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Original Experimental Research
Journal of Chinese Integrative Medicine: Volume 10, 2012   Issue 4
Effects of electroacupuncture on expression of c-fos protein and N-methyl-D-aspartate receptor 1 in the rostral ventromedia medulla of rats with chronic visceral hyperalgesia
1. De-bo Qi (Laboratory of Neural Network and Systems Biology, Shanghai Medical College, Fudan University, Shanghai 200032, China )
2. Wei-min Li (Laboratory of Neural Network and Systems Biology, Shanghai Medical College, Fudan University, Shanghai 200032, China E-mail: E-mail: wmli9110@fudan.edu.cn)
OBJECTIVE: Acupuncture has been clinically proved to be effective in treating abdominal pain in patients with irritable bowel syndrome (IBS). However, its neurobiological mechanism remains largely unexplored. The aim of this study was to investigate the effect of electroacupuncture (EA) in relieving chronic visceral hyperalgesia and the possible involvement of N-methyl-D-aspartate receptor 1 (NR1) in rostral ventromedia medulla (RVM) of the brain in an IBS rat model.
METHODS: To establish the IBS rat model, male Sprague-Dawley neonatal rats received colon mechanical irritation on a daily basis from the 9th to the 22nd day after their birth. After a resting period of another two to four weeks, behavioral tests of pain threshold pressure (PTP) and abdominal withdrawal reflex (AWR) responding to colorectal distention (CRD) stimuli were conducted to judge the colorectal sensitive situation. Then administration of EA at acupoints of Zusanli (ST36) and Shangjuxu (ST37) bilaterally in the hind limbs was repeated four times every other day, while sham-EA was done by inserting needles at similar acupoints without electrical stimulation. Immunohistochemical method was used to display the expression of proto-oncogene protein c-fos and NR1 in RVM of rats.
RESULTS: The results demonstrated that the PTP values and AWR scores, in response to the CRD stimuli, significantly decreased and increased, respectively (P<0.01, P<0.01), while the number of immunoreactive neurons of c-fos protein and NR1 significantly increased in nucleus reticularis gigantocellularis (Gi), nucleus lateralis paragigantocellulari (LPGi), nucleus reticularis gigantocellularis pars alpha (GiA) and nucleus raphe magnus (NRM) of RVM in IBS model rats compared with the normal rats (P<0.05). After EA treatment, PTP values and AWR scores significantly increased and decreased, respectively (P<0.01, P<0.05); the number of immunoreactive neurons of c-fos and NR1 significantly decreased respectively in Gi, LPGi and GiA and in Gi, LPGi, GiA and NRM (P<0.05). No such effects on PTP values, AWR scores and the number of immunoreactive neurons of c-fos and NR1 were observed after sham-EA treatment.
CONCLUSION: These data provide the evidence that EA can relieve chronic visceral hyperalgesia in rats with IBS, and such an effect may be correlated with inhibitory modulation of hyperactivity of neurons by means of down-regulating the high expression of NR1 in RVM of IBS model rats.

Received December 5, 2011; accepted December 27, 2011; published online April 15, 2012.
Full-text LinkOut at PubMed. Journal title in PubMed: Zhong Xi Yi Jie He Xue Bao.

基金项目:This study was supported by a program to facilitate clinical implementation of traditional Chinese medicine from Canada
Correspondence: Wei-min Li, MD, PhD, Professor; E-mail: wmli9110@fudan.edu.cn

  

     Visceral hypersensitivity is the main mechanism in patients who suffer from the irritable bowel syndrome (IBS) with chronic visceral hyperalgesia[1,2], while the recurrent occurrence of visceral pain is a major cause of patients seeking for medical treatment[3,4]. The lack of understanding of visceral pain has hindered specific treatment choices, and caused unsatisfactory effect and high cost of treatment consequently[5,6].
     Acupuncture, an alternative medicine, has certain curative effect and fewer side effects compared with pharmacotherapy, which might be beneficial to patients with chronic visceral pain[7,8], although a few of acupuncture trials demonstrated no efficacy or minimal superiority over the placebo control in treatment of patients or animal models[9,10].
     It has been already known that rostral ventromedia medulla (RVM) has a dual role for descending modulation of pain sensation or nociception by means of inhibiting[11-13] or facilitating[11-15] the visceral sensory input. The facilitory transmission of pain via the RVM has been implicated in the development of central sensitization; therefore, it is supposed that the disordered descending influences from the brainstem may underlie abnormal pain perception in functional pain disorders such as IBS, fibromyalgia and neuropathic pain[16].
     Al-Chaer et al[17] developed a rat model of IBS characterized by chronic visceral hyperalgesia through repetitive neonatal colorectal distention (CRD). Our previous reserch has reported that there is an analgesic effect of electroacupuncture (EA) on chronic visceral hyperalgesia in rats and the involvement of N-methyl-D-aspartate receptor 1 (NR1) in the central nervous system may underly such an effect of EA[18,19]. It is known that the glutamate neurotransmitter system in RVM is closely involved in the formation and development of visceral pain[11,12,14,15], and proto-oncogene protein c-fos has been widely used as a marker of the increased neural activity in central nervous system[20]. Therefore, c-fos and NR1 were chosen to investigate the specific response of neurons in the RVM, and to clarify the mechanism of EA in relieving chronic visceral hyperalgesia in this study.

 
  


1  Materials and methods
1.1  Animals 
Thirty-two male Sprague-Dawley neonatal rats (younger than 8 d) were obtained from the Experiment Animal Center, Chinese Academy of Sciences Shanghai Branch, China. Rats were housed in plastic cages in such a way: 10 male neonates were housed with a nursing adult female rat until they were 25 d old; the adult female rat had access to food and water ad libitum. After separating from the adult female rat, every four weaned rats were housed in one cage with accessing to food and water ad libitum. All rats in the study were used strictly in accordance with the National Institutions of Health Guide for the Care and Use of Laboratory Animals in order to minimize the number of animals used and their suffering.
1.2  Establishment of the IBS rat model  The IBS model was established as our previous reports[18,19]. Daily mechanical CRD was performed on neonatal rats (n=24) during 9 to 22 d after birth. The distention was applied using silica gel balloons (20.0 mm in length; 2.0 mm in diameter) inserted into the descending colon through the rectum of the awaked rats. The balloon was distended with 0.35 mL of air for 1 min and then deflated and withdrawn. The distention was repeated twice a day at a 30 min interval. After the distention stimulus was terminated, the rat was kept until it reached adulthood (at least six weeks old) and then experiments were conducted using behavioral test of visceral pain detection. Another eight neonatal rats were raised together with the IBS model rats; only their perianal skin was gently kneaded by balloons and served as the normal control group.
1.3  Assessment of abdominal withdrawal reflex  Behavioral responses to CRD were assessed when the rats were older than six weeks by observing the abdominal withdrawal reflex (AWR). Semi-quantitative scores were used for the judgment of the responses to CRD stimulus. After inserting a balloon into the descending colon, the rats were placed in a small cubicle (20 cm×8 cm×8 cm) on a platform and allowed to adapt for 20 min. Measurement of AWR responded to CRD at strengths of 20, 40, 60, and 80 mmHg, respectively. AWR scores were assigned according to the scale of Al-Chaer et al[17] and our improved method[18]: 0 means no behavioral response to CRD; 1 means immobile during the CRD at the onset of the stimulus; 2 means a mild contraction of the abdominal muscles, but not lifting the abdomen off the platform; 3 means a strong contraction of the abdominal muscles and lifting the abdomen off the platform, but not lifting the pelvic structure off the platform; 4 means body arching and lifting the pelvic structure and scrotum.
1.4  Evaluation of pain threshold pressure  Preparation before the test of pain threshold pressure (PTP) was performed as previously described to detect the AWR score. Briefly, CRD was applied in increments of 5 mmHg at an interval of 5 s until a visible contraction of the abdominal wall was observed by the investigator, then the pressure applied was immediately released. PTP of CRD eliciting an observable AWR of all groups was repeated for five times with at least 5-min intervals for recovering.
1.5  Administration of EA treatment  The IBS model rats were randomly divided into three groups: IBS model control group without any treatment, group of IBS model rats with EA treatment and group of IBS model rats with sham-EA treatment (n=8). EA was applied by two pairs of stainless steel needles (0.25 mm in diameter) inserted bilaterally at a depth of 5 mm into two acupoints, Zusanli (ST36, 5 mm lateral to the anterior tubercle of the tibia and 10 mm below the knee joint) and Shangjuxu (ST37, 5 mm lateral to the anterior tubercle of the tibia and 15 mm below the knee joint), of each hind limb. Each pair of needles (one in ST36 and the other in ST37) was connected with the output terminals of an EA apparatus (Model SDZ-IV, Suzhou Medical Appliance Factory, China). Alternating trains of dense to sparse frequencies (5 to 100 Hz; sparse wave time was 5 s; dense wave time was 10 s alternately) were selected. The intensity of stimulation was appropriate with mild shakes in hind limbs. Repeated EA treatments were applied to IBS model rats for 30 min every other day and lasted for 8 d. In the sham-EA group, needles were inserted bilaterally into the acupoints similar to those in the EA treatment and kept without electrical stimulation, while the IBS model control group received no applications. There was no treatment applied to normal control rats either. As revealed in our previous report[18], repeated EA treatment gradually enhanced to its maximum within 8 to 12 d, and AWR assessment was performed during 24 h after four consecutive EA treatments.
1.6  Tissue preparation  After 5 to 8 h of the CRD stimulation, distended rats were deeply anesthetized with pentobarbital and intracardially perfused with 350 mL saline followed by 400 mL of 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) at 4 ℃. The RVM was sliced at the rostral end of the Ⅳ ventricle[21] (Figure 1), and the post-fixed slice was put in 4% paraformaldehyde overnight. After subsequently allowed to equilibrate in 30% sucrose with PB for 48 h, 30 μm transverse sections were cut on a cryostat.

Figure 1  RVM in the brainstem sliced at the rostral end of the Ⅳ ventricle

RVM: rostral ventromedia medulla; Gi: nucleus reticularis gigantocellularis; NRM: nucleus raphe magnus; GiA: nucleus reticularis gigantocellularis pars alpha; LPGi: nucleus lateralis paragigantocellulari; 4V: the Ⅳ ventricle; Sol: solitary tract; Sp5: spinal trigeminal tract; Py: pyramidal tract; RPa: raphe pallidus nucleus; ml: medial lemniscus; 7n: facial nucleus.

1.7  c-fos and NR1 immuohistochemistry  The RVM sections were stained for investigating the c-fos and NR1 by strept-avidin-biotin complex (SABC) method. Free floating sections were treated in methanol containing 3% hydrogen peroxide to block the endogenous peroxidase activity for 10 min at room temperature. Then sections were blocked with 10% normal goat serum in phosphate buffered saline (PBS) for 30 min at room temperature followed by incubation with rabbit polyclonal antibody solutions against c-fos (1∶400 in PBS containing 0.2% Triton X-100, Wuhan Boster Bio-Engineering Limited Company, China) or NR1 (1∶100 in PBS, Wuhan Boster Bio-Engineering Limited Company, China) for 48 h at 4 ℃. Following primary antibody incubation, the sections were incubated for 30 min with a biotinylated secondary antibody, Biotin-Goat Anti-Rabbit IgG (1∶100 in PBS, Wuhan Boster Bio-Engineering Limited Company, China), at room temperature. Following incubation in a solution containing avidin-biotin complex (SABC kit, Wuhan Boster Bio-Engineering Limited Company, China) for 30 min at room temperature and subsequent reaction with diaminobenzidine for 4 min, sections were mounted on gelatin-coated slides, and then dehydrated in a series of graded alcohol and coverslipped. The sections were rinsed in 0.01 mol/L PBS for 35 min between the transitions of these steps. To measure the levels of intensity of c-fos and NR1 immunoreactivity, five slices for each rat were selected for the count of c-fos- and NR1-positive neurons by images at 25 magnifications.
1.8  Statistical analysis  Data were presented as mean±standard error of mean and analyzed by the statistical software SPSS, version 13.0. Differences between two groups were analyzed by independent sample t-test. Differences between before and after treatment were analyzed by paired t-test. Data was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons. P<0.05 implied statistical significance.

  

2  Results
2.1  PTP and AWR score of the IBS model rats 
By evaluating the colorectal sensitivity of the IBS model rats in response to CRD stimulation, it was found that the PTP values in IBS model rats were significantly decreased compared with the normal rats (P<0.01) (Figure 2). Parallel to the PTP changes, with graded CRD stimulation, AWR scores were significantly increased in IBS model rats compared to those in the normal rats (P<0.01) (Table 1).

Figure 2  PTP values measured in response to continuous colorectal distention

Data are represented as mean±standard error of mean, n=8; **P<0.01, vs normal control group; △△P<0.01, vs IBS model control group. PTP: pain threshold pressure; IBS: irritable bowel syndrome; EA: electroacupuncture.

Table 1  AWR score for evaluating colorectal sensitivity of the normal and IBS model rats

(Mean±standard error of mean)

Group n AWR score
20 mmHg 40 mmHg 60 mmHg 80 mmHg

Normal control

8

0.30±0.09

1.39±0.12

2.26±0.14

3.02±0.22

IBS model control

8

1.24±0.18**

2.17±0.17**

3.04±0.11**

3.78±0.08**

**P<0.01, vs normal control group. AWR: abdominal withdrawal reflex; IBS: irritable bowel syndrome.

2.2  EA reversed abnormal PTP values and AWR scores of the IBS rats  The second step was to observe the effect of EA treatment on those changed PTP values and AWR scores in response to CRD in IBS rats. After four consecutive EA treatments to the IBS rats, PTP values were significantly increased compared with IBS rats without EA treatment (P<0.01) (Figure 2). Parallel to the PTP changes, AWR scores of the EA-treated IBS rats were significantly decreased, when compared to those before EA treatment with distention pressures (P<0.05) (Table 2). There was no significant decrease in AWR scores of sham EA-treated rats compared to those before sham EA treatment.

Table 2  AWR score before and after EA treatment

(Mean±standard error of mean)

Group n AWR score
20 mmHg 40 mmHg 60 mmHg 80 mmHg
EA treatment          
    Before 8 1.27±0.21 2.21±0.19 2.98±0.11 3.77±0.10
    After 8 0.33±0.09▲□ 1.36±0.10▲□ 2.25±0.11▲□ 2.88±0.10▲□
Sham-EA treatment          

    Before

8

1.29±0.08

2.27±0.15

2.81±0.09

3.75±0.09

    After

8

0.69±0.10

1.87±0.11

2.48±0.13

3.15±0.21

P<0.05, vs before EA treatment; P<0.05, vs after sham-EA treatment. AWR: abdominal withdrawal reflex; EA: electroacupuncture.


2.3  EA treatment reduced hyperexpression of c-fos protein in RVM of the IBS rats  The third step was to observe the change in expression of proto-oncogene protein c-fos in the subnuclei of IBS rat’s RVM. RVM includes four subnuclei, namely, nucleus reticularis gigantocellularis (Gi), nucleus raphe magnus (NRM), nucleus reticularis gigantocellularis pars alpha (GiA), and nucleus lateralis paragigantocellulari (LPGi). Figures 3A to 3D show sample images of c-fos immunoreactive neurons in RVM in each tested group and Table 3 summarizes the data obtained from those images of each group, respectively. The numbers of c-fos-immunoreactive neurons in the Gi, LPGi, GiA and NRM of IBS model rats were significantly increased compared to those of normal rats (P<0.05). After four consecutive EA treatment, c-fos-positive neurons significantly decreased (P<0.05). There was no difference observed in the NRM of RVM (Table 3). Similarly to the result of behavioral test, there was no inhibitory effect on hyperexpression of c-fos protein after sham EA treatment.

Figure 3  Samples of microphotographs with the c-fos- (A-D) and NR1- (E-H) immunoreactive neurons of the RVM in different groups (Light microscopy, ×25)

The images of immunohistochemical results were taken under the light microscopy. The arrows show c-fos- or NR1-immunoreactive neurons. N: Normal control group; M: IBS model control group; EA: IBS model rats after EA treament; SEA: IBS model rats after sham-EA treatment; RVM: rostral ventromedia medulla; NR1: N-methyl-D-aspartate receptor 1; IBS: irritable bowel syndrome; EA: electroacupuncture.

Table 3  Number of immunoreactive neurons of c-fos in the subnuclei of RVM

(Mean±standard error of mean)

Group n c-fos
Gi LPGi GiA NRM

Normal control

8

34.62±2.02

29.13±1.25

9.52±0.80

3.23±0.45

IBS model control

8

59.66±3.00*

37.34±1.95*

13.50±1.53*

4.84±0.76*

EA treatment

8

40.84±2.28

30.18±1.51

9.12±1.20

2.68±0.56

Sham-EA treatment

8

50.70±2.52

35.76±1.98

13.86±1.33

2.46±0.34

*P<0.05, vs normal control group; P<0.05, vs IBS model group. EA: electroacupuncture; RVM: rostral ventromedia medulla; IBS: irritable bowel syndrome; Gi: nucleus reticularis gigantocellularis; NRM: nucleus raphe magnus; GiA: nucleus reticularis gigantocellularis pars alpha; LPGi: nucleus lateralis paragigantocellulari.


2.4  EA reduced hyperexpression of NR1 receptor in RVM of the IBS rats  By investigating the involvement of NR1 in RVM of IBS rats’ brain, the results revealed the significant differences in the number of NR1-positive neurons in the subnuclei of RVM among different groups. Figures 3E to 3H show sample images of NR1-immunostaining neurons in the RVM of each group and Table 4 summarizes the data obtained from those images of each group. The NR1-positive neurons of IBS model rats were significantly increased when compared to those in the Gi, LPGi, GiA and NRM of normal rats. However, NR1-positive neurons were reversed to normal levels after EA treatment (P<0.05) (Table 4). Compared with IBS model rats, there was no significant immunostaining change in NR1 after sham-EA treatment.

Table 4  Number of immunoreactive neurons of NR1 in the subnuclei of RVM

(Mean±standard error of mean)

Group n NR1
Gi LPGi GiA NRM

Normal control

8

23.05±1.75

14.28±1.28

4.85±0.75

1.73±0.31

IBS model control

8

36.44±1.99*

21.15±1.45*

8.88±0.98*

3.00±0.50*

EA treatment

8

24.03±1.38

13.93±1.20

3.23±0.61

1.08±0.28

Sham-EA treatment

8

36.45±1.40

24.20±1.50

8.10±0.83

3.08±0.48

*P<0.05, vs normal control group; P<0.05, vs IBS model control group. EA: electroacupuncture; NR1: N-methyl-D-aspartate receptor 1; RVM: rostral ventromedia medulla; Gi: nucleus reticularis gigantocellularis; NRM: nucleus raphe magnus; GiA: nucleus reticularis gigantocellularis pars alpha; LPGi: nucleus lateralis paragigantocellulari.

  


3  Discussion
    
The main clinical features of IBS are onset of chronic intermittent abdominal pain and changes in bowel habits with the lack of obviously abnormal changes in morphology and laboratory indicators. Along with sexual, physical, or verbal abuse in childhood, it might result in sensitization of the central nervous system at a vulnerable state to develop functional abdominal pain in adulthood[22]. Irritating to neonatal rats by factors such as colorectal inflammation[17,23], mechanical expansion[17-19], maternal separation[24], can lead to the visceral hyperalgesia in their adulthood. In this study, it was found that the pain threshold of IBS rats decreased significantly according to PTP data, and the AWR scores of IBS rats were significantly higher than those of normal rats with not only noxious (40, 60 and 80 mmHg) but also innoxious (20 mmHg ) CRD stimulation. Consistent with other reports[17,18], these results suggest that rats received colon mechanical irritation at early stage of the birth can result in visceral allodynia and visceral hyperalgesia in their adulthood.
    There is considerable evidence suggesting that the descending facilitatory influences from RVM are important to the maintenance of visceral hyperalgesia and the activation of NR in RVM plays a key role in such influences in animal models of visceral pain[11,12,14,15]. Micro-injection of L-glutamate or N-methyl-D-aspartate (NMDA), the agonist of NR, into the RVM facilitated the responses of spinal neurons and visceral motor reflex to CRD in normal rats[11,12,15], while micro-injection of DL-2-amino-5-phosphonovaleric acid[14], a selective NMDA antagonist, or MK-801[15], a non-selective antagonist, into RVM, respectively attenuated the visceral hyperalgesia in a rat model of visceral hyperalgesia induced by colonic instillation of zymosan. In this study, the result of c-fos immunostaining revealed that there were more neurons activated and more NR1 immunoreactive neurons recruited in the RVM of IBS rats with a situation of visceral pain as compared with the normal control rats. This may reflect a fact that abnormally high neuronal excitability, especially the increased NR1 expression in the RVM, may be the important reason underlying visceral hyperalgesia in IBS model rats.
     Invasive sham treatment was employed similarly in our previous report[18], because it enabled better blinding for investigators. After EA treatment, it was found that PTP values and AWR scores of IBS rats with EA treatment significantly decreased and increased, respectively, while sham-EA could not produce the similar effects. These results prove that selection of invasive sham EA treatment is suitable. It was also found that abnormally high neuronal excitability especially the NR1-immunoreactive neurons in RVM of IBS model rats was suppressed by EA but not sham-EA treatment. It suggests that the above changes in RVM after EA treatment maybe one of the mechanisms underlying the genuine effects rather than diffuse noxious inhibitory control effects produced by sham-EA.
     In conclusion, data of the present study suggest that EA treatment can relieve chronic visceral hyperalgesia in IBS rats, and such an effect might be correlated with inhibitory modulation of hyperactivity of neurons by means of down-regulating the high expression of NR1 in the RVM of the rats’ brain.

  


4  Competing interests
    
The authors declare that they have no competing interests.

  
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