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Acupuncture-Stimulated Activation of Sensory Neurons
J Acupunct Meridian Stud 2012;5(4):148e155
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com
- RESEARCH
ARTICLE
-
Acupuncture-Stimulated Activation of Sensory Neurons* Min-Hee Kim, Yang-Chun Park, Uk Namgung* Department of Oriental Medicine, Daejeon University, Daejeon, Republic of Korea Available online Jun 16, 2012 Received: Dec 22, 2011 Revised: Mar 9, 2012 Accepted: Mar 16, 2012 KEYWORDS acupuncture; electroacupuncture; GAP-43; sensory neuron; vagus nerve; zusanli
Abstract Acupuncture is one of the key therapeutics in clinical oriental medicine, and recent studies using experimental animals have begun to provide the pathophysiological basis for the efficacy of acupuncture. Here, we investigated neuronal responses in rodent models given acupuncture stimulation. In both mice and rats, acupuncture stimulation at zusanli (ST36) generated an increased expression of axonal growth-associated protein (GAP-43) in the sensory neurons of the dorsal root ganglion (DRG). Electroacupuncture stimulation at ST36 in rats induced GAP-43 mRNA and protein expression in DRG neurons at the levels of lumbar 4 and 5. Stimulation on a non-acupuncture site as a sham control induced GAP-43 expression as well, but the induction level was lower than it was with acupuncture. We further found that acupuncture stimulation upregulated phosphoErk1/2 signals in DRG neurons. Electroacupuncture stimulation induced c-Fos expression in the neurons of the dorsal motor nucleus of the vagus nerve (DMV), which was identified by retrograde tracing. These data suggest that acupuncture stimulation may generate physiological effects on the autonomic nervous system via the activation of a somatosensory pathway.
1. Introduction Acupuncture is one of the key therapeutics in clinical oriental medicine and its efficacy has become more widely accepted worldwide than before. According to the philosophical theory *
in oriental medicine, acupuncture stimulation relieves the dysfunctional blockage of meridian channels through which ‘qi,’ the life energy, flows. A growing body of evidence shows that acupuncture stimulation can engender pathophysiological consequences which alleviate disease symptoms for
Declaration of any source of financial income: None. * Corresponding author. Department of Oriental Medicine, Daejeon University, 96-3 Yongun-dong, Daejeon 300-716, Republic of Korea. E-mail: [email protected]
Copyright ª 2012, International Pharmacopuncture Institute http://dx.doi.org/10.1016/j.jams.2012.05.002
Neuronal activation by using acupuncture several organs, including those in the endocrine, immune and nervous systems [1,2]. Several lines of theoretical and experimental studies, including brain imaging studies, implicate that the nervous system may be involved in transmitting acupuncture signals into the target organ, in which traditional nerve-reflex theory, gate control theory, and neurotransmitter release theory are considered [2,3]. Most widely demonstrated is the pain relief, in which the acupuncture stimulation regulates the neuronal pathway responsible for pain suppression [4]. Emerging evidence reveals that acupuncture therapy is effective for treating disorders such as gastritis, vasomotor syndrome in breast cancer patients, and neuronal death as shown in Parkinson’s animal model [5e7]. A recent study showed that stimulation at the zusanli (ST36) acupuncture point (acupoint) in mice induces purinergic receptor activation, which inhibits the transmission of pain signals to the brain [8]. Interestingly, purinergic receptor activation in the sciatic nerve increases the synthesis of axonal growth-associated protein (GAP-43) in dorsal root ganglion (DRG) sensory neurons [9]. GAP-43 is the neural-specific protein known to play a role in neuronal development and activity-dependent synaptic plasticity [10,11]. However, whether and/or how acupuncture stimulation can activate a neuronal pathway resulting in physiological consequences is largely unknown. In some studies, acupuncture stimulations on non-meridian, as well as meridian, sites are similarly effective, raising the issue of the physiological identity of the acupoint and the placebo effects [12,13]. Here, we investigated the effects of acupuncture stimulation on nervous system activation in mice and rats. In order to determine acupoint-specific neuronal responses, we examined the effects of sham acupuncture at the same time, and both classic needle acupuncture and electroacupuncture were employed. Our data show that acupoint stimulation generates neuronal responses in terms of increased expression of GAP-43 and Erk1/2 activation in DRG sensory neurons and induction of c-Fos expression in neurons of the dorsal vagal complex (DVC) area.
2. Materials and methods
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2.2. Acupuncture stimulation Acupuncture needles (0.20 7 mm) were purchased from Haeng Lim Seo Won Acuneedle Company (Seoul, Korea). Rats and mice were anesthetized with 80 mg/kg of ketamine and 5 mg/kg of xylazine, and acupuncture therapy was performed similarly as described [4,8]. In mice, the needle was inserted in the zusanli point (ST36; 3e4 mm below and laterally 1e2 mm for the midline knee to a depth of 2e3 mm). For rats, the needle was inserted in the corresponding ST36 (6e7 mm below and laterally 2e3 mm to a depth of 4e5 mm). For sham stimulations, the needle was inserted at the points 5 mm and 10 mm lateral to ST36 in mice and rats, respectively. These locations are known to be free from classical acupoints. Acupuncture needles were inserted for 30 minutes, with a slow rotation every 5 minutes. Animals were subjected to acupuncture therapy for 3 days and sacrificed at day 4. Electroacupuncture was performed at the ST36 point by using the same kinds of needles as above. After the needle had been inserted into the shaven skin, electric stimulation was given at 1 Hz, using an electroacupuncture stimulator (Pulse Generator PG-306, Suzuki Iryoki, Oita-Shi, Japan) for 30 minutes, with the lowest and the intermediate modes of intensity for mice and rats, respectively. Stimulation was given once per day for 3 days, and the animals were sacrificed 30 minutes or 24 hours after the last acupuncture treatments.
2.3. Retrograde tracing and c-Fos analysis Mice were anesthetized with ketamine and xylazine, and fluorogold (FG; 2 mL of 5% saline, Molecular Probes, Eugene, OR, USA) was injected into the exposed vagus nerve at the cervical level by using a Hamilton syringe (Innovative Labor System, Stutzerbach, Germany). Some animals were subjected to sciatic nerve injury immediately after FG injection, and some other animals were given electroacupuncture on the 5th day after FG injection for 3 days thereafter and were sacrificed 30 minutes after the last acupuncture. All animals were sacrificed 7 days after FG injection, and coronal brain sections (30 mm sections) were collected. Fluorescence images of FG-labeled neurons in the brain sections were captured and analyzed by using Photoshop software. The brain sections on the slides were subjected to immunofluorescence staining with anti-c-Fos antibody (Santa Cruz Biotech, Santa Cruz, CA, USA 1:400).
2.1. Materials 2.4. Immunofluorescence staining Sprague-Dawley rats (male, 200e250 g, Dae Han Biolink, Eumseong-gun, Chungcheongbuk-do, Korea) and albino ICR mice (male, 20e25 g Dae Han Biolink, Eumseong-gun, Chungcheongbuk-do, Korea) were maintained in an animal room with regulated temperature (22 C), 60% humidity, and a 12-hour light and 12-hour dark cycle. Animals were allowed to eat commercial chow (Dae Han Biolink) and drink water ad libitum. All protocols involving live and postoperative animal care were approved by the Daejeon University Institutional Animal Use and Care Committee, and were in accordance with the Animal-Use Statement and Ethics Committee Approval Statement for Animal Experiments provided by Daejeon University (Daejeon, Korea).
DRG tissues were embedded and frozen at -20 C. Sections (20 mm thickness) were cut on a cryostat and mounted on positively charged slides. Immunofluorescence staining was performed as described previously [14]. Briefly, sections were fixed with 4% paraformaldehyde and 4% sucrose in PBS at room temperature for 40 minutes, permeabilized with 0.5% Nonidet P-40 in phosphate-buffered saline (PBS), and blocked with 2.5% horse serum and 2.5% bovine serum albumin for 4 hours at room temperature. Sections were incubated with anti-GAP-43 antibody (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-Erk1/2 antibody (1:400; Cell Signaling, Danvers, MA, USA), or anti-
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bIII-tubulin antibody (1:200, TUJ1, Covance, Princeton, New Jersey, USA), after which they were incubated with rhodamine-labeled goat anti-rabbit secondary antibody (1:400, Molecular Probes, Eugene, OR, USA) or fluoresceinlabeled sheep anti-mouse antibody (1:400, Molecular Probes) in 2.5% horse serum and 2.5% bovine serum albumin for 1 hour at room temperature and cover-slipped with gelatin mount medium. We included control sections treated with secondary antibody alone. In cases when the nonspecific signals were high, the data were excluded for further analysis. Sections were viewed with a fluorescence microscope (Nikon, Tokyo, Japan) and the images were captured using a Nikon camera. The merged images were produced by using the layer blending mode options of the Adobe Photoshop.
2.5. RT-PCR Total RNA was isolated from the DRGs at lumbar levels 4 and 5 by using Easy-BLUE reagent (Intron, Sungnam, Korea). cDNA was prepared using 2 mg of total RNA as a template for a reverse transcription (RT) reaction with MMLV reverse transcriptase (Promega, Madison, WI, USA) and random primer (Bioneer, Daejeon, Korea) for 1 hr at 37 C. For PCR amplification of GAP-43 and actin cDNA, the RT reaction was diluted 4-fold in H2O, and 5 mL of cDNA in 80 mL of total reaction volume was used for PCR with Taq DNA polymerase (Takara, Ohtsu, Japan). For quantitative comparison of GAP-43 mRNA expression among samples, 30 cycles of amplification was optimal for both GAP-43 and actin RTPCR. The primer sequences used for PCR were forward primer (50 -GATGCAGCCCCAGCCACCAG-30 ) and reverse primer (50 -TCAGGTGGGGGCAACGTGGA-30 ) for GAP-43 mRNA, and forward primer (50 -CACACTGTGCCCATCTATGA30 ) and reverse primer (50 -TACGGATGTCAA CGTCACAC-30 ) for actin mRNA. The amplified DNA sizes were 454 bp and 409 bp for GAP-43 and actin, respectively. PCR-amplified DNA products for individual samples were analyzed on agarose gels. The images of these gels were transferred to Photoshop images and quantified using the i-Solution software (Image & Microscope Technology, Daejeon, Korea).
2.6. Statistical analysis Data were presented as mean standard error of mean (sem). The mean numbers of data in individual groups were compared by using the one-way ANOVA test followed by the Tukey test (SPSS computer software version 12.0), and statistically significant differences were reported as *p < 0.05, **p < 0.01, or ***p < 0.001.
Figure 1 Regulation of GAP-43 expression in mice given acupuncture stimulation. (A) RT-PCR of GAP-43 mRNA in DRG tissues. Mice were subjected to acupuncture stimulation by inserting the needle on ST36 (ACU) and to sham stimulation and sciatic nerve injury (SNI). A DRG from an intact animal was used as the control. (Upper) Representative data on RT-PCR for GAP-43 and actin as an internal loading control. (Bottom) Quantification of the band intensity in a ratio of GAP-43 to actin (number of independent experiments Z 4). Data denote mean sem (**p < 0.01, ***p < 0.001 vs. intact group). (B) Immunofluorescence staining of GAP-43 of the DRG of animal groups with different treatments. Scale bar: 200 mm.
3. Results To determine whether acupuncture stimulation on ST36 triggers neuronal responses, we investigated GAP-43 mRNA expression by using RT-PCR in the DRG sensory neurons of mice. The basal level of GAP-43 mRNA was found in the intact tissue, and its level was slightly elevated by sham acupuncture stimulation and was significantly increased by ST36 acupuncture (Fig. 1A). A quantitative comparison of GAP-43 mRNA between the acupuncture and the sham groups showed GAP-43 expression to be 34% higher in the
acupuncture group; however, the difference was not statistically significant. A robust increase in GAP-43 mRNA was caused by injury stimulation on the nerve. Immunofluorescence staining showed that GAP-43 signals were detected in the DRG sensory neurons of intact mice and that the overall signal intensities in the sham, acupuncture, and injury groups were elevated in a proportional manner as mRNA increased (Fig. 1B). To examine whether GAP-43 expression was similarly induced in the rat model, we gave needle stimulation to
Neuronal activation by using acupuncture ST36, and GAP-43 expression was investigated in the DRG. As shown in Fig. 2A, both acupuncture and sham stimulations generated significant increases in GAP-43 mRNA expression compared to the intact control, but the effects were more significant for the acupuncture group than for the sham group (Fig. 2A). Immunofluorescence staining showed more intense protein signals in animal groups given acupuncture stimulation and nerve injury than in the intact control (Fig. 2B). We noted that more neurons in the acupuncture-stimulated groups were GAP-43-positive than in the intact control group. In rodents, sensory neurons for the sciatic nerves are distributed in the DRGs at lumbar levels 4e6. To localize the DRG neurons specifically responding to ST36 and to examine the reproducibility of acupuncture effects, we
151 performed electroacupuncture at ST36 in the rats and analyzed GAP-43 expressions in the DRGs at lumbar levels 4 and 5 separately. In both DRGs, GAP-43 mRNA was increased by sham stimulation, but further increased by acupuncture stimulation (Figs. 3A and 3B). Immunofluorescence staining revealed that among the different preparations, consistent GAP-43 signals were found in the DRG neurons at lumbar level 4 in the acupuncture group, and a similar pattern was also observed in the sham group, although the overall protein level was lower than it was in the acupuncture group (Fig. 3C). In the intact animals, GAP43 signals were much weaker than they were in the acupuncture group. We further investigated whether electroacupuncture stimulation activated phospho-Erk1/2 signals in the rat DRG neurons. As shown in Fig. 4A, basal levels of phospho-Erk1/ 2 signals were observed in the intact group, and in the sham control, some neurons showed moderately elevated signals. More intense signals were found in both the acupuncture and the sciatic nerve injury groups. Phospho-Erk1/2 signals were co-localized with neuronal marker protein bIII-tubulin (Fig. 4B), indicating that acupuncture stimulation induced Erk1/2 activation as well as GAP-43 in DRG sensory neurons. To explore the possibility that acupuncture stimulation on ST36 had any neurophysiological interaction with the autonomic nervous system, we investigated the activation of neurons in the DVC of the mouse brain where the vagal afferent and efferent fibers communicate with the visceral organs. Electroacupuncture on ST36 and sciatic nerve injury induced c-Fos signals over the DMV and the nucleus of the solitary tract (NST) (Fig. 5AeC). Retrograde labeling of DMV neurons after the injection of FG into the peripheral vagus nerve revealed that some, but not all, of the c-Fos signals were co-localized with FG-labeled DMV neurons (Fig. 5B). Additionally, weak c-Fos signals were scattered over the NST area after electroacupuncture stimulation (Fig. 5C).
4. Discussion
Figure 2 GAP-43 expression in rat DRG after acupuncture stimulation. (A) RT-PCR analysis of GAP-43 mRNA in the DRG. Animals were treated with acupuncture stimulation by inserting needles on ST36 (ACU) and with sham stimulation and sciatic nerve injury (SNI). (Upper) Representative data on RTPCR for GAP-43 and actin as an internal loading control. (Bottom) Quantification of the band intensity in a ratio of GAP43 to actin (number of independent experiments Z 4). Data denote mean sem (*p < 0.05, **p < 0.01 vs. intact group). (B) Immunofluorescence staining of GAP-43 in the DRG of animal groups with different treatments. Scale bar: 100 mm.
A growing body of evidence shows that acupuncture stimulation in an experimental animal generates pathophysiological responses, which may reflect a clinical correlation to acupuncture therapy. However, a mechanistic basis on how the acupuncture generates physiological responses is not known, although the philosophical theory states that acupoint stimulation invigorates the meridian flow of ‘qi’ thus balancing yin-yang. As an initial step to elucidate the potential role of the nervous system in mediating acupuncture stimulation, we examined the responsiveness of DRG sensory neurons following ST36 stimulation in mice and rats, where all of the peripheral somatosensory inputs are transmitted to the spinal cord via DRG sensory neurons. Our data show that acupuncture stimulation clearly induces signals of GAP-43 and phospho-Erk1/2 in DRG neurons. To characterize acupuncture-specific responsiveness, we analyzed DRG neuronal responses to sham acupuncture in parallel. Our data show that a certain level of neuronal response to sham stimulation is generated, although the extent of the responsiveness is weaker than that of acupuncture stimulation. We further demonstrated that acupuncture-mediated ascending signals induced c-Fos
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Figure 3 GAP-43 expression in the rat DRG after electroacupuncture. RT-PCR analyses of GAP-43 mRNA in the DRG at (A) lumbar level 4 and (B) lumbar 5. Animals were treated with electroacupuncture stimulation by inserting the needle on ST36 (EACU) and with sham stimulation and sciatic nerve injury (SNI). (Upper) Representative data on RT-PCR for GAP-43 and actin as an internal loading control. (Bottom) Quantification of the band intensity in a ratio of GAP-43 to actin (number of independent experiments Z 4). Data denote mean sem (*p < 0.05, **p < 0.01, ***p < 0.001 vs. intact group). (C) Electroacupuncture at ST36 (EACU) and sham stimulation were given to individual animals (#1 and #2 per experimental group) and these animals were used for the GAP-43 analyses of the DRG at lumbar levels 4 and 5 by immunofluorescence staining. Scale bar: 100 mm.
signals in the DVC, some of which were localized to the DMV neurons. As the primary target molecule determining acupuncture-specific neuronal responses, we selected GAP43. GAP-43 is expressed in developing neurons and in some adult neurons which are involved in activity-dependent synaptic plasticity [11]. Possibly, GAP-43 is most clearly induced at the gene expression level after peripheral nerve injury. Injuries on either peripheral nerves or central nervous system axons generate retrograde signals which are transmitted into the cell body and trigger regenerative responses in the cell nucleus [15]. Purinergic receptor (P2Y2) activation by ATP-gS injection in the sciatic nerve
has been reported to upregulate GAP-43 expression in DRG sensory neurons [9]. Interestingly, acupuncture stimulation on ST36 in mice has induced focal increases in ATP, ADP, AMP, and adenosine, as well as adenosine A1 receptor activation, which is known to generate antinociceptive action [8]. Thus, we speculate that purines released by ST36 stimulation may initiate retrograde signaling events such as GAP-43 production in the soma. Our data showed that GAP-43 mRNA expression was increased by 130% and 30% by ST36 acupuncture in mice and rats, respectively. There were some increases caused by sham stimulation, but the induction levels were consistently lower than those caused by acupuncture, implying
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Figure 4 Phospho-Erk1/2 signals in rat DRG after electroacupuncture. (A) After acupuncture treatments, phospho-Erk1/2 signals were analyzed by immunofluorescence staining in the DRG of individual animals. (B) Double immunofluorescence staining of DRG sections with anti-phospho-Erk1/2 antibody and anti-bIII-tubulin antibody (TUJ1). The merged image shows that phospho-Erk1/2 and bIII-tubulin signals are highly colocalized (in yellow). The images in (B) are representatives from DRG sections prepared from the rats given electroacupuncture. Scale bar: 100 mm.
acupuncture-specific effects. When we applied an alternative stimulation paradigm with electroacupuncture to the same acupoint, overall responses in terms of GAP-43 induction were similar to those of conventional acupuncture in the rats; GAP-43 mRNA levels were comparable between the DRGs at lumbar levels 4 and 5, although the induction was more consistent in the DRG at lumbar level 4. Although further studies are required to confirm whether GAP-43 is a reliable indicator for acupuncture-specific neuronal responses, our experimental approach to exploring two rodent species has provided convincing evidence that nervous system activation appears to mediate acupuncture stimulation. As another indicator of neuronal responses, Erk1/2 activation was investigated. Phospho-Erk1/2 is known to be increased in response to diverse external stimulations and to be associated with activation of neurons in culture and in vivo systems [16]. At this moment, whether GAP-43 and phospho-Erk1/2 are functionally linked to each other in DRG neurons is not known. Presumably, a retrograde signal of phospho-Erk1/2 may induce cell-body responses, including GAP-43 expression in the nucleus [17], which is consistent with pharmacological demonstration of Erk1/2dependent GAP-43 regulation [9]. How can acupuncture-mediated somatic nerve activation lead to physiological and pathological consequences
for visceral organs? While pathological responses such as gastric motility changes following acupuncture support the notion that vagus nerve activation may be one of the major targets for acupuncture stimulation [18,19], underlying neurophysiological mechanisms are still elusive. Previous studies showed that the peripheral somatosensory signals could be integrated into the DVC neuronal circuits composed of the NST, the DMV, and the area postrema [20e22]. Our study shows that c-Fos signals after electroacupuncture at ST36 were detected in the DMV and the NST, but the signal was much stronger in the DMV neurons as identified by FG dye retrogradely traced from the peripheral vagus nerve. The distribution patterns of c-Fos in the NST and the DMV neurons are somewhat different from those in the previous reports [23e25], which may reflect variations in the experimental paradigms, including the stimulation protocol. NST neurons receive visceral afferent signals via the solitary tract and communicate with numerous ascending and descending neural pathways between the vagus nerve and the brain. The DMV integrates ascending somatosensory inputs, as well as NST neurons, and relays visceral outputs to internal organs [26]. Previous studies reported the generation of an evoked potential in the NST area or the neurons in the rostro ventrolateral medulla (RVLM) following electric stimulation on the periphery [20,27]. Interestingly, acupuncture at ST25 was
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Figure 5 Co-localization of c-Fos signals with FG-labeled DMV motor neurons in mice after electroacupuncture. (A) Hematoxylin and eosin staining of coronal brain sections. Fluorescence images of FG-labeled neurons and c-Fos signals in the (B) DMV and the (C) NST areas. While FG-labeled neurons by retrograde tracing were observed exclusively in the DMV, but not the NST, areas, c-Fos signals were seen in both the DMV and the NST areas. AP Z area postrema; NST Z nucleus of the solitary tract; DMV Z dorsal motor nucleus of the vagus nerve; CBL Z cerebellum. The scale bars in (A) and (B) represent 300 mm and 100 mm, respectively.
reported to increase c-Fos signals in RVLM [23]. Thus, acupuncture-mediated somatosensory signals may affect DMV neurons via indirect neuronal circuits in the lower brain stem areas. In summary, we found that acupuncture stimulation on ST36 activates DRG sensory neurons and DMV neurons. Future studies on characterizing the neuronal circuitry in the DVC that innervates vagal efferents are critical in understanding the pathophysiological bases for acupuncture-specific neural mechanisms.
Acknowledgment This work was supported by Daejeon University Research Fund.
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3. Dhond RP, Yeh C, Park K, Kettner N, Napadow V. Acupuncture modulates resting state connectivity in default and sensorimotor brain networks. Pain. 2008;136:407e418. 4. Zhao ZQ. Neural mechanism underlying acupuncture analgesia. Prog Neurobiol. 2008;85:355e375. 5. Lux G, Hagel J, Ba ¨cker P, Ba ¨cker G, Vogl R, Ruppin H, et al. Acupuncture inhibits vagal gastric acid secretion stimulated by sham feeding in healthy subjects. Gut. 1994;35:1026e1029. 6. Walker EM, Rodriguez AI, Kohn B, Ball RM, Pegg J, Pocock JR, et al. Acupuncture versus venlafaxine for the management of vasomotor symptoms in patients with hormone receptorpositive breast cancer: a randomized controlled trial. J Clin Oncol. 2010;28:634e640. 7. Park HJ, Lim S, Joo WS, Yin CS, Lee HS, Lee HJ, et al. Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model. Exp Neurol. 2003;180:93e98. 8. Goldman N, Chen M, Fujita T, Xu Q, Peng W, Liu W, et al. Adenosine A1 receptors mediate local anti-nociceptive effects of acupuncture. Nat Neurosci. 2010;13:883e888. 9. Arthur DB, Akassoglou K, Insel PA. P2Y2 receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation. Proc Natl Acad Sci U S A. 2005;102: 19138e19143. 10. Aigner L, Arber S, Kapfhammer JP, Laux T, Schneider C, Botteri F, et al. Overexpression of the neural growth-
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associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice. Cell. 1995;83:269e278. Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 1997;20:84e91. Schneider A, Enck P, Streitberger K, Weiland C, Bagheri S, Witte S, et al. Acupuncture treatment in irritable bowel syndrome. Gut. 2006;55:649e654. Ernst E. Acupunctureea critical analysis. J Intern Med. 2006; 259:125e137. Han IS, Seo TB, Kim KH, Yoon JH, Yoon SJ, Namgung U. Cdc2mediated Schwann cell migration during peripheral nerve regeneration. J Cell Sci. 2007;120:246e255. Skene JH. Axonal growth-associated proteins. Annu Rev Neurosci. 1989;12:127e156. Segal RA, Greenberg ME. Intracellular signaling pathways activated by neurotrophic factors. Annu Rev Neurosci. 1996; 19:463e489. Hanz S, Fainzilber M. Retrograde signaling in injured nerveethe axon reaction revisited. J Neurochem. 2006;99:13e19. Iwa M, Matsushima M, Nakade Y, Pappas TN, Fujimiya M, Takahashi T. Electroacupuncture at ST-36 accelerates colonic motility and transit in freely moving conscious rats. Am J Physiol Gastrointest Liver Physiol. 2006;290:G285eG292. Yin J, Chen J, Chen JD. Ameliorating effects and mechanisms of electroacupuncture on gastric dysrhythmia, delayed emptying, and impaired accommodation in diabetic rats. Am J Physiol Gastrointest Liver Physiol. 2010;298:G563eG570.
155 20. Zagon A. Sciatic and vagal sensory inputs converge onto nonbaroreceptive neurones of the rostral ventrolateral medulla. Brain Res. 2001;896:64e68. 21. Zagon A. Does the vagus nerve mediate the sixth sense? Trends Neurosci. 2001;24:671e673. 22. Potts JT, Paton JF, Mitchell JH, Garry MG, Kline G, Anguelov PT, et al. Contraction-sensitive skeletal muscle afferents inhibit arterial baroreceptor signalling in the nucleus of the solitary tract: role of intrinsic GABA interneurons. Neuroscience. 2003; 119:201e214. 23. Iwa M, Tateiwa M, Sakita M, Fujimiya M, Takahashi T. Anatomical evidence of regional specific effects of acupuncture on gastric motor function in rats. Auton Neurosci. 2007; 137:67e76. 24. Liu JH, Li J, Yan J, Chang XR, Cui RF, He JF, et al. Expression of c-fos in the nucleus of the solitary tract following electroacupuncture at facial acupoints and gastric distension in rats. Neurosci Lett. 2004;366:215e219. 25. Kim SK, Kim J, Woo HS, Jeong H, Lee H, Min BI, et al. Electroacupuncture induces Fos expression in the nucleus tractus solitarius via cholecystokinin A receptor signaling in rats. Neurol Res. 2010;32:116e119. 26. Travagli RA, Hermann GE, Browning KN, Rogers RC. Brainstem circuits regulating gastric function. Annu Rev Physiol. 2006;68: 279e305. 27. Person RJ. Somatic and vagal afferent convergence on solitary tract neurons in cat: electrophysiological characteristics. Neuroscience. 1989;30:283e295.
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com
- RESEARCH
ARTICLE
-
Acupuncture-Stimulated Activation of Sensory Neurons* Min-Hee Kim, Yang-Chun Park, Uk Namgung* Department of Oriental Medicine, Daejeon University, Daejeon, Republic of Korea Available online Jun 16, 2012 Received: Dec 22, 2011 Revised: Mar 9, 2012 Accepted: Mar 16, 2012 KEYWORDS acupuncture; electroacupuncture; GAP-43; sensory neuron; vagus nerve; zusanli
Abstract Acupuncture is one of the key therapeutics in clinical oriental medicine, and recent studies using experimental animals have begun to provide the pathophysiological basis for the efficacy of acupuncture. Here, we investigated neuronal responses in rodent models given acupuncture stimulation. In both mice and rats, acupuncture stimulation at zusanli (ST36) generated an increased expression of axonal growth-associated protein (GAP-43) in the sensory neurons of the dorsal root ganglion (DRG). Electroacupuncture stimulation at ST36 in rats induced GAP-43 mRNA and protein expression in DRG neurons at the levels of lumbar 4 and 5. Stimulation on a non-acupuncture site as a sham control induced GAP-43 expression as well, but the induction level was lower than it was with acupuncture. We further found that acupuncture stimulation upregulated phosphoErk1/2 signals in DRG neurons. Electroacupuncture stimulation induced c-Fos expression in the neurons of the dorsal motor nucleus of the vagus nerve (DMV), which was identified by retrograde tracing. These data suggest that acupuncture stimulation may generate physiological effects on the autonomic nervous system via the activation of a somatosensory pathway.
1. Introduction Acupuncture is one of the key therapeutics in clinical oriental medicine and its efficacy has become more widely accepted worldwide than before. According to the philosophical theory *
in oriental medicine, acupuncture stimulation relieves the dysfunctional blockage of meridian channels through which ‘qi,’ the life energy, flows. A growing body of evidence shows that acupuncture stimulation can engender pathophysiological consequences which alleviate disease symptoms for
Declaration of any source of financial income: None. * Corresponding author. Department of Oriental Medicine, Daejeon University, 96-3 Yongun-dong, Daejeon 300-716, Republic of Korea. E-mail: [email protected]
Copyright ª 2012, International Pharmacopuncture Institute http://dx.doi.org/10.1016/j.jams.2012.05.002
Neuronal activation by using acupuncture several organs, including those in the endocrine, immune and nervous systems [1,2]. Several lines of theoretical and experimental studies, including brain imaging studies, implicate that the nervous system may be involved in transmitting acupuncture signals into the target organ, in which traditional nerve-reflex theory, gate control theory, and neurotransmitter release theory are considered [2,3]. Most widely demonstrated is the pain relief, in which the acupuncture stimulation regulates the neuronal pathway responsible for pain suppression [4]. Emerging evidence reveals that acupuncture therapy is effective for treating disorders such as gastritis, vasomotor syndrome in breast cancer patients, and neuronal death as shown in Parkinson’s animal model [5e7]. A recent study showed that stimulation at the zusanli (ST36) acupuncture point (acupoint) in mice induces purinergic receptor activation, which inhibits the transmission of pain signals to the brain [8]. Interestingly, purinergic receptor activation in the sciatic nerve increases the synthesis of axonal growth-associated protein (GAP-43) in dorsal root ganglion (DRG) sensory neurons [9]. GAP-43 is the neural-specific protein known to play a role in neuronal development and activity-dependent synaptic plasticity [10,11]. However, whether and/or how acupuncture stimulation can activate a neuronal pathway resulting in physiological consequences is largely unknown. In some studies, acupuncture stimulations on non-meridian, as well as meridian, sites are similarly effective, raising the issue of the physiological identity of the acupoint and the placebo effects [12,13]. Here, we investigated the effects of acupuncture stimulation on nervous system activation in mice and rats. In order to determine acupoint-specific neuronal responses, we examined the effects of sham acupuncture at the same time, and both classic needle acupuncture and electroacupuncture were employed. Our data show that acupoint stimulation generates neuronal responses in terms of increased expression of GAP-43 and Erk1/2 activation in DRG sensory neurons and induction of c-Fos expression in neurons of the dorsal vagal complex (DVC) area.
2. Materials and methods
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2.2. Acupuncture stimulation Acupuncture needles (0.20 7 mm) were purchased from Haeng Lim Seo Won Acuneedle Company (Seoul, Korea). Rats and mice were anesthetized with 80 mg/kg of ketamine and 5 mg/kg of xylazine, and acupuncture therapy was performed similarly as described [4,8]. In mice, the needle was inserted in the zusanli point (ST36; 3e4 mm below and laterally 1e2 mm for the midline knee to a depth of 2e3 mm). For rats, the needle was inserted in the corresponding ST36 (6e7 mm below and laterally 2e3 mm to a depth of 4e5 mm). For sham stimulations, the needle was inserted at the points 5 mm and 10 mm lateral to ST36 in mice and rats, respectively. These locations are known to be free from classical acupoints. Acupuncture needles were inserted for 30 minutes, with a slow rotation every 5 minutes. Animals were subjected to acupuncture therapy for 3 days and sacrificed at day 4. Electroacupuncture was performed at the ST36 point by using the same kinds of needles as above. After the needle had been inserted into the shaven skin, electric stimulation was given at 1 Hz, using an electroacupuncture stimulator (Pulse Generator PG-306, Suzuki Iryoki, Oita-Shi, Japan) for 30 minutes, with the lowest and the intermediate modes of intensity for mice and rats, respectively. Stimulation was given once per day for 3 days, and the animals were sacrificed 30 minutes or 24 hours after the last acupuncture treatments.
2.3. Retrograde tracing and c-Fos analysis Mice were anesthetized with ketamine and xylazine, and fluorogold (FG; 2 mL of 5% saline, Molecular Probes, Eugene, OR, USA) was injected into the exposed vagus nerve at the cervical level by using a Hamilton syringe (Innovative Labor System, Stutzerbach, Germany). Some animals were subjected to sciatic nerve injury immediately after FG injection, and some other animals were given electroacupuncture on the 5th day after FG injection for 3 days thereafter and were sacrificed 30 minutes after the last acupuncture. All animals were sacrificed 7 days after FG injection, and coronal brain sections (30 mm sections) were collected. Fluorescence images of FG-labeled neurons in the brain sections were captured and analyzed by using Photoshop software. The brain sections on the slides were subjected to immunofluorescence staining with anti-c-Fos antibody (Santa Cruz Biotech, Santa Cruz, CA, USA 1:400).
2.1. Materials 2.4. Immunofluorescence staining Sprague-Dawley rats (male, 200e250 g, Dae Han Biolink, Eumseong-gun, Chungcheongbuk-do, Korea) and albino ICR mice (male, 20e25 g Dae Han Biolink, Eumseong-gun, Chungcheongbuk-do, Korea) were maintained in an animal room with regulated temperature (22 C), 60% humidity, and a 12-hour light and 12-hour dark cycle. Animals were allowed to eat commercial chow (Dae Han Biolink) and drink water ad libitum. All protocols involving live and postoperative animal care were approved by the Daejeon University Institutional Animal Use and Care Committee, and were in accordance with the Animal-Use Statement and Ethics Committee Approval Statement for Animal Experiments provided by Daejeon University (Daejeon, Korea).
DRG tissues were embedded and frozen at -20 C. Sections (20 mm thickness) were cut on a cryostat and mounted on positively charged slides. Immunofluorescence staining was performed as described previously [14]. Briefly, sections were fixed with 4% paraformaldehyde and 4% sucrose in PBS at room temperature for 40 minutes, permeabilized with 0.5% Nonidet P-40 in phosphate-buffered saline (PBS), and blocked with 2.5% horse serum and 2.5% bovine serum albumin for 4 hours at room temperature. Sections were incubated with anti-GAP-43 antibody (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-Erk1/2 antibody (1:400; Cell Signaling, Danvers, MA, USA), or anti-
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bIII-tubulin antibody (1:200, TUJ1, Covance, Princeton, New Jersey, USA), after which they were incubated with rhodamine-labeled goat anti-rabbit secondary antibody (1:400, Molecular Probes, Eugene, OR, USA) or fluoresceinlabeled sheep anti-mouse antibody (1:400, Molecular Probes) in 2.5% horse serum and 2.5% bovine serum albumin for 1 hour at room temperature and cover-slipped with gelatin mount medium. We included control sections treated with secondary antibody alone. In cases when the nonspecific signals were high, the data were excluded for further analysis. Sections were viewed with a fluorescence microscope (Nikon, Tokyo, Japan) and the images were captured using a Nikon camera. The merged images were produced by using the layer blending mode options of the Adobe Photoshop.
2.5. RT-PCR Total RNA was isolated from the DRGs at lumbar levels 4 and 5 by using Easy-BLUE reagent (Intron, Sungnam, Korea). cDNA was prepared using 2 mg of total RNA as a template for a reverse transcription (RT) reaction with MMLV reverse transcriptase (Promega, Madison, WI, USA) and random primer (Bioneer, Daejeon, Korea) for 1 hr at 37 C. For PCR amplification of GAP-43 and actin cDNA, the RT reaction was diluted 4-fold in H2O, and 5 mL of cDNA in 80 mL of total reaction volume was used for PCR with Taq DNA polymerase (Takara, Ohtsu, Japan). For quantitative comparison of GAP-43 mRNA expression among samples, 30 cycles of amplification was optimal for both GAP-43 and actin RTPCR. The primer sequences used for PCR were forward primer (50 -GATGCAGCCCCAGCCACCAG-30 ) and reverse primer (50 -TCAGGTGGGGGCAACGTGGA-30 ) for GAP-43 mRNA, and forward primer (50 -CACACTGTGCCCATCTATGA30 ) and reverse primer (50 -TACGGATGTCAA CGTCACAC-30 ) for actin mRNA. The amplified DNA sizes were 454 bp and 409 bp for GAP-43 and actin, respectively. PCR-amplified DNA products for individual samples were analyzed on agarose gels. The images of these gels were transferred to Photoshop images and quantified using the i-Solution software (Image & Microscope Technology, Daejeon, Korea).
2.6. Statistical analysis Data were presented as mean standard error of mean (sem). The mean numbers of data in individual groups were compared by using the one-way ANOVA test followed by the Tukey test (SPSS computer software version 12.0), and statistically significant differences were reported as *p < 0.05, **p < 0.01, or ***p < 0.001.
Figure 1 Regulation of GAP-43 expression in mice given acupuncture stimulation. (A) RT-PCR of GAP-43 mRNA in DRG tissues. Mice were subjected to acupuncture stimulation by inserting the needle on ST36 (ACU) and to sham stimulation and sciatic nerve injury (SNI). A DRG from an intact animal was used as the control. (Upper) Representative data on RT-PCR for GAP-43 and actin as an internal loading control. (Bottom) Quantification of the band intensity in a ratio of GAP-43 to actin (number of independent experiments Z 4). Data denote mean sem (**p < 0.01, ***p < 0.001 vs. intact group). (B) Immunofluorescence staining of GAP-43 of the DRG of animal groups with different treatments. Scale bar: 200 mm.
3. Results To determine whether acupuncture stimulation on ST36 triggers neuronal responses, we investigated GAP-43 mRNA expression by using RT-PCR in the DRG sensory neurons of mice. The basal level of GAP-43 mRNA was found in the intact tissue, and its level was slightly elevated by sham acupuncture stimulation and was significantly increased by ST36 acupuncture (Fig. 1A). A quantitative comparison of GAP-43 mRNA between the acupuncture and the sham groups showed GAP-43 expression to be 34% higher in the
acupuncture group; however, the difference was not statistically significant. A robust increase in GAP-43 mRNA was caused by injury stimulation on the nerve. Immunofluorescence staining showed that GAP-43 signals were detected in the DRG sensory neurons of intact mice and that the overall signal intensities in the sham, acupuncture, and injury groups were elevated in a proportional manner as mRNA increased (Fig. 1B). To examine whether GAP-43 expression was similarly induced in the rat model, we gave needle stimulation to
Neuronal activation by using acupuncture ST36, and GAP-43 expression was investigated in the DRG. As shown in Fig. 2A, both acupuncture and sham stimulations generated significant increases in GAP-43 mRNA expression compared to the intact control, but the effects were more significant for the acupuncture group than for the sham group (Fig. 2A). Immunofluorescence staining showed more intense protein signals in animal groups given acupuncture stimulation and nerve injury than in the intact control (Fig. 2B). We noted that more neurons in the acupuncture-stimulated groups were GAP-43-positive than in the intact control group. In rodents, sensory neurons for the sciatic nerves are distributed in the DRGs at lumbar levels 4e6. To localize the DRG neurons specifically responding to ST36 and to examine the reproducibility of acupuncture effects, we
151 performed electroacupuncture at ST36 in the rats and analyzed GAP-43 expressions in the DRGs at lumbar levels 4 and 5 separately. In both DRGs, GAP-43 mRNA was increased by sham stimulation, but further increased by acupuncture stimulation (Figs. 3A and 3B). Immunofluorescence staining revealed that among the different preparations, consistent GAP-43 signals were found in the DRG neurons at lumbar level 4 in the acupuncture group, and a similar pattern was also observed in the sham group, although the overall protein level was lower than it was in the acupuncture group (Fig. 3C). In the intact animals, GAP43 signals were much weaker than they were in the acupuncture group. We further investigated whether electroacupuncture stimulation activated phospho-Erk1/2 signals in the rat DRG neurons. As shown in Fig. 4A, basal levels of phospho-Erk1/ 2 signals were observed in the intact group, and in the sham control, some neurons showed moderately elevated signals. More intense signals were found in both the acupuncture and the sciatic nerve injury groups. Phospho-Erk1/2 signals were co-localized with neuronal marker protein bIII-tubulin (Fig. 4B), indicating that acupuncture stimulation induced Erk1/2 activation as well as GAP-43 in DRG sensory neurons. To explore the possibility that acupuncture stimulation on ST36 had any neurophysiological interaction with the autonomic nervous system, we investigated the activation of neurons in the DVC of the mouse brain where the vagal afferent and efferent fibers communicate with the visceral organs. Electroacupuncture on ST36 and sciatic nerve injury induced c-Fos signals over the DMV and the nucleus of the solitary tract (NST) (Fig. 5AeC). Retrograde labeling of DMV neurons after the injection of FG into the peripheral vagus nerve revealed that some, but not all, of the c-Fos signals were co-localized with FG-labeled DMV neurons (Fig. 5B). Additionally, weak c-Fos signals were scattered over the NST area after electroacupuncture stimulation (Fig. 5C).
4. Discussion
Figure 2 GAP-43 expression in rat DRG after acupuncture stimulation. (A) RT-PCR analysis of GAP-43 mRNA in the DRG. Animals were treated with acupuncture stimulation by inserting needles on ST36 (ACU) and with sham stimulation and sciatic nerve injury (SNI). (Upper) Representative data on RTPCR for GAP-43 and actin as an internal loading control. (Bottom) Quantification of the band intensity in a ratio of GAP43 to actin (number of independent experiments Z 4). Data denote mean sem (*p < 0.05, **p < 0.01 vs. intact group). (B) Immunofluorescence staining of GAP-43 in the DRG of animal groups with different treatments. Scale bar: 100 mm.
A growing body of evidence shows that acupuncture stimulation in an experimental animal generates pathophysiological responses, which may reflect a clinical correlation to acupuncture therapy. However, a mechanistic basis on how the acupuncture generates physiological responses is not known, although the philosophical theory states that acupoint stimulation invigorates the meridian flow of ‘qi’ thus balancing yin-yang. As an initial step to elucidate the potential role of the nervous system in mediating acupuncture stimulation, we examined the responsiveness of DRG sensory neurons following ST36 stimulation in mice and rats, where all of the peripheral somatosensory inputs are transmitted to the spinal cord via DRG sensory neurons. Our data show that acupuncture stimulation clearly induces signals of GAP-43 and phospho-Erk1/2 in DRG neurons. To characterize acupuncture-specific responsiveness, we analyzed DRG neuronal responses to sham acupuncture in parallel. Our data show that a certain level of neuronal response to sham stimulation is generated, although the extent of the responsiveness is weaker than that of acupuncture stimulation. We further demonstrated that acupuncture-mediated ascending signals induced c-Fos
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Figure 3 GAP-43 expression in the rat DRG after electroacupuncture. RT-PCR analyses of GAP-43 mRNA in the DRG at (A) lumbar level 4 and (B) lumbar 5. Animals were treated with electroacupuncture stimulation by inserting the needle on ST36 (EACU) and with sham stimulation and sciatic nerve injury (SNI). (Upper) Representative data on RT-PCR for GAP-43 and actin as an internal loading control. (Bottom) Quantification of the band intensity in a ratio of GAP-43 to actin (number of independent experiments Z 4). Data denote mean sem (*p < 0.05, **p < 0.01, ***p < 0.001 vs. intact group). (C) Electroacupuncture at ST36 (EACU) and sham stimulation were given to individual animals (#1 and #2 per experimental group) and these animals were used for the GAP-43 analyses of the DRG at lumbar levels 4 and 5 by immunofluorescence staining. Scale bar: 100 mm.
signals in the DVC, some of which were localized to the DMV neurons. As the primary target molecule determining acupuncture-specific neuronal responses, we selected GAP43. GAP-43 is expressed in developing neurons and in some adult neurons which are involved in activity-dependent synaptic plasticity [11]. Possibly, GAP-43 is most clearly induced at the gene expression level after peripheral nerve injury. Injuries on either peripheral nerves or central nervous system axons generate retrograde signals which are transmitted into the cell body and trigger regenerative responses in the cell nucleus [15]. Purinergic receptor (P2Y2) activation by ATP-gS injection in the sciatic nerve
has been reported to upregulate GAP-43 expression in DRG sensory neurons [9]. Interestingly, acupuncture stimulation on ST36 in mice has induced focal increases in ATP, ADP, AMP, and adenosine, as well as adenosine A1 receptor activation, which is known to generate antinociceptive action [8]. Thus, we speculate that purines released by ST36 stimulation may initiate retrograde signaling events such as GAP-43 production in the soma. Our data showed that GAP-43 mRNA expression was increased by 130% and 30% by ST36 acupuncture in mice and rats, respectively. There were some increases caused by sham stimulation, but the induction levels were consistently lower than those caused by acupuncture, implying
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Figure 4 Phospho-Erk1/2 signals in rat DRG after electroacupuncture. (A) After acupuncture treatments, phospho-Erk1/2 signals were analyzed by immunofluorescence staining in the DRG of individual animals. (B) Double immunofluorescence staining of DRG sections with anti-phospho-Erk1/2 antibody and anti-bIII-tubulin antibody (TUJ1). The merged image shows that phospho-Erk1/2 and bIII-tubulin signals are highly colocalized (in yellow). The images in (B) are representatives from DRG sections prepared from the rats given electroacupuncture. Scale bar: 100 mm.
acupuncture-specific effects. When we applied an alternative stimulation paradigm with electroacupuncture to the same acupoint, overall responses in terms of GAP-43 induction were similar to those of conventional acupuncture in the rats; GAP-43 mRNA levels were comparable between the DRGs at lumbar levels 4 and 5, although the induction was more consistent in the DRG at lumbar level 4. Although further studies are required to confirm whether GAP-43 is a reliable indicator for acupuncture-specific neuronal responses, our experimental approach to exploring two rodent species has provided convincing evidence that nervous system activation appears to mediate acupuncture stimulation. As another indicator of neuronal responses, Erk1/2 activation was investigated. Phospho-Erk1/2 is known to be increased in response to diverse external stimulations and to be associated with activation of neurons in culture and in vivo systems [16]. At this moment, whether GAP-43 and phospho-Erk1/2 are functionally linked to each other in DRG neurons is not known. Presumably, a retrograde signal of phospho-Erk1/2 may induce cell-body responses, including GAP-43 expression in the nucleus [17], which is consistent with pharmacological demonstration of Erk1/2dependent GAP-43 regulation [9]. How can acupuncture-mediated somatic nerve activation lead to physiological and pathological consequences
for visceral organs? While pathological responses such as gastric motility changes following acupuncture support the notion that vagus nerve activation may be one of the major targets for acupuncture stimulation [18,19], underlying neurophysiological mechanisms are still elusive. Previous studies showed that the peripheral somatosensory signals could be integrated into the DVC neuronal circuits composed of the NST, the DMV, and the area postrema [20e22]. Our study shows that c-Fos signals after electroacupuncture at ST36 were detected in the DMV and the NST, but the signal was much stronger in the DMV neurons as identified by FG dye retrogradely traced from the peripheral vagus nerve. The distribution patterns of c-Fos in the NST and the DMV neurons are somewhat different from those in the previous reports [23e25], which may reflect variations in the experimental paradigms, including the stimulation protocol. NST neurons receive visceral afferent signals via the solitary tract and communicate with numerous ascending and descending neural pathways between the vagus nerve and the brain. The DMV integrates ascending somatosensory inputs, as well as NST neurons, and relays visceral outputs to internal organs [26]. Previous studies reported the generation of an evoked potential in the NST area or the neurons in the rostro ventrolateral medulla (RVLM) following electric stimulation on the periphery [20,27]. Interestingly, acupuncture at ST25 was
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Figure 5 Co-localization of c-Fos signals with FG-labeled DMV motor neurons in mice after electroacupuncture. (A) Hematoxylin and eosin staining of coronal brain sections. Fluorescence images of FG-labeled neurons and c-Fos signals in the (B) DMV and the (C) NST areas. While FG-labeled neurons by retrograde tracing were observed exclusively in the DMV, but not the NST, areas, c-Fos signals were seen in both the DMV and the NST areas. AP Z area postrema; NST Z nucleus of the solitary tract; DMV Z dorsal motor nucleus of the vagus nerve; CBL Z cerebellum. The scale bars in (A) and (B) represent 300 mm and 100 mm, respectively.
reported to increase c-Fos signals in RVLM [23]. Thus, acupuncture-mediated somatosensory signals may affect DMV neurons via indirect neuronal circuits in the lower brain stem areas. In summary, we found that acupuncture stimulation on ST36 activates DRG sensory neurons and DMV neurons. Future studies on characterizing the neuronal circuitry in the DVC that innervates vagal efferents are critical in understanding the pathophysiological bases for acupuncture-specific neural mechanisms.
Acknowledgment This work was supported by Daejeon University Research Fund.
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