The digestive system (DS) consists of the upper digestive tract and the lower digestive tract. The upper digestive tract is mainly regulated by the vagus nervous system, especially the vagovagal reflex [
Traditional body surface-stimulating therapies such as acupuncture and moxibustion are widely used to treat gastrointestinal disorders [
In the study, we hypothesized EA at Neiguan (PC6) influences gastric motility via brainstem vagovagal neurocircuits. To test this hypothesis, we measured intragastric pressure in vagus nerve and splanchnic nerve excision model to observe the role of autonomic nerve pathway of EA at PC6. We also measured the activity of the autonomic nerve using electrophysiology. Finally, we studied the brainstem vagovagal neurocircuits by microinjecting GABA, L-Glu into DMV; then, we investigated the intragastric pressure and vagus nerve activity.
Sprague-Dawley rats (male, 250–300 g; Model Animal Research Center of Nanjing Medical University, China) were used in this study. All animals were housed under controlled environmental conditions (22°C, 40–60% relative humidity, 12/12 h light/dark cycle) and were given free access to water and food. All animals were allowed 2 weeks of feeding adaptation. All experimental manipulations were undertaken in accordance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, published by the National Science Council, China.
Among the drugs used in the experiments, urethane (U2500; Sigma, St. Louis, MO, USA) was intraperitoneally injected to anesthetized rats, and L-Glu (G1251-100G; Sigma), GABA (A2129-10G; Sigma), and artificial cerebrospinal fluid (R22153; Yuan Ye Biological Co., Ltd., Shanghai, China) were administered via microinjection prior to surgery, whereas penicillin (2011; Shandong Shengwang Pharmaceutical Co., Ltd., Shandong, China) was administered after the surgery.
All rats in the study underwent fasting for 12 h, but were given free access to water. A small incision (approximately 1 cm) was made under the xiphoid process. A small balloon (approximately 2 mm in diameter) made of flexible rubber was inserted into the duodenum and placed in the gastric antrum. The balloon was filled with warm water (0.05–0.1 ml), to keep the pressure at about 0.1KPa H2O.
Pressure in the balloon was measured by a transducer (YP201; Chengdu Instrument Factory, Chengdu) through a polyethylene tube and then transmitted into a multipurpose polygraph (RM6240; Chengdu Instrument Factory). The signal was captured by RM6240 software. An electric heating board was used to maintain the animals’ body temperature at 37°C ± 0.5°C during the test. At the end of experiments, the animals were killed with an overdose of urethane.
To examine nerve discharge, rats were anesthetized with 8 mL/kg of 20% urethane intraperitoneally, an incision was made in the midline of the abdomen, and the posterior branch of gastric vagus nerve or the left splanchnic nerve was identified and isolated. The positive electrode was kept in contact with the nerve, and the negative electrode was kept in contact with the surrounding tissue. The liquid paraffin wax, preheated to 37°C, was used to cover the nerve to protect it. Nerve discharges were recorded using a preamplifier (NL100; CED, UK) and Micro1401-3 Bioelectric Module (NL125NL126; CED, UK), both of which were connected to biological signal acquisition and analysis system (Microl1401-3; CED, UK). Spike2 software was used to analyze the nerve discharge data. Discharge during acupuncture was compared to that before the treatment. A difference of >5% in these values was considered as an increase in nerve discharge. Equation (
For vagotomy, rats were anesthetized with 8 mL/kg of 20% urethane intraperitoneally, an incision was made in the midline of the abdomen, and parasympathetic nerves on both sides were dissected. For sympathectomy, rats were anesthetized with 8 mL/kg of 20% urethane intraperitoneally, an incision was made in the midline of the abdomen, and the left greater splanchnic nerve was dissected. The animals in the sham control group underwent the same surgical procedure, but without nerve dissection. After surgery, the animals received penicillin (0.2 mL/d of 800 IU penicillin in 5 mL saline per rat, intramuscular) and were allowed 3 days of postoperative recovery.
The animals were placed on a stereotaxic apparatus (Kopf Instruments, US) and kept in the prone position. The location of the dorsal nucleus of the vagus nerve was determined using Paxinos and Watson rat brain mapping (AP 13.8 mm, RL 0.5 mm, H 7.8 mm) [
At the end of the experiment, the rats were microinjected with pontamine sky blue (0.2
PC6 (Neiguan) is located between the palmar tendon and flexor carpi ulnaris. A pair of stainless-steel acupuncture needles (diameter, 0.3 mm) were inserted to a depth of 3 mm into the muscle layer at the left PC6. The needles were connected to Han’s EA instrument (LH402A; Beijing Huawei Co., Ltd., China). The stimulating intensity was 2 mA, the frequency was 2/15 Hz, and stimulation time was 2 min.
Since EA interferes with nerve discharge, hand stimulation was used for the nerve discharge experiments. The frequency of stimulation was 2–3 Hz, and the stimulation time was 2 min.
The intragastric pressure during EA was compared with that before needle insertion. If the percentage difference in the intragastric pressure before and after needle insertion was >5%, gastric motility was considered to have increased. Similarly, if the percentage difference in intragastric pressure before and after drug microinjection into the dorsal nucleus of the vagus nerve was >5%, gastric motility was considered to have increased. Equations (
In the first experiment, we recorded the gastric pressure to describe the characteristics of gastric motility during EA. The rats were divided into two groups (parasympathetic nerve discharge and sympathetic nerve discharge), and the nerve discharges were measured during EA.
In the second experiment, the rats were divided into four groups (control, sham control, sympathectomy, and vagotomy groups). We first cut the gastric vagal nerves of the rats and recorded gastric pressure during EA to evaluate the role of the parasympathetic pathway. Then, we cut the splenic greater splanchnic nerve and recorded gastric pressure during EA to evaluate the role of the sympathetic pathway in different rats.
In the last experiment, we divided animals into three groups (CSF group, L-Glu group, and GABA group) and microinjected CSF, L-Glu, and GABA into DMV. Then we measured the gastric pressure and parasympathetic discharge used EA PC6 to explore the mechanism of DMV pathway. There were 8 animals per group in all experiments.
Data were analyzed using SPSS 23.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Any 2 groups were compared using the independent sample
The gastric motility was measured by intragastric pressure in anesthetized rats. The pressure was maintained at approximately 0.1kPa as baseline by expanding the volume of the balloon with warm water, and rhythmic contractions were recorded at a rate of 4–6/min with 0.2–0.3 kPa in amplitude.
To investigate the effect of EA at PC6 on gastric motility, the intragastric pressure was recorded at different time points. The results showed that EA at PC6 can increase gastric motility; in fact, after 30 s of EA stimulation at PC6, the gastric motility was significantly different as compared to that before needle insertion (
Effect of EA at PC6 on gastric motility. (a) Wave patterns of gastric motility changes in Sprague-Dawley rats that received EA stimulation at PC6. (b) Changes in the intragastric pressure in Sprague-Dawley rats that received EA stimulation at PC6 at different time intervals of stimulation (60 s before EA, and after EA for 30 s, 30–60 s, 60–90 s, and 90–120 s).
The discharge from the parasympathetic and sympathetic nerves before and after EA was examined to assess the effects of acupuncture on the autonomic nerves. The results showed that parasympathetic nerve discharge after EA at PC6 was significantly higher than that before needle insertion, although there was no significant increase in splanchnic nerve discharge (n = 8; Figures
Effect of EA at PC6 on autonomic nerve discharge. (a) Percentage increase of parasympathetic nerve discharge and sympathetic discharge during EA.
To investigate whether EA at PC6 affects gastric motility via the sympathetic pathway or the parasympathetic pathway, the left splanchnic nerve and the parasympathetic nerves on both sides were severed in the rats. The results showed a significant increase in gastric motility in the control, sham control, and sympathectomy groups during EA, but no significant increase in gastric motility in the vagotomy group. The percentage increase in gastric motility via EA decreased significantly in the vagotomy group and differed significantly between the control and sham groups (P < 0.05). However, there are no differences among the control, sham control, and the sympathectomy groups (P>0.05). Furthermore, the baseline intragastric pressure reduced significantly after vagotomy. These results indicate that the parasympathetic pathway but not the sympathetic pathway plays an important role in the effect of EA at PC6.
To further study the relationship between the central nervous system and the vagal nerve pathways, L-Glu and GABA were microinjected into the DMV, and the consequent changes in gastric motility were analyzed. The results showed that the gastric motility increased after L-Glu microinjection, but decreased after GABA microinjection (n = 8; Figure
To confirm the effects of the drugs, artificial cerebrospinal fluid was microinjected into the DMV in the control group. The results showed no significant difference in gastric motility before and after treatment in this group.
Furthermore, we examined the effects of EA at PC6 on rats that received the drug microinjections. L-Glu and GABA were microinjected separately into the DMV. The results showed that EA at PC6 significantly reverse the inhibition of gastric motility by microinjection GABA into DMV (
Based on above data, GABA reverses the efficiency of EA at PC6. Therefore, we determined to verify the output of DMV by EA at PC6. The results showed that microinjection GABA in DMV inhibited vagus nerve discharge, and EA at PC6 promoted vagus nerve discharge (n = 8; Figure
EA therapy has been promoted by the World Health Organization (WHO) and National Institutes of Health (NIH) as a surface stimulation therapy [
Effect of EA at PC6 on gastric motility via the sympathetic and the parasympathetic pathway. (a) Gastric motility waveforms in the control, sham control, sympathectomy, and vagotomy groups induced by EA at PC6. (b) Percentage increase in gastric motility in the control, sham control, sympathectomy, and vagotomy groups induced by EA at PC6.
Increase/decrease in gastric motility after the microinjection of different drugs into the dorsal nucleus of the vagus nerve. (a) Wave patterns of gastric motility before and after the injection of artificial cerebrospinal fluid, L-Glu, and GABA into the dorsal nucleus of the vagus nerve. (b) Increase/decrease in gastric motility after the microinjection of artificial cerebrospinal fluid, L-Glu, and GABA into the dorsal nucleus of the vagus nerve. Percentage reduction,
Gastrointestinal movement is mainly dominated by vagus nerve regulation, and brainstem vagovagal neurocircuits are crucial for the regulation of the upper digestive tract [
To investigate the effect of EA at PC6 in the brainstem vagovagal neurocircuits, the rats in the present study that received L-Glu and GABA microinjections into the DMV also received EA at PC6. As observed in Figure
Effect of EA at PC6 on gastric motility after the microinjection of different drugs into the dorsal nucleus of the vagus nerve. (a) Artificial cerebrospinal fluid, L-glutamic acid, and GABA were microinjected to the dorsal nucleus of the vagus nerve, and the wave patterns of gastric motility during EA at PC6 were recorded. (b) Increase in intragastric pressure induced by EA at PC6 after the microinjection of artificial cerebrospinal fluid, L-Glu, and GABA into the dorsal nucleus of the vagus nerve.
Effect of EA at PC6 on vagus nerve discharge via microinjection GABA in DMV. (a) Percentage increase of parasympathetic nerve discharge during microinjection GABA and EA.
In conclusion, EA at PC6 reverse the inhibition of efferent vagal motor fibers to the DMV—mainly through the inhibition of GABA transmission—and thus promotes the activity of efferent vagus nerves and increases gastric motility.
Electroacupuncture
Dorsal motor nucleus of the vagus
Nucleus tractus solitarii
Gamma-aminobutyric acid
L-glutamic acid
Nitric oxide
Vasoactive intestinal polypeptide.
The Excel data used to support the findings of this study are included within the Supplementary Materials files (available
Mechanisms of EA in adjusting gastric motility via brainstem vagovagal neurocircuits remain largely unknown. The aim of this study was to investigate mechanisms of EA in adjusting gastric motility. EA at PC6 improves gastric motility via brainstem vagovagal neurocircuits and GABA transmission. This study further proves the effects and mechanisms of EA for adjusting gastric motility. It will help complete the biological mechanism of EA.
The study was reviewed and approved by the Institutional Review Board of Nanjing University of Chinese Medicine.
All experimental manipulations were undertaken in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health), and the study was approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine.
The authors declare that there are no conflicts of interest to disclose.
Yu Z and Xu B conceived and designed the experiments; Lu MJ performed the experiments and wrote the paper; Chen CC and Li W performed the experiments; Lu MJ and Yu analyzed the data; all authors read and approved the final version of the article to be published.
The paper is supported by the National Natural Science Foundation of China, No. 81373749, No. 81574071, No. 81673883, and No. 81873238.
Sheet1 (gastric pressure) was used to prove the effect of EA at PC6 on gastric motility (control group, vagotomy group, and sympathectomy group). Sheet2 (nerve discharge) was used to prove the effect of EA at PC6 on autonomic nerve discharge. Sheet3 (microinjection) was used to prove the effect of L-Glu and GABA microinjection into DMV on gastric motility and the effect of EA at PC6 on gastric motility after microinjection of L-Glu and GABA into the DMV.