (Ma, 2004; Han and Terenius, 1982). Activated spinal neurons can convey acupuncture signals to the brain and activate a descending inhibitory system including initiation of its

opioidergic, adrenergic and serotonergic components, which in turn inhibits hyperalgesia (Lao et al., 2001). Moreover, the analgesic effect of EA in the spinal cord level may be linked with the activation of enkephalin-interneurons, which induce 5-HT release (Tsai et al., 1989). 5-HT and 5-HT1,2,3 receptors are involved in EA-induced analgesia in the spinal trigeminal nucleus, which is closely associated with the perception and transmission of sensory information (Takagi and Yonehara, 1998). It is well known, however, that visceral pain differs from somatic pain, because it is difficult to localize and is referred to somatic areas (Ness and Gebhart, 1990). Pathophysiology of visceral pain is not fully understood, partially because of the lack of a valid animal model. Recently, some models successfully produce the characteristics of chronic visceral hypersensitivity (Al-Chaer et al., 2000; Coutinho et al., 2002), providing a valuable tool to unravel the underlying mechanism of visceral

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pain and to investigate the effect and related mechanism of EA treatment on visceral hypersensitivity. Previous studies showed that neonatal adverse experience results in not only chronic visceral hyperalgesia (De Deurwaerdere et al., 1998) but also altered physiological responses to stress in adults in animals and humans (Bakshi and Kalin, 2000; Heim et al., 2000), especially sensitizing colonic responses to subsequent stress. Furthermore, stressinduced colorectal motor abnormalities in adults with early life events are secondary to visceral hyperalgesia (Bonaz and Tache, 1994; Coutinho et al., 2002). Consistent with these results, our research found that WAS greatly enhanced the fecal pellet output in CVH rats compared with the control normal rats, indicating a close relationship between CVH and the pronounced increase in stress-induced defecation. This evidence may emphasize the significance of CVH in the increased susceptibility of colorectal motility to stress in IBS, although we cannot exclude the possibility that the increased amount of WAS-induced defecation in the CVH group may have also been associated with an increase pellet fluid content. Previous studies showed that 5-HT plays an important role in the dysmotility and hypersensitivity of the bowel. The 5-HT medicates gastrointestinal functions via activation of secretory cells, stimulation of afferent and efferent neurons as well as direct effects on gut smooth muscle leading to contraction or relaxation (Kim and Camilleri, 2000). Majority of the body's 5-HT is stored in the enterochromaffin (EC) cells that scattered in the epithelium of intestine and colon, while minority of 5-HT is distributed in the myenteric neurons that serves as neurotransmitter of the enteric nervous system (Crowell, 2004), synapsing with the ascending and descending interneurons, resulting in circular muscle contraction and relaxation to initiate peristaltic reflex, associated by the releasing of substance P, calcitonin gene-related peptide (CGRP), nitric oxide, vasoactive intestinal peptide, ATP, etc. (Pan and Gershon, 2000). However, available research data have not come to consensus about the importance of 5-HT in the pathophysiology of IBS. Moreover, results from different studies about the relationship between 5-HT and IBS were not consistent. One study showed that EC cell abundance is increased in the colon of patients with diarrhea and in patients with postdysenteric IBS (Spiller et al., 2000), and 5-HT concentration increased in the colon mucosa of constipation-predominant IBS patients compared with diarrhea-predominant IBS patients and subjects with normal bowel habit (Miwa et al., 2001). However, another study showed that mucosal 5-HT, tryptophan hydroxylase 1 mRNA, SERT mRNA and immunoreactivity are significantly reduced in both constipationpredominant IBS and diarrhea-predominant IBS patients, suggesting some intrinsic molecular defects of the bowel in IBS (Coates et al., 2004). In our study, data showed that 5-HT concentration in CVH rats was significantly higher than that of normal rats. This result indicates that 5-HT is involved in the mechanism of visceral hypersensitivity in the rat IBS model. After EA treatment, 5-HT concentration decreased while the pain threshold of CVH rats increased. WAS significantly elevated the 5-HT concentration accompanied with more fecal pellet output. These data indicate that 5-HT

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is also involved in the mechanism of increased susceptibility of colorectal motility to stress. The signaling of 5-HT is composed by secretion of 5-HT by EC cells as the first messenger, which acts on the 5-HTresponsive nerve fibers in the lamina propria, and then the response of the colon. Several 5-HT receptors involved in the process, and two major 5-HT receptors, 5-HT3 and 5-HT4, have already been targeted in this decade to develop new drugs, such as 5-HT3 receptor antagonist alosetron, and 5-HT4 receptor agonist tegaserod (Crowell, 2004). Data suggest that 5-HT4a is involved in the activation of peristaltic reflex in human, rat and guinea pig intestine, in which the submucosal IPANs involved are a calcitonin gene-related peptide neuron activated by 5-HT acting on 5-HT4 receptors (Grider et al., 1998). In contrast, 5-HT3 receptors activate extrinsic sensory nerves, thus mediate transmission of signals from the gut to the central nervous systems, and also expressing in myenteric IPANs, where intestinal motility can be slowed by interfering with serotonergic neurotransmission and blocking the initiation of reflexes within the enteric nervous system (Bertrand et al., 1998). Our results demonstrated that expression of 5-HT4a receptors in CVH rats is lower than that of normal rats in the colon mucosa, and EA increased its expression, thus the level of 5-HT4a is reciprocal to 5-HT concentration among the same groups. Acute stress such as WAS also influences the effect of EA treatment. The expression of 5-HT3 receptor that is also a potential marker was not tested in this study. After the release by enterochromaffin cells, serotonin is reuptaken from the mucosa into the nerve fibers, which is mediated by SERT in the enterocytes (Camilleri, 2002). Recently, it was hypothesized that, in at least a subset of IBS patients, a genetic defect in the SERT gene might be responsible for IBS symptoms. Indeed, a larger sample study suggested that within the female IBS population there is an increased association between diarrhea-predominant IBS and the SERT homozygous s/s genotype (Yeo et al., 2004). Our study showed that a slightly decreased SERT in CVH groups corresponding to an increased 5-HT concentration, indicating decreased 5-HT uptake, which is consistent to the previous study that showed mucosal SERT expression, is decreased in both constipation-predominant and diarrheapredominant IBS patients, in which SERT mRNA and protein expression significantly reduced, while no change of 5-HT release is observed (Crowell, 2004). Similarly, the potentiation of serotonergic signaling induced by the deletion of SERT gene can cause increase of extracellular 5-HT and protracted 5-HT clearance, resulting in the increased availability of 5-HT that can increase watery stool and motility in SERT knockout mice (Chen et al., 2001; Kim et al., 2005). Therefore, increased mucosal availability of 5-HT, which is caused by altered 5-HT synthesis and removal, might be related to the hypersensitivity and dysmotility of IBS patients. In conclusion, our present findings provide evidence for the inhibitory effect of EA on chronic visceral hypersensitivity. EA might be possible to modulate the availability of 5-HT to restore the balance between 5-HT synthesis and removal. These findings suggest the possibility of EA as a useful method for the treatment of IBS.

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4.

Experimental procedures

4.1.

Animals

All rats were obtained from the Experimental Animal Center, Chinese University of Hong Kong. Forty-two adult male Sprague–Dawley rats (250–300 g) were used; 30 rats received colon irritation as neonates and 12 rats served as controls. All studies were performed in accordance with the proposals of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (Zimmermann, 1983) and were approved by the Committee on the Use of Human and Animal Subjects in Teaching and Research, Hong Kong Baptist University.

4.2.

Study design

To examine the effect of EA on rats with CVH, neonatal rat pups were given daily mechanical colon distention beginning 8–21 days after their birth. After the distention, stimulus was terminated, the rats were kept until they reached adulthood (75th day, 250–300 g), and then experiments were conducted using behavioral testing or measurement to visceral pain induced by acute colorectal distention stimulus. EA was administered to CVH rats (n = 6) to detect whether EA can attenuate the visceral pain induced by noxious CRD. CVH rats (n = 6) without EA and normal rats (n = 6) were chosen as the control. The second set of experiments was to examine the effect of EA on WAS-induced colon transit in CVH rats. CVH rats (n = 6, 75th day, 250–300 g) were given EA for half hour first, and then WAS was administered for 1 h. The fecal pellet output was monitored to detect the colon transit. CVH rats (n = 6) with EA and WAS, normal rats (n = 6) without WAS and EA, and CVH rats plus WAS without EA (n = 6) were chosen as the control. After the detection of AWR and pain threshold pressure (PTP) in the first set and counting of fecal output in the second set, whole colon was harvested, and serotonin concentration in colon segment was assayed and SERT, 5-HT4a expression in colon was examined in two sets of experiments.

4.3.

Neonatal colon irritation

Neonatal rats of 8 days old received CRD on a daily basis between the ages of 8 and 21 days (the procedure was modified from previous reports (Saab et al., 2004; Al-Chaer et al., 2000). Briefly, angioplasty balloon (length 20.0 mm; diameter 3.0 mm) was inserted rectally into the descending colon at postnatal days 8 to 21. The balloon was distended with air, exerting a pressure of 60 mm Hg as measured with a sphygmomanometer for 1 min, and then deflated and withdrawn. The distention was repeated 2 times (separated by 30 min) within an hour.

4.4. Colon stimulation and testing of abdominal withdrawal reflex Colon stimulation in adult rats and the testing of related abdominal withdrawal reflex were measured with the proce-

dure modified from previous reports (Saab et al., 2004; AlChaer et al., 2000). Briefly, rats were lightly anesthetized with diethyl ether, and the balloon (4 cm in length, made of the finger of a latex glove) was inserted through the anus into the rectum and descending colon, attaching with a fogarty catheter. The open end of the balloon was secured to the catheter with thread and wrapped with tape (1 cm wide). Prior to use, the balloon was inflated and left overnight so that the latex stretched and the balloon became compliant. The balloon was inserted so that the thread was approximately 1 cm proximal to the anal sphincter, and was held in place by taping the tubing to the tail. The catheter was attached via a Tconnector to a sphygmomanometer pump and a pressure gauge. The rats were then housed in small Lucite cubicle (20 × 8 × 8 cm) on an elevated Plexiglas platform and allowed to wake up and adapt for 30 min. CRD was produced by rapidly inflating the balloon to the desired pressure (20, 40, 60 or 80 mm Hg) for a duration of 10 s. Stimuli were applied in an ascending graded manner (spaced by 4 min). To decrease the “human factor” bias to the minimum possible, the same stimulation paradigm was used in every rat. Abdominal withdrawal reflex (AWR), an involuntary motor reflex similar to the visceromotor reflex, was recorded. Measurements of the AWR by visual observation were reproduced by 2 blinded observers. The PTP is defined as the stimulus pressure that evokes a visually identifiable contraction of the abdominal wall. Increasing pressure was applied in steps of 5 mm Hg lasting 30 s until pain behavior was displayed or until 80 mm Hg was reached in order to avoid invincible damage to the animal.

4.5.

EA treatment

Prior to the EA treatment, rats were kept in small Lucite cubicle (12 × 5 × 5 cm). EA was applied by two pairs of stainless steel needles (0.25 mm in diameter) inserted bilaterally at a depth of approximate 5 mm into two acupoints, in the Traditional Chinese medicine, ST-36 (5 mm lateral to the anterior tubercle of the tibia and 10 mm below the knee joint) and SP-6 (3 mm proximal to the medial melleolus, at the posterior border of the tibia) of each hind leg. Each pair of needles (one in ST-36 and the other in SP-6) was connected with the output terminals of an EA apparatus (Model G-68052, Shanghai Medical Electronic Apparatus Company, P. R. China) was selected. Stimulation with continuous wave of 2 Hz (the intensity 0.3 mA) was applied and lasted for 30 min each time.

4.6.

Water-avoidance stress (WAS)

The water avoidance stress (WAS) was conducted with the procedures modified from previous reports (Million et al., 2003; Bonaz and Tache, 1994). Briefly, the animals were placed on the plastic platform (8 × 6 × 6 cm), which was fixed in the center of tank filled with flesh tap water up to 7 cm (within 1 cm of the top of the block). The animals were placed on the block for a period of 1 h. Stress sessions were performed between 10:00 AM and 13:00 PM to minimize diurnal variations in response. The fecal pellet output was countered every 15 min over 60 min.

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4.7.

Detection of 5-HT concentration

5-HT content of colon was assayed with the procedure modified from previous report (Farmer and Laniyonu, 1984). Briefly, the whole colon was harvested and one segment of colon (5 cm in length) was homogenized in glass tube in 5 ml of acidified butanol (850 μl HCl in 1 l butanol) by a motordriven pestle (Ultra-Turrax T8, IKA-Werke, Germany). The volume of each homogenate was adjusted to 25 ml and centrifuged at 3000 × g, 4 °C for 10 min. A 2 ml aliquot of each supernatant was transferred to a tube containing 5 ml of Nheptane and 600 μl of acidic cysteine (0.1% w/v in 0.1 M HCl); the contents of each tube were mixed for 2 min at 4 °C and centrifuged at 3000×g for 5 min. From each tube the upper, organic phase together with the disc at the organic/aqueous inter-phase was removed by aspiration and discarded. A 200 μl aliquot of the lower aqueous phase was incubated at 77 °C for 15 min with 20 μl 1% cysteine and 800 μl conc. HCL containing 0.004% o-phthalaldehyde. When the tube was cooled to room temperature, the fluorescence that developed was measured in an Aminco-Bowman spectro-photo-fluorimeter at an activation wavelength of 365 nm, emission wavelength 470 nm. Standards were prepared by dissolving 5-HT in the distilled water such that 200 μl volumes containing 50–200 ng 5-HT could be added to tissue extracts to serve as the internal standards. These were prepared throughout the assay procedure.

4.8.

SERT and 5-HT4a Imunohistochemistry

After the measurement of AWR and PTP, rats were deeply anesthetized with hydrate chloride (7%, i.p. at 0.5 ml/100 g, Uni-chem.) and perfused via the ascending aorta with 50 ml of normal saline, followed by 500 ml ice-cold 4% paraformaldehyde in 0.1 M PBS (pH 7.4, Sigma). The Lumbosacral spinal cord (L6-S1) was dissected out and post-fixed in the same fixative overnight and cryoprotected by immersion in 10% sucrose for 1 day (data not reported in this paper). The colon section was processed with ABC techniques with slight modifications. At first, section was deparaffined to water, and then endogenous peroxidase was quenched in methanol: 30%H2O2 (9:1) for 10 min, and microwaved for 10 min in citrate buffer (10 mM, pH 6.0) for antigen retrieval. Histomouse-Max Kit (Zymed Laboratories, Inc) was applied for immunohistochemistry. Sections were incubated overnight at 4 °C with mouse anti-rat monoclonal IgG to SERT (Advanced Targeting Systems, San Diego, USA) at 1:500 dilution, and rabbit anti-rat polyclonal IgG for 5-HT4a (Oncogene, Merck, Darmstadt, Germany) at 1:500 dilution separately, followed by incubation with 2nd antibody-peroxidase conjugate for 10 min. PBS was used for washing after each step. Subsequent visualization used diaminobenzidine (DAB) as chromogen. The 1st antibody was omitted on one section of each reaction as a negative control. The number of SERT or 5-HT4a immunoreactive cells in colon was observed using bright field microscopy at the magnification of 10 × 20. Immunoreactive cells were captured by Axiotron microscope (Zeiss, Germany) and a 3CCD KY-F30B camera (JVC, Yokohama, Japan). Three areas were randomly selected the mucosa of each specimen and gray levels were

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counted and processed by IBAS 2.0 automatic image analysis system (Kontron, Germany).

4.9.

Data analysis

Results are expressed as means ± SE unless otherwise stated. Groups were compared with ANOVA or unpaired t test as applicable. Differences with P < 0.05 were considered significant.

Acknowledgment This study was supported by Faculty research grants, Hong Kong Baptist University (FRG/03-04/II-79).

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